As technologies advance and applications for uncrewed aircraft increase, the capability to conduct automated air-to-air refueling becomes increasingly important. This paper provides a review of required sensors to enable automated air-to-air refueling for an uncrewed aircraft, as well as a review of published research on the topic. Automated air-to-air refueling of uncrewed aircraft eliminates the need for ground infrastructure for intermediate refueling, as well as the need for on-site personnel. Automated air-to-air refueling potentially supports civilian applications such as weather monitoring, surveillance for wildfires, search and rescue, and emergency response, especially when airfields are not available due to natural disasters. For military applications, to enable the Air Wing of the Future to strike at the ranges required for the mission, both crewed and uncrewed aircraft must be capable of air-to-air refueling. To cover the sensors required to complete automated air-to-air refueling, a brief history of air-to-air refueling is presented, followed by a concept of employment for uncrewed aircraft refueling, and finally, a review of the sensors required to complete the different phases of automated air-to-air refueling. To complete uncrewed aircraft refueling, the uncrewed receiver aircraft must have the sensors required to establish communication, determine relative position, decrease separation to astern position, transition to computer vision, position keep during refueling, and separate from the tanker aircraft upon completion of refueling. This paper provides a review of the twelve sensors that would enable the uncrewed aircraft to complete the seven tasks required for automated air-to-air refueling. The concept of air-to-air or aerial refueling has existed for over a century and it provides significant benefits by increasing the range and flight time of the aircraft . A serieGiven the strategic advantage aerial refueling provides, aerial refueling has been used for military operations for decades. Due to the cost of integration of aerial refueling systems, the requirement for close formation flying, and the safety risks associated with the task, aerial refueling has predominately been limited to military operations only. During operations in support of Desert Storm, the United States Air Force (USAF) flew nearly 15,000 sorties dedicated to aerial refueling in which their fleet refueled approximately 46,000 aircraft with approximately 700 million pounds of fuel . Th. Th18]. To conduct probe-and-drogue refueling, the receiver pilot must visually identify the drogue within the domain to maneuver the receiver aircraft’s probe tip into the coupler of the drogue .During air-to-air refueling, the human pilot must perform object detection and localization within the pilot’s field of view (FOV). After identifying the drogue in the astern position and determining the location relative to the probe tip installed on the receiver aircraft, the pilot must then reduce the separation between the coupler and probe tip by increasing closure. The nominal closure rates range from approximately 1 to 3 knots of closure.During the approach, the pilot must continually input small flight path adjustments via the control stick to account for the drogue movement in free air. The movement of the drogue can be attributed to multiple factors to include wing flex of the tanker wing, environmental conditions such as turbulence, and finally the creation of a bow wave, which may be attributed to different aspects of the receiver airframe, as it interacts with the drogue during the final moments before engagement . AdditioThroughout the approach, the pilot must assess the probability of successful engagement. Outside of the nominal outcome of a successful engagement of the probe tip into the drogue coupler, sub nominal outcomes include “lipping the basket” by contacting the outer ringer of the drogue or the drogue impacting a portion of the receiver aircraft, such as the nose cone or canopy. These conditions may result in damage ranging from minimal to safety of flight depending on the severity of the incident.If the pilot assesses the engagement will not be successful, the pilot must discontinue the approach by decreasing the closure rates and increasing the separation between the receiver aircraft and the drogue in a safe and controlled manner. The decision to reattempt engagements after a failed attempt will be made after an assessment of the root cause of the failed attempt. Based on any damage during the failed attempt, identification of root cause of the error, probability of error correction, and on mission planning factors to include acceptable airfield availability, allowable level of risk for success of the mission, and physiological state  of the pilot, the pilot may reattempt the engagement if deemed necessary.Once the pilot has successfully engaged the probe tip and the coupler of the drogue, the pilot must continue to decrease the range between the tanking aircraft and receiver aircraft by approximately 5 to 25 feet to enter the Fuel Transfer Zone (FTZ), as annotated on the long hose markings. The FTZ represents a corridor in space determined by the length of hose extended from the tanker aircraft. When the receiving aircraft is in the FTZ, fuel will begin to transfer from the tanker aircraft to the receiver aircraft. When the hose is fully extended prior to engagement, no fuel is being transferred. As the receiver aircraft engages the coupler with the probe tip and begins to decrease the range between the tanking aircraft and receiver, the hose will begin to retract, but fuel will not transfer until the hose reel retracts to a predetermined distance, which represents the FTZ. The pilot receives visual confirmation upon entering the FTZ through a combination of lights installed on either the fuselage of the tanker aircraft or the ARS connected to the wing, as well as the hose markings being in the correct positions. Once established in the FTZ, the pilot then transitions to formation position keeping. Directional position keeping uses the tanker aircraft wing, fuselage, or other marking to help maintain appropriate vertical and horizontal separation. Forward/aft positioning within the FTZ is done using the hose markings on the tanker hose by keeping the first long marking within the tanker hose outlet.The amount of time required to complete aerial refueling depends on a multitude of variables to include: the tanker’s fuel delivery rate, the maximum fuel rate the receiver can accept, and the total amount of fuel to be transferred. The receiver can confirm fuel flow through the static green light on the drogue system signal light or on their fuel quantity management indications in the receiver cockpit. Two methods exist for determining that the receiver aircraft received the desired amount of fuel. Either the tanker aircraft programs the desired amount of fuel to transfer or the receiver pilot monitors the fuel quantity to determine the desired amount has been transferred.Once complete with the transfer of fuel, the receiver will begin separation by decreasing the energy state of the receiver to increase the distance between the tanker and receiver. As the receiver begins increasing the range from the tanker, the hose will begin to unreel to increase the length available until the hose reaches the maximum length attainable. Upon reaching the maximum length of the hose, the probe tip will disengage from the coupler and the receiver aircraft begins to transition to right echelon to either hold until the wingmen are complete or complete final administrative procedures prior to transitioning to the next phase of the mission .Aerial refueling requires experienced pilots to identify objects in free space, such as the drogue, probe tip, and coupler, prior to maneuvering the receiver aircraft into close proximity of the tanker aircraft to complete an engagement of an object approximately 3 to 4 inches in diameter into a coupler of slight larger proportions. This challenging task can only be completed by adapting the inputs that are dependent on a multitude of environmental factors. While not impossible to automate, a UA capable of A3R would require multiple sensors to provide information to the UA. As of the date of this publication, only demonstrations of this capability have been accomplished.While no UA currently exists in the Department of Defense (DoD) that has been authorized to conduct the A3R portion of a mission, the task has been accomplished on both crewed aircraft and UA as a demonstration. Extensive research has been conducted on the algorithms required to complete the task from as a receiver, specifically the computer vision (CV) portion of A3R ,34,35,36The first crewed aircraft to demonstrate A3R resulted from a combined research effort between the National Aeronautics and Space Administration (NASA) and the Defense Advanced Research Projects Agency (DARPA) in 2005 and 2006 . Phase 1On 22 April 2015, the USN, using the Northrop Grumman built X-47B Unmanned Carrier Air Vehicle Demonstrator (UCAS-D), demonstrated the A3R portion of the mission using the X-47B as the receiver and an Omega Aerial Refueling Services Boeing 707 tanker . The demSince the completion of the X-47B demonstration program, the USN has not conducted additional A3R testing with a UA acting as the receiver aircraft. In 2021, during the initial testing of the Boeing produced MQ-25, the USN did demonstrate the capability of a UA to serve as the tanker aircraft when a crewed F/A-18F , E-2D 4, and F-3Establish communications. Prior to initiation of refueling, communications and datalinks must be established between the UA and the tanker, and any supporting entities that are involved, such as the Ground Control Station (GCS) and Aerial Vehicle Operator (AVO). The GCS receives all pertinent data related to the A3R and the AVO monitors the performance of the UA . As technology advances, the role of the AVO will likely diminish as additional assurance in the system becomes available.Determine relative position. Determine the initial position of the UA relative to the tanker.Phase 1. Transitioning from Execution Portion to A3R Portion of Mission.3.Decrease separation to astern proximity. Once cleared by the tanker, decrease the separation of the two aircraft in a safe and predictable manner.Phase 2. UA Receiver Joins on Tanker.4.Transition to computer vision (CV). Once cleared by the tanker, arrive at the astern position to transition from position keeping provided by a data link or navigation aid to position keeping provided by a CV system. Object identification is required to provide location information to the guidance, navigation, and control (GNC) system; this information will then be translated by the UA to decrease the distance between the probe tip and the coupler, once cleared to contact by the tanker.5.Position keeping during refueling. Transition back to relative position keeping upon successful engagement to allow fuel to transfer within the FTZ.Phase 3. UA Transitions from Astern to Engagement.6.Initiate separation after refueling. Once refueling is complete and cleared by the tanker, the receiver aircraft decreases airspeed to begin to increase the distance between the receiver and tanker. Upon reaching the limits of the hose reel, the probe tip will disconnect from the coupler and the receiver returns to astern position prior to transitioning to right echelon, when cleared by the tanker, for final administrative procedures.Phase 4. UA Receiver Separates from Tanker.7.Final separation to enable independent flight. Once cleared by the tanker, safely maneuver to increase separation between the two aircraft such that the UA can transition away from relative position keeping to continue the next mission task.Phase 5. UA Receiver Proceeds on Mission.Aerial refueling has been an integral capability supporting both strategic and tactical aircraft within the DoD for decades . HistoriThe relative location of the receiver and tanker begins at maximum separation in the bottom right of Technology, sensors, and supporting control systems may be located on the UA, on the drogue, on the tanker, or on peripheral sites; for simplicity, this discussion will assume that the technology is on the UA and tanker only. It is worth noting that advances in guidance control systems for other applications, such as landing UAs on moving platforms  and landThe UA must be able to complete all guidance and control tasks throughout the entire operational design domain (ODD) or the domain in which the system is intended to operate. The ODD of the UA will be determined by the desired flight envelope  capabilities of both aircraft, the need to operate in different environmental conditions  and the requirements of the intended mission of the platform . The ODD will drive not only the sensor requirements of the platform, but also the assurance requirements of the test program. An example ODD was built from guidance provided by the National Highway Safety and Transportation Administration (NHSTA) ; the ODDIn early deployments and during system development, it is common to limit the ODD to evaluate new technologies. For example, most systems are initially evaluated in clear, daylight conditions with unrestricted visibility and limited wind. As systems are matured and refined, operations in different portions of the ODD expand to encompass more challenging environments, such as night or reduced visibility operations, that reflect broader operating conditions. The operational constraints may reflect characteristics of the aircraft, as well as the sensing, control, and communications hardware and software.Removing the human from the aircraft eliminates system capabilities , but it also removes the constraints in that system. Without a human, the system can operate in harsher conditions, and safety considerations may change significantly when human life in the aircraft is not at risk. Although not listed explicitly in the ODD or the tasks, safety remains a top priority for all aviation activities, including air-to-air refueling. Safety includes the safety of any aircraft occupants, safety of other aircraft in the vicinity, and safety of people on the ground.The previous sections provided a description of A3R and a conceptual framework for the required steps for A3R for a UA. This section provides a discussion of the sensor requirements for UA A3R. These sensors support the tasks listed in The framework for A3R described in this paper was developed using the current NATO standards for crewed United States Aerial Refueling  and uncrWhen determining types of sensors required for specific tasks, if a transmit or receive requirement exists, knowing the required frequency band will help determine the size, weight, power, and cost (SWaP-C) . TherefoMany of the sensors discussed are installed on platforms currently or previously included in the NATO inventory. Due to potential distribution limitations, only open-source articles were included in this analysis of sensors that could complete the required task. While the Lockheed Martin website lists that the F-35, the newest fifth-generation fighter in the NATO inventory, includes an active electronically-scanned array (AESA) radar, distributed aperture system (DAS), and Electro Optical Targeting System (EOTS) , informaTo complete A3R, reliable data links that support both voice and data transfer are critical. Data transfer is important for automation functions and monitoring; voice transfer is needed for coordination and communication to support humans that are monitoring and/or interacting with the system. Communications are needed between the tanker and the receiver for automated operations, and the requirements for apertures include both transmission (T) and receiver (R) capabilities.In addition to communication between the tanker and the UA, there must be communication with the GCS for the AVO to monitor the UA. Communication with the AVO enables a human or team of humans to monitor the operation of the tanker and potentially multiple receivers. Similarly, multiple GCSs controlling multiple UAs must be able to transmit both data and voice for humans to coordinate operations. A GCS serves as the command and control (C2) node for a UA or team of UAs while providing flight information to the AVO in a similar fashion to that of a cockpit instrument display panel in a crewed aircraft .Depending on the level of autonomy onboard the UA, a data link may or may not be required to the AVO. The airworthiness officials certifying the UA at the platform level may not require a human either in (interacting with operations) or on the loop (monitoring operations) to conduct A3R if additional assurance is provided by a certified Run Time Assurance (RTA) . A RTA sA data link to an AVO may be installed for redundancy and/or oversight, but it may not be required for operation. Unless the UA employs a computer vision (CV)-only solution with no fusion of additional location information, such as differential global positioning system (DGPS) data , a data To transmit data and voice between multiple GCSs, if the GCSs are geographically located in a close area, a fiber optic cable or ethernet may be routed to pass information between GCSs . AlternaTo transmit data between the GCS and UA, either a LOS or BLOS data link will be required depending on the anticipated range between the GCS and UA . OperatiIn the event the UA achieves a distance where the radar horizon prohibits communication, a BLOS waveform generator and aperture would need to be installed on both the UA and GCS. BLOS communications could occur between multiple aircraft at high altitudes and long distances or between Satellite constellations through Satellite Communication (SATCOM), which operate in the K Band (18 to 27 GHz) . In the The tanker and receiver aircraft must have an accurate assessment of the relative position throughout refueling. To calculate the relative position, the receiver aircraft could employ a blended solution between the aircraft’s inertial navigation system (INS), Global Navigation Satellite System (GNSS), and the CV system . In 2001Modern day INS are dead reckoning systems that utilize the initial velocity and position information, as well as information from three-ring laser gyro (RLG) systems mounted on orthogonal axes . After iThe ability to integrate GNSS information through a receiver attached to a UA is a critical component of a UA’s guidance, navigation, and control (GNC) system, since it is necessary to address INS drift and since accurate location information is critical for all aspects of uncrewed flights. The space segment of GNSS is comprised of 24 satellites in medium Earth orbit (MEO), the user segment consists of a receiver and ground module, and the control segment for monitoring proper functionality of the constellation. GNSS provides the UA position and time information when in LOS of four or more satellites . AdditioThe operational environment is an important consideration when determining the optimal sensors for relative position. GNSS jammers may threaten the ability of an INS/GNSS system onboard the UA . In a coCurrently in the research phase, SOPs include signals not normally associated with Precision Navigation Timing (PNT) and include AM/FM radio, cellular, digital television, and low Earth orbit (LEO) satellites . UA-mounHaving solved for their own ship position, determining the relative position at range comes with the inclusions of different airborne sensors to include A/A TACAN, the A/A radar system, an infrared search and track (IRST) system, or an electro-optical/infrared (EO/IR) system to a UA. As SWaP-C decreases with increases in technology, consideration should be given to adding an A/A radar, an IRST, or an EO/IR system to a UA. Along with providing ranging information to aircraft within the operating area, the target aspect, bearing angle, speed, and closure rates could also be calculated using the same sensors with the assistance of a fusion engine to correlate the information . These sTactical Air Navigation (TACAN) systems have been in service for decades for both surface navigation and A/A ranging and bear information with reports dating back to 1977 covering the accuracy A/A range and information . OperatiMultiple UAs already have a X frequency band radar for Synthetic Aperture Radar mapping in support of ISR-T missions to include Predator B, Global Hawk, and Gray Eagle ,69. WhilAdditionally known as Infrared Surveillance Systems (IRSS), IRST systems have been employed by military aircraft for decades, such as the system that was installed on the F-14D Super Tomcat. In July 1984, the USN signed a full scale development contract with Grumman to upgrade the F-14A with additional avionics to include the introduction of an IRST system for long-range air-to-air detection . IRST syThe last sensor, the EO/IR system, will be discussed to support decreasing the range of the receiver to the tanker to arrive at the astern position. When operating multiple UAs with crossing flight paths, collision avoidance systems become a critical safety component for safe and efficient operations. In terms of perception, EO/IR systems and tracking algorithms have been researched to assist active sensors, such as sonar or radar, in providing perception for a collision avoidance system to ensure safe deconfliction ,75. UndeFusing all the available information into a blended solution will require synchronization among all contributing sensors ,68,77. TAs the separation between the aircraft decreases, the requirement for more precise location and time data increases. This is analogous to the requirement that commercial airliners have an increased need for accuracy as they approach landing: airliner accuracy requirements are 2.0 nautical mile (NM) enroute, decreasing to 1.0 NM for the terminal approach, and further decreasing to 0.3 NM for the final approach . A similAs the receiver UA decreases the range separation to the tanker, DGPS can be used to support the increased accuracy for the UA. DPGS has a multitude of applications for air navigation , includiTransitioning from a possible contested environment  to a more permissive environment to conduct A3R, the UA must be able to transmit and receive position information both (1) to the GCS over BLOS or LOS data links and (2) to the tanker aircraft to create a P-RELNAV solution. To complete the P-RELNAV solution, this requires GPS receivers on both the tanker and UA in L-Band  and a daOnce the UA and the tanker are in closer proximity, as defined by the astern position (10 to 15 ft. aft of the drogue), it is necessary to transition to CV to provide guidance information to the GNC for the placement of the probe tip into the drogue coupler. Given the limitations associated with DGPS, demonstrations to date ,38,89, aFor the camera, either an EO or IR-based system provides benefits and drawbacks. Research has addressed use of a single monocular camera , a 3D FlThe purpose of a CV system installed on a UA is to detect, classify, and localize objects within the domain. A key aspect of the CV system is that it must be trained on data collected and labeled prior to the system being able to detect, classify, and locate objects within the domain. While organizations have begun to develop frameworks to provide assurance of machine learning algorithms, such as neural networks , that coAfter detecting objects within the domain, the CV system, trained on data collected during the development of the system, can begin to classify the object detected and provide the information to the GNC. The resulting information is then fused into the UA’s GNC to maneuver the probe tip of the UA into the coupler portion of the drogue attached to the tanking aircraft. In many cases, their capabilities have been tested through modeling ,94; scalA variety of methods have been attempted to detect the required objects within the domain, mainly the drogue, coupler, and probe tip. Some of the most common methods include color information, visible markers, or IR markers . The useLess commonly, acoustic methods have been suggested for object identification, as well as for air navigation applications . AlthougWhile transitioning from astern to contact, using DGPS to form a corridor of autonomy (COA) as described in ; this COWhen considering the camera system installed on the UA, many options exist, depending on the expected domain of operation. Using an EO system coupled with an illumination source capable of illuminating objects within the domain from the astern position represents one of the many different combinations available for satisfying the perception requirement of A3R. Enabling a UA to exercise higher levels of autonomy by utilizing a CV system within the COA enables completion of the positional transition from astern to contact and currently represents the best method available to complete this portion of A3R.Once the probe tip has connected to the coupler portion of the drogue, the UA must continue to push the drogue towards the fuselage of the tanker aircraft, if the drogue is installed on the centerline, to enter into the FTZ . If the Once A3R is complete, as measured by the fuel system onboard the UA or the computer that programs fuel transfer on the tanker, the UA must increase separation between the probe tip and the tanker aircraft. This should be accomplished by using DGPS information to maneuver the UA aft of the tanker aircraft to the maximum length of the hose reel attached to the drogue . Note thOnce the UA receives the command for final separation or autonomously decides to separate based on criteria established via preflight planning factors, the UA will execute the same procedure used for joining but in reverse order. Following the separation procedures outlined in , the UA The tasks required for A3R are conducted in the context of the ODD presented in Consider the physical domain of the ODD. This physical domain includes the type of tankers, refueling stores, position of the basket (wing or fuselage mounted), altitude, and airspeed, which will be driven by the intended tanker and informed by NATO SRDs . The maiFor the connectivity portion of the ODD, the CV system may necessitate a HOTL for additional assurance, especially in early deployments. Therefore, if the CONEMPS for early operations require a human monitor, a BLOS or LOS data link would be a requirement for the UA to facilitate the required monitoring. Therefore, even if operational deployment would not require a BLOS or LOS data link, early development may require the inclusion of that capability.Column five of the ODD , environThe environmental conditions portion of the ODD will consist of multiple layers to account for all the permutations of conditions the UA may encounter conducting A3R. For example, the weather may include wind, rain, snow, or sleet , if the The ODD presented provides one example of the factors that need to be defined, the example ODD would be appropriate for a system design and deployment to support the collection of training data and for evaluating the system performance throughout the anticipated domain. The ODD should be designed prior to defining sensor requirements for the UA to ensure that limitations associated with different sensors covered in this paper do not result in unintended limitations of the platform.Safety is a key consideration for all aircraft operations. The sensors onboard the UA support safety directly by ensuring critical functions and indirectly by providing redundancy to operational and monitoring capabilities. For example, the COA defined by DGPS information ensures that, while the UA is in the COA, the UA can exercise a higher level of autonomy by navigating based on information provided by the CV system. The COA also helps ensure that the UA does not inadvertently navigate towards an incorrectly identified object. By confining the approach to a DGPS defined corridor, additional assurance can be provided to airworthiness officials that the UA will operate in a safe manner when a human cannot monitor operation in real time.Depending on the criticality of the system, additional redundancy may be required for sensors onboard the UA. For example, if the mission of the UA requires a combat radius that exceeds the unrefueled range of the platform, A3R becomes a safety critical function to the success of the flight. For a mission that requires A3R to succeed, redundancy may be required for specific sensors, such as the camera system, to ensure that a single failure does not eliminate the capability for the platform to receive fuel airborne. This requirement will be derived from the CONEMPS and will inform requirements for redundancy throughout the system.In training, crewed refueling is typically conducted in special use airspace (SAU), as defined by the FAA, to include restricted areas, warning areas, or military operations areas (MOA) containing a published tanker track . SimilarThe extended flight time enabled by aerial refueling may introduce additional safety considerations for UAs. Previous research has explored the use of sensors and measurements for UA safety and advocates for standard testing to ensure reliability and assess performances .While SWaP-C decreases for many of the sensors discussed in this paper and the capabilities of CV systems continue to increase, the possibility of a UA conducting A3R in the future is not a question of if but when. The current DoD acquisition lifecycle consisting of requirements, contracts, development, test and evaluation (T&E), deployment, and sustainment is not optimized to expeditiously acquire a LES, such as the CV system required for a UA to complete A3R. In 2022, the Department of Air Force/Massachusetts Institute of Technology (DAF/MIT) AI Accelerator program published a guidebook to AI Acquisitions, which represented one of the first DoD guidebooks to address many of the concerns related to acquiring a LES .Specifically addressing the T&E portion of the acquisition lifecycle, many challenges exist for adequately evaluating the performance of a UA conducting A3R to include the complexity of the system; safety; security; and lack of methods, tools, and infrastructure, policy, standards, or metrics . In 2020Luckily, different government organizations, such as the European Union Aviation Safety Agency (EASA), have begun publishing concepts for assuring neural networks, which could be used to provide perception to a UA . AdditioWhile, from a sensor standpoint, a UA capable of A3R could very well happen in the near future, extensive research must be completed, and challenges addressed prior to the successful fielding of a UA capable of A3R. Given the severity to mission success of a UA failing to complete A3R, resources must be committed immediately to begin to address the challenges noted above and to ensure a successful acquisition.While demonstrated multiple times by DARPA/NASA and the USN, a UA that has achieved Initial Operational Capability (IOC), capable of conducting A3R, does not exist as of time of this publication. Extensive research has been conducted on different aspects of the missions, such as DGPS for close formation flying and CV for object detection within the domain. As technologies advance, new sensors bring additional capabilities that were not available during previous demonstrations. In this paper, a discussion of the sensors required to complete the different phases of A3R was covered, and they are shown in As discussed in In conclusion, all sensors required to complete A3R are available as of the date of this publication. There are numerous benefits from A3R, and the future of military and commercial aviation will necessitate that UAs become capable of completing A3R. This paper introduces and reviews the sensors required for a UA to complete A3R. Suggested future research to support A3R includes the deployment of scaled models, demonstrations of the application of sensors to accomplish the tasks and phases required for A3R, and the investigation of new sensor technologies to support A3R requirements.


Chronic infection with human T‐cell lymphotropic virus type‐1 (HTLV‐1) may result in aggressive adult T‐cell leukaemia/lymphoma (ATL) in 4‐6% carriers. The majority of this risk arises in carriers infected during infancy, and so each infant has ∼25% lifetime risk. Other risk factors include a family history of ATL. Antenatal HTLV‐1 screening is not undertaken in the UK.Here we describe four cases of ATL diagnosed during pregnancy and describe strategies to minimise HTLV‐1 transmission to neonates.These cases highlight undiagnosed HTLV‐1 in pregnancy which allows ongoing mother to child vertical transmission and risk of future ATL. We recommend the UK National Screening Committee incorporate HTLV‐1 serology into antenatal screening. In spitSince the 1987 implementation of antenatal screening in the Nagasaki region of Japan, HTLV‐1 seroprevalence in mothers has reduced from ∼5% to 1% and vertical transmission from 20.3% to 2.5% . This suATL in pregnancy has been reported five times as individual case reports since first described in 1977, two cases from USA and three from Japan , 11, 12.Here we describe four cases of ATL presenting during pregnancy in the UK  including cases managed with ZDV/IFN‐α, with a focus on the strategies used to treat the mothers to reduce vertical HTLV‐1 transmission, and highlight an undiagnosed HTLV‐1 population in a non‐endemic country. Cases are summarised in Table 1.19/L , lactate dehydrogenase (LDH) 1382 IU/L , normal haemoglobin and platelets. Her blood film showed >5% abnormal pleomorphic lymphocytes with typical flower cells, and flow cytometry revealed a clonal CD3+CD4+CD25+CD7− population with positive HTLV serology. She was commenced on steroids to control her WCC and to mature foetal lungs, and with oral ZDV as part of ATL treatment and to reduce vertical transmission. She underwent emergency caesarean section 9 days later for maternal indications (WCC 172 × 109/L). The maternal HTLV‐1 proviral load (PVL) (proportion of infected mononuclear cells per 100 cells) was unknown, and the mother died shortly after delivery due to sepsis during first CHOP  chemotherapy. The infant was formula‐fed and treated with ZDV post‐exposure prophylaxis for 6 weeks (the first 2 weeks were administered intravenously). At birth and age 7 months, HTLV‐1 PVL was not detected but was detected (PVL 0.01%) at 15 months, and she became HTLV‐1 seropositive. The child is now aged 13 years old and is clinically well with a stable low PVL 0.01‐0.03%.A 40‐year‐old pregnant female was diagnosed in 2006 with acute ATL at 28 weeks’ gestation. Her full blood count (FBC) showed: white cell count (WCC) 76.4 × 101.29/L , normal haemoglobin and platelets, LDH 516 IU/L  and normal calcium. Film morphology showed occasional flower cells and frequent atypical lymphocytes. Flow cytometry and T‐cell receptor gene rearrangement studies revealed a clonal CD3+CD4+CD25+CD7‐ population with positive HTLV serology and HTLV‐1 PVL 94%. She was commenced on ZDV 250 mg twice daily and subcutaneous interferon‐α (IFN‐α) three million units three times per week with successful normalization of the lymphocyte count within weeks. From 24 weeks gestation onwards maternal HTLV PVL was stable low load between 6‐9%. Raltegravir (integrase inhibitor) 400 mg twice daily was added to ZDV/IFN‐α at 34 weeks’ gestation to further reduce the risk of vertical transmission. There were no pregnancy complications, and the infant was born by planned pre‐labour caesarean section at 39 weeks’ gestation. The infant was formula‐fed and received 6 weeks oral ZDV. HTLV‐1 deoxyribonucleic acid (DNA) was not detected at day 1, 6 weeks or 6 months. Six months post‐delivery, the mother's ATL accelerated to an acute ATL subtype and was refractory to both ZDV/IFN‐α and salvage ESHAP  chemotherapy and died. The baby returned to family overseas and is lost to follow up.A 38‐year‐old asymptomatic woman was diagnosed in 2016 with chronic ‘unfavourable’ ATL at 7 weeks’ gestation with FBC: WCC 16.8 × 101.3A 34‐year‐old female was diagnosed with ATL lymphoma subtype overseas and treated there with chemotherapy (eight cycles of CHOP). Two years later in 2016, she presented in the UK with a gastric bleed, and a gastric biopsy revealed relapsed ATL lymphoma diagnosed at 24 weeks’ gestation. She was treated with two cycles of ESHAP chemotherapy, with concurrent ZDV 250 mg twice a day and subcutaneous IFN‐α 3 million units three times per week. A caesarean section was performed at 29 weeks’ gestation for foetal growth cessation (PVL 1.2% at time of delivery). The infant was severely unwell after birth with renal failure, cardiac failure and pancytopenia, attributed to maternal chemotherapy. The infant was formula‐fed and received 6 weeks of oral ZDV. HTLV‐1 DNA was not detected at day 1 or week 6. The mother died from refractory ATL 2 months post‐delivery, and the baby returned to family overseas and is lost to follow up.1.49/L , normal haemoglobin and platelets, corrected calcium and LDH. Her blood film showed >5% abnormal pleomorphic lymphocytes with positive HTLV‐1 serology. Diagnostic Immunophenotyping showed CD3+CD4+CD25‐CD7‐ population and HTLV‐1 PVL 45%. She was commenced on ZDV 250 mg twice daily and subcutaneous pegylated IFN‐α‐2b pegasys 1.5 mcg/kg weekly at the start of the second trimester. Raltegravir 400 mg twice daily was added at 32 weeks. There was not a significant response to therapy despite escalating doses of ZDV to 500 mg twice daily and IFN‐α 6mcg/kg, and so an elective caesarean was expedited at 38 weeks’ gestation. The infant was formula‐fed and completed 6 weeks oral ZDV syrup. At delivery neonatal HTLV PVL was undetectable and remained undetectable at 6 weeks and 3 months. The baby remains well and is under active follow‐up with repeat testing planned at 18 months. Following delivery, the mother was treated with mogamulizumab 1 mg/kg due to lack of response to ZDV/IFN‐ α. Following one dose, haematological remission was achieved, and PVL was reduced to 0.06%. She has received a total of three doses, currently stopped due to mild skin rash and remains in a persistent haematological remission 8 months later.A 35‐year‐old female was diagnosed with chronic ‘favourable’ ATL at 12 weeks’ gestation following a routine blood count. Her FBC revealed: WCC 17.2 × 102Two of these patients were diagnosed with chronic leukaemic ATL in the first trimester of pregnancy following the incidental finding of lymphocytosis (cases 2 and 4). Both mothers were treated with ZDV/IFN‐α which are known to be relatively safe in pregnancy, the aim being to induce remission and reduce the risk of vertical HTLV‐1 transmission. In both cases raltegravir, which inhibits HTLV‐1 integrase in vitro and readily crosses the placenta, was added in the 3rd trimester to protect foetal mononuclear cells exposed to maternal cells during the perinatal period . Both inThe diagnoses of both cases of chronic ATL early in pregnancy, whilst asymptomatic, allowed time to undertake strategies to reduce transmission and reflects the ‘tip of the HTLV‐1 iceberg’ since unscreened pregnant women in the UK with asymptomatic HTLV‐1 infection are unknowingly transmitting infection, chiefly through breastfeeding which is promoted by healthcare bodies in the UK. It is notable and reassuring that breastmilk banks do screen their donors for HTLV‐1.In the UK, HIV, hepatitis B and syphilis are the only infectious diseases routinely screened for in pregnancy, and the exclusion of HTLV‐1 from this list is difficult to justify given its associated fatal diseases. Additionally, a recent meta‐analysis revealed that asymptomatic HTLV‐1 infection results in 57% increased risk of premature death that is independent of ATL and HTLV‐1 associated myelopathy, the two diseases commonly associated with HTLV‐1 .HTLV‐1 remains neglected by health agencies globally, has not been recognised as an oncogenic virus by the National Cancer Institute and only very recently acknowledged by the WHO as a sexually transmitted infection . With al


Inflammatory environments provide vital biochemical stimuli  for triggered drug delivery in a controlled manner. Inflammation alters the local pH within the affected tissues. As a result, pH-sensitive nanomaterials can be used to effectively target drugs to the site of inflammation. Herein, we designed pH-sensitive nanoparticles in which resveratrol (an anti-inflammatory and antioxidant compound (RES)) and urocanic acid (UA) were complexed with a pH-sensitive moiety using an emulsion method. These RES-UA NPs were characterized by transmission electron microscopy, dynamic light scattering, zeta potential, and FT-IR spectroscopy. The anti-inflammatory and antioxidant activities of the RES-UA NPs were assessed in RAW 264.7 macrophages. The NPs were circular in shape and ranged in size from 106 to 180 nm. The RES-UA NPs suppressed the mRNA expression of the pro-inflammatory molecules inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages in a concentration-dependent manner. Incubation of LPS-stimulated macrophages with RES-UA NPs reduced the generation of reactive oxygen species (ROS) in a concentration-dependent manner. These results suggest that pH-responsive RES-UA NPs can be used to decrease ROS generation and inflammation. Osteoarthritis (OA), the most common degenerative joint disease, is characterized by degradation of the extracellular matrix (ECM), synovial inflammation, subchondral bone sclerosis, and regression of articular cartilage, and is a major cause of disability among the elderly worldwide ,2,3. TheVeratrum grandiflorum in 1940, is a polyphenolic compound found in many fruits and vegetables such as peanut bean sprouts, grapes, and peanuts  × 100, where Abscon is the absorbance of the control, and Abssam is the absorbance of the sample in PBS with different pH values .To evaluate the antioxidant capacities of various concentrations of RES-UA NPs in PBS with different pH conditions , we performed the ABTS ) assay, as described previously . L-ascor4 on cover glasses, and these were then placed into the wells of 24-well plates followed by a 24-h incubation. Cells were washed three times with PBS and pre-exposed to RES-UA NPs  in serum-free medium for 1 h, followed by stimulation with LPS (100 ng/mL) for 24 h. Cells were stained with DCFDA (25 μM) in the dark for 30 min and then fixed with 3.7% paraformaldehyde  for 30 min. After fixation, 4′,6-diamidino-2-2phenylidole  and Alexa® Fluor 594-labeled wheat-germ agglutinin (Thermo Fisher Scientific Inc.) were used to stain cell nuclei and membranes, respectively. Each sample was examined under a confocal laser scanning microscope . Fluorescence intensity was measured using a multimode plate reader at excitation and emission wavelengths of 485 and 535 nm, respectively, to further quantify ROS formation.To assess whether RES-UA NPs could protect cells against oxidative stress, we investigated intracellular ROS generation using an ROS-responsive fluorescence indicator, 2,7-dichlorofluorescein diacetate . Macrophages were deposited at a density of 5 × 10p values less than 0.01 were considered statistically significant.All data are reported as means ± standard deviations. Statistical analyses were performed using a one-way ANOVA with the post hoc Holm–Sidak test in SigmaPlot 12.0 . In this study, pH-responsive self-assembled RES-UA nanoparticles were successfully fabricated using single-emulsion solvent evaporation and ultrasonication. The release pattern of RES-UA NPs was faster in slightly acidic environments than in neutral environments. The RES-UA NPs had anti-oxidative and anti-inflammatory effects in LPS-stimulated RAW 264.7 macrophages. The RES-UA NPs also significantly reduced the production of NO in LPS-activated cells in a concentration-dependent manner. In addition, RES-UA NPs remarkably diminished the mRNA expression of pro-inflammatory molecules . Furthermore, RES-UA NPs effectively scavenged ROS in cells in a concentration-dependent fashion. Accordingly, RES-UA nanoparticles are promising materials for the treatment of inflammatory conditions such as osteoarthritis.


The development of the embryonic lung demands complex endodermal–mesodermal interactions, which are regulated by a variety of signaling proteins. Hedgehog (Hh) signaling is vital for lung development. It plays a key regulatory role during several morphogenic mechanisms, such as cell growth, differentiation, migration, and persistence of cells. On the other hand, abnormal expression or loss of regulation of Hh signaling leads to airway asthmatic remodeling, which is characterized by cellular matrix modification in the respiratory system, goblet cell hyperplasia, deposition of collagen, epithelial cell apoptosis, proliferation, and activation of fibroblasts. Hh also targets some of the pathogens and seems to have a significant function in tissue repairment and immune-related disorders. Similarly, aberrant Hh signaling expression is critically associated with the etiology of a variety of other airway lung diseases, mainly, bronchial or tissue fibrosis, lung cancer, and pulmonary arterial hypertension, suggesting that controlled regulation of Hh signaling is crucial to retain healthy lung functioning. Moreover, shreds of evidence imply that the Hh signaling pathway links to lung organogenesis and asthmatic airway remodeling. Here, we compiled all up-to-date investigations linked with the role of Hh signaling in the development of lungs as well as the attribution of Hh signaling in impairment of lung expansion, airway remodeling, and immune response. In addition, we included all current investigational and therapeutic approaches to treat airway asthmatic remodeling and immune system pathway diseases. The adult human lung is a tree-like structure consisting of branches of airway coils connected to a single tube, the trachea. The latter delivers breathed air through bronchi and bronchioles into the highly vascularized gas exchange units, the alveoli. The lung is among the human organs that possess a low steady-state cell turnover yet can enormously respond to repair damaged cell safter injury . For theHedgehog (Hh), the family of a morphogenic protein, is one of the highly regulated signaling pathways, which is significant in organogenesis, proliferation, and differentiation ,6. In inDeactivation of Hh signaling pathway may lead to genetic developmental defects, i.e., holoprosencephaly, whereas mutation-induced over-expression of this pathway may result in cancer of various organs, including the lungs, breast, pancreas, and prostate ,16. MoreAsthmatic airway remodeling, described for the first time in 1922, is characterized by irreversible structural changes in the airway wall ,21,22,23We propose that differential Hh activation is a promising mechanism for maintaining compartmental-specific identity as well as function of the lungs. When endogenous inhibitors of Hh activation are lost, they can disrupt the physiological asymmetry of Hh and result in the altered compartmental identity and structural changes observed in lung disorders.In this study, we discuss the molecular basis and current investigations linked with Hh signaling and its role in lung organogenesis. We also present the contribution of abnormal stimulation or removal of Hh signaling to the induction or development of pulmonary alteration, along with its influence in linking embryonic lung development and asthmatic remodeling and immune diseases. Finally, all current investigational and potential therapeutic approaches used to treat airway asthmatic remodeling and repair immune system pathway diseases are highlighted.The vertebrate Hh signaling pathway is composed of the following major constituents: (a) three different Hh ligands, including Shh, Ihh, and Dhh; (b) trans-membrane patched receptors (Ptch), having two isoforms encoded as Ptch-1 and Ptch-2; (c) smoothened receptors ; and (d) a cytoplasmic complex that controls the transcription factors, including Cubitus interruptus (Ci) in insects or the GLI family of the zinc-finger DNA-binding domain in vertebrates  ,28. AddiHh signaling is propagated by the binding of the Hh ligand to Ptch and Smo receptors. In the absence of the Hh ligand, the transmembrane Ptch blocks the formation of active Smo-C from transmembrane Smo-B , while in the presence of the Hh ligand, Smo gets activated, which results in cascade of events dealing with the translocation and binding of GLI with DNA, via the zinc-finger domain, where GLI transmits transcriptional information in response to the Hh ligand. The GLI family acts as a transcriptional activator or repressor. GLI-2 and GLI-3 contain activator (carboxyl-) and repressor (amino-) terminals, respectively ,33, wher, or negative (hedgehog-interacting protein (Hhip) and Ptch-1), creating a feedback loop for Hh signaling . Wh. Wh45]. The Hh signaling mostly occurs in embryogenesis, but it also occurs postnatally in the lungs, gut, hemopoietic, skin, immune system, and various other tissues. Postnatal signaling has played an effective role in maintaining the stem cell and tissue homeostasis as well as regulating the lymphopoiesis and hematopoiesis. It also has been stated that the Hh signaling showed their activation in response to pathogen, damaging, and wounds agents. Thus, Hh signaling has been shown to be a target of various pathogens .Here is Hepatitis B (HBV) and Hepatitis C (HCV) are the main causes of liver cirrhosis and hepatocellular carcinoma. The infected liver from HBV and HCV patients demonstrated that an excessive production of Hh ligands in hepatocytes and Hh-responsive cells accumulation at higher levels have more dire outcomes ,111. It H. pylori)” is prevalent in the stomach and is considered as the main cause of gastric cancer and gastric atrophy [H. pylori infection, the Hh upregulation was shown in parietal cells (secreting HCL) in vivo (mouse model) as well as in vitro . The upregulated signaling appeared due to inflammation or repair process as well as recruitment of macrophages to the infection site along with NFκB induction. It was analyzed that prolonged exposure to H. Pylori in the gastric epithelium, together with the depletion of parietal cells (secreting HCL) in the digestive glands, resulted in a significant reduction in Hh expression in both H. Pylori animal models and chronic gastritis H. Pylori-positive patients [H. Pylori infection, and Hh signaling could be reduced by macrophages secreting regulatory cytokines [H. pylori-dependent protein upregulation  is capable of blinding the Shh promoter and inhibiting Hh pathway expression [A Gram-negative bacterium “Helicobacter Pylori , a cytokine implicated in tissue repair or healing, reducing either Hh signaling or diminished OPN. GLI and GLI-binding sites were assumed to have a strong link with the OPN promotor. Furthermore, infections such as HBV and HCV are also known for causing liver fibrosis, which is thought to be mediated by increased Hh signaling .H-pylori infection [The Hh pathway performs an essential function in immune system, which is conserved from flies to human, although the relevant signaling output and how it acts as a defense system differ between invertebrates and vertebrates. A study was designed in Drosophila in which Hh signaling established a first-line protection in the gut against detrimental uracil-secreting pathogens by provoking microbicidal reactive oxygen species (ROS) production ,130,131.nfection ,135. Shhnfection . Hence, nfection . Likewisnfection . It was nfection . Taken tnfection .The ultimate targeting of canonical/non-canonical Hh pathways at downstream levels can inhibit fibroproliferative disorders, mainly tumors, but these antagonistic agents still need to be evaluated in asthmatic animal models to ensure that the physiological participation of Ptch, Smo, GLI, and other factors in the Hh pathway will open broad research avenues for the remedy of asthmatic airway remodeling and immune diseases. Airway tissue fibrosis, ASMC proliferation, airway hyper-reactivity, and overexpression of collagen, fibronectin, ECM, mesenchyme, and IL-4 are all hallmarks of Shh overexpression and/or aberrant signaling at various levels; all of these actions are similar to be seen in airway remodeling.Shh expression is upregulated by JAK/STAT6 signaling triggered by IL-4/13, which promotes allergic airway epithelial remodeling. Whereas, in animal models with ovalbumin- or house dust mite allergen-induced airway inflammation, intratracheal suppression of STAT6 by AS-1517499 dramatically reduced not only the allergen-induced Shh overexpression, but also the hyper-responsiveness and goblet cell metaplasia . MoreoveAs previously described, Smo is overexpressed during hyperplastic epithelium, fibroblasts, and interstitial inflammatory cells’ activation. Cyclopamine, a specific Smo antagonist , is wideVarious signaling pathways, such as TGF-β, Notch, and Wnt/β-catenin, develop complex interactions with Shh during pulmonary fibrosis. The Shh pathway attenuated the expression of collagen-1 and fibronectin, while blocking the TGF-β effect on these proteins ,146, indFurthermore, different recent FDA-approved treatments, such as CDC-0449 and LDE225, inhibit SMO, but arsenic trioxide inhibits GLI-1/2. As a result, it makes perfect sense that FDA-approved medicines that suppress Hh signaling could be used as powerful inhibitors to treat a variety of pathogenic illnesses. Despite currently existing medicines, such as antivirals and vaccinations, which target specific viral proteins that rapidly mutate, therapies that target host targets could provide stronger protection over a wide range of strains . RemarkaThis may require a synergistic paradigm where numerous medications are targeted to various Hh-dependent processes, allowing for signaling to be strengthened or reduced in a context-dependent manner. This could be accomplished by combining medications that target signaling pathway elements that have beneficial and harmful effects. Furthermore, establishing the suitable technique by which viral elements link with the pathway would almost certainly limit the investigation into effective treatments.From these data, it can be inferred that Hh signaling regulates the indications associated with airway remodeling and immune system pathway diseases either directly or through other pathways; thereby, a combination of genetic/personalized and pharmacological analysis of this pathway will provide an efficient approach in future research.To date, many research investigations have been published to investigate the role of Hh signaling in numerous airway diseases, but clinical evidence to confirm the role of the Hh signaling pathway is still lacking in asthmatic subjects. The facts and figures of experimental animal data support the pivotal role of Hh signaling in embryonic lung expansion, initiation, and the development of asthmatic airway remodeling, which is linked with impaired early lung development with asthmatic remodeling. Moreover, Hh signaling acts as a target for pathogens, and many Hh antagonists have been identified that might be effective in the treatment of wound repair and pulmonary fibrosis through overcoming the aberrant expression and overproduction of Hh signaling. We postulate that the significant postnatal contribution in wound repair and immune response disease is a key factor for pathogenic control. These findings could lead to new therapies for asthmatic airway remodeling, lung injury, and immune system pathway diseases involving Hh-stimulatory/inhibitory drugs or combined medications  that target signaling aspects that have both beneficial and adverse effects, which interrupt/reverse these connections, suggesting a promising new research direction.


Because of the potential therapeutic benefit, considerable attention has been devoted to the synthesis of new TRH analogs that may have some advantageous properties compared with TRH. In this context, it may be interesting to consider the phenomenon of biased agonism and signaling at the TRH receptor. This possibility is supported by some recent findings. Although knowledge about the mechanisms of TRH receptor-mediated signaling has increased steadily over the past decades, there are still many unanswered questions, particularly about the molecular details of post-receptor signaling. In this review, we summarize what has been learned to date about TRH receptor-mediated signaling, including some previously undiscussed information, and point to future directions in TRH research that may offer new insights into the molecular mechanisms of TRH receptor-triggered actions and possible ways to modulate TRH receptor-mediated signaling.Thyrotropin-releasing hormone (TRH) is an important endocrine agent that regulates the function of cells in the anterior pituitary and the central and peripheral nervous systems. By controlling the synthesis and release of thyroid hormones, TRH affects many physiological functions, including energy homeostasis. This hormone exerts its effects through G protein-coupled TRH receptors, which signal primarily through G Its main role is to maintain thyroid hormone homeostasis through regulation of thyroid-stimulating hormone secretion (Thyrotropin-releasing hormone (TRH), also termed thyroliberin, is a tripeptide  335-337 in the C-terminus of mouse TRH-R are important for ligand driven receptor internalization . The samBecause mutations in some GPCRs have been shown to be involved in pathological conditions in humans , TRH-R wGPCR-mediated signaling has been shown to be modulated by posttranslational modifications of a receptor, such as glycosylation, phosphorylation, and palmitoylation . No detaq/11-coupled receptors  method using the construct of rat TRH-R1 with bioluminescent enzyme . This stqα and G11α need not dissociate from receptors during internalization at the plasma membrane, and they confirmed that TRH-R and the G11α subunit are internalized together in clathrin-coated vesicles in GH4C1 cells. Later studies based on the use of clear native polyacrylamide gel electrophoresis revealed the presence of preassembled complexes of rat TRH-R1 and Gq/11α P2) to IP3 and DAG. These 2 s messengers initiate the mobilization of calcium from the cell’s internal stores and the activation of PKC, respectively. Intracellular calcium and PKC then trigger a number of cellular responses, including activation of MAP kinase signaling cascades or regulation of ion channels  in GH4C1 prolactin-producing cells, thereby activating cAMP-dependent PKA  that regulate specific downstream effectors and trigger specific pathways through a variety of membrane-bound receptors. Besides that, Gβγ dimers also contribute to the regulation of various effectors. Previous studies have shown that TRH-R is quite promiscuous, coupling predominantly to Gq/11 but also interacting with Gs and Gi proteins in GH3 cells  and C-terminal domains. It interacts exclusively with members of the Gq/11α protein family. It binds directly to activated Gqα subunits, inhibiting the activation of downstream effectors. In addition to its function in desensitizing receptors, GRK2 additionally regulate signal transduction by binding to the Gqα subunit  are other proteins that possess the RGS domain. There are seven GRKs (GRK 1-7) . GRKs ar subunit . The bineins use . Besides1α class . And as lass -dependent kinases (CAMK). Activation of the PLC/PKC pathway and possibly the AC/PKA pathway by TRH-R has been frequently described in different animal and cellular models /Akt and Car models . Interesr models . Endocrimulation . Much leq/11α-coupled receptors trigger various signals involved in the control and regulation of all major members of the MAPK signaling pathways. To increase signaling efficiency between successive kinases in the cascade, increase signal fidelity by restricting cross-talk between two parallel kinase cascades, and target MAPKs to specific subcellular locations, MAPKs bind to scaffold proteins. One of the most representative scaffold molecule is β-arrestin, which can serve as a GPCR-regulated scaffold for MAPK activation. Interestingly, in addition to serving as downstream effectors, MAPKs can also act as negative regulators of GPCRs. Two phosphosites (Ser14 and Thr276) in β-arrestin were found to be critical for ERK1/2-triggered intracellular sequestration of the receptor  as well as PKC in GH3 cells  channels and that this inhibition relies on Gqα and PLC  and possibly Rho kinases (q/11 protein does not appear to be involved in TRH-induced inhibition of endogenous ERG currents in rat pituitary cells and that Gs (or a Gs-like protein), G13 and Gβγ may participate in modulating these currents triggered by TRH  to which the agonist can bind  axis, which plays a critical role in development, growth, and cell metabolism . TRH is TRH mimetics have been shown to be useful in the treatment of spinocerebellar degeneration , amyotroin vivo neuroprotective effects of TRH likely involves several factors, including inhibition of the effects of glutamate, endogenous opioids, platelet activating factor, or leukotrienes, among others, which have a role in neurodegenerative damage. TRH may be able to help restore cellular bioenergetics and increase blood flow to the brain (There is growing preclinical evidence that activation of TRH-R can protect cells from a variety of deleterious influences. There are some indications that TRH and its analogs may have certain neuroprotective effects , but thehe brain . The neuhe brain . Additiohe brain . Similarhe brain  and hepahe brain .Possible antiapoptotic effects of TRH have also been reported. They were observed in both cellular and animal models . The mecTRH is known to exert both neuroendocrine and extrahypothalamic effects, which are of great importance for the normal functioning of all vertebrates. Intensive research in recent decades yielded numerous publications on the biochemistry of TRH receptor-mediated signaling, as well as on the physiology, pharmacology, and potential therapeutic effects of TRH and its analogs. Most of the pioneering studies on the molecular mechanisms of TRH-R signaling have been conducted in the 1990s and the first decade of the 21st century. Although the basic principles of TRH-R-mediated signaling are known, there are still many unresolved issues. The role of di- and/or oligomerization of the receptor and the formation of protein complexes of TRH-R with other signaling partners should be further investigated in future studies to describe more precisely the molecular mechanisms of TRH-R-mediated signal transduction. The principles governing TRH-R trafficking are not well understood, and it is unclear how phosphorylated and dephosphorylated receptors are sorted in endosomes. TRH-R was originally considered to be a prototypical “calcium-mobilizing” GPCR. However, the signaling pathways triggered by TRH-R have proven to be more complex than anticipated. In addition to its interaction with different classes of G proteins, TRH-R apparently can also transduce signals through its diverse β-arrestin interactions in ways not yet fully recognized. Activation of TRH-R can apparently initiate various intracellular signaling pathways and lead to different responses depending on the cell type and specific experimental conditions. Some recent data suggest that biased agonism should be taken into consideration when characterizing TRH-R function and post-receptor signaling. These findings may open new avenues for the development of novel TRH analogs capable of controlling specific aspects of receptor function and potentially useful for therapeutic purposes. Research into the fundamental principles of TRH-R-mediated signaling deserves continued attention because of the important role this signaling system plays in maintaining a normal state of health. In the long term, these efforts are likely to have significant translational value, as the medical use of TRH and related drugs appears rational and warranted.


Ursolic acid (UA) is a pentacyclic triterpene carboxylic acid which produces various effects, including anti-cancer, hepatoprotective, antioxidant and anti-inflammatory. However, UA demonstrates poor water solubility and permeability. Niosomes have been reported to improve the bioavailability of low water-soluble drugs. This study aimed to investigate the protective action of UA-niosomes with chitosan layers against liver damage induced by N-Nitrosodiethylamine (NDEA). UA niosomes were prepared using a thin layer hydration method, with chitosan being added by vortexing the mixtures. For the induction of liver damage, the mice were administered NDEA intraperitoneally (25 mg/kgBW). They were given niosomes orally (11 mg UA/kgBW) seven and three days prior to NDEA induction and subsequently once a week with NDEA induction for four weeks. The results showed that chitosan layers increased the particle sizes, PDI, and ζ-potentials of UA niosomes. UA niosomes with chitosan coating reduced the SGOT and SGPT level. The histopathological evaluation of liver tissue showed an improvement with reduced bile duct inflammation and decreasing pleomorphism and enlargement of hepatocyte cell nuclei in UA niosomes with the chitosan coating treated group. It can be concluded that UA niosomes with chitosan coating improved the efficacy of preventive UA therapy in liver-damaged mice induced with NDEA. Chemically-induced liver damage results from the metabolic transformation of chemicals into reactive intermediate compounds with the potential to change the structure and function of cellular macromolecules3. There are several causes of liver damage, one being exposure to carcinogenic chemicals such as N-nitrosodiethylamine (NDEA) which produces reactive oxygen species (ROS) causing oxidative stress and cellular destruction4. Reactive products and free radicals cause an increase in the serum index of liver function such as alanine transaminase (ALT) or serum glutamic-pyruvic transaminase (SGPT), aspartate aminotransferase (AST) or serum glutamic-oxaloacetic transaminase (SGPT), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), and total bilirubin. In cases of severe histopathological lesions they cause neoplastic transformation5.Liver damage is the leading global cause of death. In 2017, 1.32 million deaths worldwide or 2–4% of the annual total were due to liver cirrhosis7. As an antioxidant, UA reduces oxidative stress, modulates the Receptor for Advanced Glycation End Products (RAGE) and decreases NADPH oxidase to prevent the formation of ROS8. UA also produces a hepatoprotective effect by maintaining the structural integrity of the liver, reducing high levels of bilirubin, stabilizing serum protein concentrations, and suppressing oxidative stress, inflammation, and apoptosis in the liver10. Oral administration of a 500 mg/kgBW dose of UA to subjects resulted in a reduction in SGOT and SGPT as well as improvement in liver histopathology11.UA, a natural pentacyclic triterpenoid compound, has various pharmacological properties including anticancer, hepatoprotective, anti-angiogenesis, apoptosis induction, antioxidant and anti-inflammatory12, result from poor solubility and absorption. An effective drug delivery system is required to increase its solubility and dissolution. Niosomes represent a vesicular bilayer system composed of non-ionic surfactants and cholesterol in the aqueous phase which can increase drug half-life, enhance stability, and deliver drugs to target organs in a controlled release13.However, limitations on the oral use of UA, which belongs to class IV Biopharmaceutics Classification System (BCS)14 derived from the exoskeleton of crustaceans15 and is widely employed because of its intrinsic polycation properties, low toxicity, and excellent biocompatibility. Modification of UA liposomes with chitosan coating can increase bioavailability, slow drug release in tumor tissue and reduce both dose and side effects. Chitosan can open the tight junctions of epithelial cells, thereby enabling a drug to pass easily through the epithelial membrane via the paracellular pathway15. Chitosan also possesses mucoadhesive properties as a result of ionic interactions between positively charged amino groups and negatively charged functional groups on the surface of epithelial cells provide a controlled release while also enhancing absorption in the gastrointestinal tract and intestinal permeability16. Therefore, it is expected that the modification of chitosan on the niosomal surface will enhance absorption in the gastrointestinal tract, promote UA niosome accumulation in the liver and increase bioavailability.Chitosan, a natural polysaccharide, is a product of alkaline deacetylation of chitin17. Characterization of UA reported that the presence of chitosan showed an increase in the physical stability of UA niosomes. Chitosan coating on UA niosomes affects their physicochemical properties which, in turn, causes an increase in particle size and a more positive zeta potential. Biodistribution evaluation with coumarin-6 labeling revealed that high fluorescence intensity of coumarin-6 indicates high levels of UA in plasma and liver, together with an increase in bioavailability.In our previous study, optimization of the UA niosome formula found the optimum physical stability in the span 60-cholesterol-UA formula with a mol percent ratio of 3:2:10In this study, the evaluation of the effectiveness of UA niosomes with chitosan coating as an orally administered in vivo therapy for the prevention of liver damage in NDEA-induced subjects was by means of serum levels of SGOT, SGPT, and liver tissue histopathology.Characteristic UA niosomes parameters include particle size, polydispersity index, and ζ–potential. Measurements were taken from Nio-UA and Nio-UA-CS preparations. A graph of the characteristics of AU niosomes can be seen in Fig. UA niosomes with chitosan coating (Nio-UA-CS) experienced an increase in particle size from 211.7 ± 1.7 nm (Nio-UA) to 257.4 ± 4.3 nm. A significant difference also occurred in the PDI parameters where the presence of chitosan coating increased the PDI from 0.337 ± 0.018 to 0.393 ± 0.021. The ζ-potential parameter of chitosan coating can also alter the charge from UA niosomes which was initially − 26.6 ± 0.2 mV to − 24.1 ± 0.4 mV. Based on a statistical analysis of the Independent T-Test conducted, the results were p < 0.001 on the particle size parameter, p = 0.03 on the PDI parameter, and p = 0.001 on the ζ-potential parameter, all three of which indicated a significant difference between Nio-UA and Nio.-UA-CS.The weight of the subjects in the five groups was recorded every week prior to treatment commencing. The average differences in their weight gain and loss can be seen in Fig. 18. NDEA metabolism in the liver can produce ROS that induce oxidative stress resulting in DNA damage33.The body weight profiles of the normal group subjects that had not been induced by NDEA were compared with those of the other four groups that were subjected to NDEA induction on four occasions. The normal group subjects were observed to have experienced the most significant weight gain, while those in the negative control group that had been administered NDEA, but did not undergo UA treatment, demonstrated the smallest difference in body weight. Previous studies of liver inflammation using an NDEA-induced subject model also yielded a weight loss profileEach organ was photographed post-surgery to determine the qualitative comparison of the morphological organs of subjects in the normal group, the negative control group, the group that received UA, Nio-UA, and Nio-UA-CS suspension treatment. Pictures of complete organs of the normal group subjects, the negative control group subjects induced by NDEA, and the group subjects that received the suspension treatment of UA, Nio-UA, and Nio-UA-CS can be seen in Fig. Quantitatively, all the organs of each subject were weighed with each group members’ results being subsequently compared to determine if there was a significant difference. Data on the absolute and relative weight of each organ post-UA treatment and total NDEA induction for 28 days can be seen in Fig. The results of measuring the levels of SGOT and SGPT in the blood serum of subjects in the normal group, negative control, UA suspension, Niosom UA (Nio-UA), and Niosom UA with chitosan coating (Nio-UA-CS) can be seen in Fig. The results of microscope observation of liver tissue can be seen in Fig. Parameters observed in this liver tissue include lobulation, bleeding, neutrophil infiltration and dysplastic hepatocytes. Figure The comparative observation results relating to spleen tissue viewed through a microscope of the normal group, the negative control group, suspensions of AU, Nio-UA, and Nio-UA-CS can be seen in Fig. 19. Although the particle size increased, coating chitosan on UA niosomes can enhance its effectiveness. It is estimated that, in the presence of chitosan, drug transport can be effected through two pathways, namely; direct cell membranes and paracellular pathways15. However, with the addition of chitosan, the value of the polydispersity index (PDI) also increased. The homogeneity criteria for samples with lipid-based carriers was that of PDI < 0.320. The PDI value of Nio-UA remained approximately 0.3 which indicated a relatively homogeneous size distribution. However, chitosan coating significantly increased the PDI value possibly due to the addition of chitosan forming a polymer layer on the surface of the random vesicles21. Zeta potential is a detection index of electric charge on the particle surface. In vivo, it can influence the distribution of niosomes, while it is thought that in vitro it might contribute to the physical stability of niosomes by reducing the rate of aggregation and fusion15. The addition of chitosan can significantly mitigate the negative properties of Nio-UA due to the electrostatic interaction between the positive charge on chitosan and the negative charge on UA21. Surface charge has been reported as affecting in vivo drug distribution. Several studies have revealed that positively charged nanoparticles show higher phagocytic and cellular uptake than negatively, neutrally charged, and PEGylated nanoparticles23. The positively charged nanoparticle will be endocytosized through clathrin receptors, while the negatively charged nanoparticles are primarily internalized via caveolin receptors23. However, other research into the bioavailability studies of nanoparticles has indicated that their negative charge increases the macrophage uptake more significantly than that of positively charged nanoparticles, thereby potentially reducing the effectiveness of nanodrug delivery24. Opsonin serum protein binding with negatively charged nanoparticles seems to occur to a higher degree than that of positively charged nanoparticles. Consequently, negatively charged nanoparticles are covered more extensively by opsonin proteins with greater stimulation of the phagocytosis by macrophages25.The increase in particle size of chitosan-coated UA niosomes was due to the fact that chitosan had formed a hydrophilic shell on the niosomal surface through electrostatic interaction18. A significant difference in relative liver weight occurred in the normal group compared to the UA and Nio-UA suspensions. In previous in vivo studies, administration of UA was known to reduce liver weight. UA can effectively relieve hepatic steatosis and reduce adipocyte size in the epididymis and decrease total cholesterol and triglycerides in the liver and plasma of subjects27. In this study, NDEA-induced subjects did not present a difference in relative spleen weight compared to members of the normal group.Data on the weight of each organ indicated a reduced mean relative weight of the liver in the members of the four NDEA-induced groups compared to those of the normal group. Induction of NDEA causes hepatic degeneration that generally reflects loss of function associated with hepatocellular atrophy and injury28. Moreover, positively charged nanoparticles are also more easily taken up by lung cells, compared to neutral or negatively charged nanoparticles with the result that they can accumulate extensively in the lungs29. This may underlie the significant differences in the pulmonary organs, while in the heart, no changes were observed possibly due to differences in cell types and characteristics. However, further analysis of these organs is required.NDEA is a well-known carcinogen that induces cancer of various organs in experimental animal subjects. Inducing liver cancer, NDEA can also result in lung adenocarcinoma30.The SGOT and SGPT levels in serum in the negative control group were recorded as higher than that in normal group. This indicates that the administration of NDEA 25 mg/kgBW to negative control group members on four occasions caused liver damage characterized by increased levels of SGOT and SGPT in blood serum. SGOT and SGPT are enzymes sensitive to liver cell damage which are predominantly contained in liver cells and, to a lesser extent, in muscle cells. Exposure to toxic substances causes a change in the permeability of the liver cell membrane resulting in damage or leakage, as a result of which the liver cells will release the enzymes they contain into the blood circulation, thereby increasing the levels of SGOT and SGPT and signaling liver disease31. Oral use of niosomes can improve permeation and bioavailability, solubility of hydrophobic drugs, drug accumulation in the liver and controlled and targeted drug release32.The levels of SGOT and SGPT in the negative control group were also higher than those in the Nio-UA and Nio-UA-CS groups. SGOT levels showed a significant difference (P < 0.05) while SGPT levels did not demonstrate a significant difference (P > 0.05) in the Nio-AU and Nio-UA-CS groups compared to the negative control group. This indicates that the administration of Nio-UA and Nio-UA-CS produces a hepatoprotective effect by reducing the release of SGOT and SGPT into the blood compared to UA suspension. A previous study of in vivo test results relating to paclitaxel niosomes indicated that the plasma drug concentration was higher in the paclitaxel niosome group than in the paclitaxel suspension group15. Chitosan also induces mucosal adhesion through ionic interactions between positively charged amino groups and negatively charged functional groups on the surface of epithelial cells, thereby providing controlled release and absorption in the gastrointestinal tract16. Chitosan has good mucoadhesive properties that can prolong the residence time of the drug in the gastrointestinal tract. Under acidic conditions, chitosan will trigger the opening of tight junctions between epithelial cells and facilitate paracellular transport of niosomes15. Therefore, the nanoparticle system in the presence of chitosan coating can effectively improve oral absorption. There is still no information regarding the effect of chitosan on tight junctions in hepatocytes.The SGOT level in the Nio-UA-CS group was lower than that of the Nio-UA group. The presence of chitosan can induce a greater effect marked by the release of fewer SGOT enzymes. This finding supports those of previous studies regarding the modification of UA liposomes with chitosan coating increasing bioavailability, slowing drug release in tumor tissue, and reducing dosage and potential side effects. This can happen because chitosan opens tight junctions in epithelial cells and allows drug to pass freely through epithelial cells via paracellular pathways33. Previous research into the use of UA in the prevention of liver fibrosis due to CCl4 induction found optimal protection through the administration of UA at a dose of 50 mg/kgBW in distilled water containing 0.1% Tween 8034. Moreover, this is feasible due to the difference in the amount of UA taken because the UA suspension is insoluble. Consequently, there is a possibility that the preparation is not homogeneous, while the niosomes are more evenly dispersed than the suspension.The levels of SGOT and SGPT in the UA suspension group were higher than in the negative control group, although they did not differ significantly. This is possible because the dose of 11 mg UA/kgBW administered is less effective if in the form of a suspension. The use of niosomes can overcome the problem of low drug solubility in water, thereby reducing drug dosage35. In terms of further research, if experimental subjects are used, it is preferable to complete a sampling to check the levels of SGOT and SGPT before the subjects are treated to ensure that their initial condition is healthy.An analysis of the study results confirmed that the levels of SGOT and SGPT parameters in the Nio-UA and Nio-UA-CS groups were lower than in the normal group, although not significantly different. The lower the level, the healthier the condition of the liver11. This finding is consistent with those of previous studies regarding CS modification of liposomes which resulted in increased drug activity of UA liposomes and enhanced antitumor drug efficacy15. Liver histopathology observations were linear with the results of SGOT and SGPT levels indicating that the optimum repair of liver damage occurred in the Nio-UA-CS group followed by Nio-UA and, finally, UA suspension.It is evident from these observations that the administration of Nio-UA-CS can reduce inflammation, pleomorphism, dysplasia, and enlargement of hepatocyte cell nuclei in mice liver. These results indicate that the administration of chitosan to UA niosomes increases the anti-inflammatory and anticancer activity of UA36. Liposomes and lipid nanocarriers larger than 100–150 nm can be taken up by phagocytes. Monocytes, macrophages and neutrophils are phagocytes. The majority of these phagocytes reside in the liver and spleen for subsequent elimination20.Spleen histopathology was also observed in the course of this study. Conventional nanoparticles are known to be trapped by RES, most of which will migrate to the liver and spleen37 which can protect the body from non-self-pathogens or cancer cells by destroying them38. In a previous study on UA nanoparticles with chitosan coating as folate-targeting, the preparation was shown to enhance tumor inhibition and promote an immune-boosting more effectively than free UA40.The administration of Nio-UA-CS indicates lymphoid tissue activation. Such activation is correlated with an increase in immune system activity42. Cldn4 has been recognised as a protein responsible for cell adhesion, polarity and paracellular permeability43. Intracelullar redistribution results in the weaking of the tight junction leading to the opening of the cells42. On the other hand, it has been reported that Cldn4 is not expressed in normal hepatocytes. However, its expression is increased due to fibrosis, rather than inflammatory condition, of severe liver injury44, which this gene expression correlates with differentiation of progenitor cells into mature hepatocytes. This study also reported that its expression was not found in cases of hepatocellular carcinoma. Therefore, chitosan’s effects on hepatocyte permeability and the drug’s penetration into deeper damaged liver tissue are still questionable, need to be further explored. In addition, NDEA induction has been reported to increase serum bilirubin levels45, and UA effectively reduced them, proving its potential efficacy for liver protection and promoting bile secretion47; however, this study was limited. Therefore, evaluating the serum bilirubin levels is vital to provide the information associated with the repair of liver damage and its dysfunctions48.It has been reported that Chitosan induces transient tight junction opening by translocating the membrane’s tight junction protein claudin-4 (Cldn4) into the cytoskeleton followed by its degradation in lysosomesChitosan coating on UA niosomes can improve the physical morphology of the liver, resulting in the relative weight of the liver and lung organs which are relatively the same as the normal group and there is no significant difference in the difference in body weight. Chitosan coating on UA niosomes can increase the effectiveness of UA as a therapy to prevent liver damage in subjects induced by N-Nitrosodiethylamine in terms of histopathological parameters of liver tissue which are relatively more normal than negative controls. Chitosan coating on UA niosomes can increase the effectiveness of UA as a therapy to prevent liver damage in mice induced by N-Nitrosodiethylamine in terms of decreasing serum levels of SGOT and SGPT.17. UA  solution in methanol, span 60 , and cholesterol  in chloroform  were mixed in a round bottom flask. The organic solvents were then heated in a rotary vacuum evaporator at a temperature of 60 °C until they had all evaporated and a thin lipid layer was formed. This layer was hydrated using 2 ml PBS solution pH 7.4 at 60 °C for 1 h17. Sonication was carried out with a water bath sonicator to form niosomes in order to reduce the size of the vesicles. Dissolving chitosan  in 0.1 M acetic acid produced 0.1% chitosan solution which was subsequently diluted using distilled water to obtain a solution of 0.005% v/v chitosan which was added to the UA niosomal suspension. The addition was completed by mixing 40 µl of chitosan solution with 400 µl of niosomal samples before vortexing for ten seconds.Preparation of niosomes was conducted using a thin layer hydration method with a formula composition referred to previous studies as shown in Table 3Approximately 100 µL niosomes was diluted in 2 mL aqua demineralization with particle size and PDI measurements subsequently being completed by the Dynamic Light Scattering method using Malvern Zetasizer Instruments . Furthermore, 100 µL niosomes were also taken diluted in 2 mL aqua demineralization ζ-potential measured using the Electrophoresis Light Scattering method with Malvern Zetasizer Instruments . The evaluation was completed three times for each of the Nio-UA and Nio-UA-CS samples.. All methods were performed in accordance with ARRIVE guidelines and relevant regulations49. In this study, 6-week-old male mice (Mus musculus) Balb/c represented the subjects. Determination of the number of sample replications employed the Federer’s Formula. Five randomly selected subjects formed the members of each treatment group. The negative control group was treated by means of NDEA i.p. injection for four weeks, while PBS pH 7.4 was administered orally during sample treatment.The use of experimental animals in this research was approved following an ethical feasibility test conducted on April 1, 2022 at the Faculty of Veterinary Medicine, Universitas Airlangga by the Faculty’s Research Ethics Commission through the issuance of Certificate of Ethics Eligibility No. 2.KEH.035.04.202250 once a week for four weeks. Evaluation of the resulting liver damage was effected by recording the subjects’ body weight on a weekly basis during the test period to identify any increase or decrease.Induction of liver damage in subjects was achieved through the intraperitoneal administering of a 25 mg/kgBW dose of NDEA 40. The drug was administered orally using a needle probe seven and three days before NDEA induction and was continued once a week together the intraperitoneal induction of NDEA at a dose of 25 mg/kgBW for the subsequent four weeks.Subjects were given drugs, including UA suspension in 0.5% CMC Na, Nio-UA, and Nio-UA-CS, according to whichever group they belonged. The UA dose was equivalent to 11 mg UA/kgBWg × force for 15 min at 4 °C to obtain serum whose levels of SGOT and SGPT was then determined using the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) 37 method. The decrease in SGOT and SGPT levels was determined from comparisons between each treatment group and the control group. The SGOT and SGPT levels were determined by enzymatic reaction kinetic method. The reagents used were ready-to-use reagents consisting of AST (GOT) and ALT (GPT) reagents51.After the final UA preparation had been administered, the subjects were left for seven days before their organs were surgically removed. Having been given intraperitoneal anesthesia in the form of a 10 mg/kgBW dose of ketamine, a blood sample was taken from the inferior vena cava, inserted into test tubes and centrifuged at 600011. Changes in lobular architecture, bleeding, neutrophilic infiltration, and dysplastic hepatocytes on histopathological preparations of liver tissue were observed by means of light microscopy52. To evaluate the organ weight of the subjects, quantitatively each organ of mice in each group was weighed. Because overall body weight affects the weight of individual organs, the relative weight of the livers was calculated using the formula53:Following extraction of the blood sample, the subjects’ spines were dislocated. The subjects were dissected and their livers immediately removed, rinsed with normal saline, and dry wiped with a tissue or filter paper, before finally being weighed, photographed and morphologically examined. The liver sections were fixed in 10% neutral buffered formalin and then stained with haematoxylin and eosin (H&E staining) for further histological analysis of the differences in appearance between the livers of the normal and treated subjectsThe calculation results relating to the relative weight of the organs in the treatment group were then compared with those of the normal and negative control groups to determine whether significant differences existed.P value < 0.05 is considered as a significant difference between the results.The quantitative data represent the average and standard deviation of sample measured in replications. A statistical analysis was performed using the one-way variant analysis (ANOVA) method followed by a Post Hoc Tukey HSD test. The The animal study procedures were performed in accordance with the ethical clearance issued by The Ethics Commission of Faculty of Veterinary Medicine, Universitas Airlangga .


Stroke is one of the leading causes of mortality, and survivors experience serious neurological and motor behavioral deficiencies. Following a cerebral ischemic event, substantial alterations in both cellular and molecular activities occur because of ischemia/reperfusion injury. Wnt signaling is an evolutionarily conserved signaling pathway that has been manifested to play a key role in embryo development and function maintenance in adults. Overactivation of Wnt signaling has previously been investigated in cancer-based research studies. Recently, abnormal Wnt signaling activity has been observed in ischemic stroke, which is accompanied by massive blood–brain barrier (BBB) disruption, neuronal apoptosis, and neuroinflammation within the central nervous system (CNS). Significant therapeutic effects were observed after reactivating the adynamic signaling activity of canonical Wnt signaling in different cell types. To better understand the therapeutic potential of Wnt as a novel target for stroke, we reviewed the role of Wnt signaling in the pathogenesis of stroke in different cell types, including endothelial cells, neurons, oligodendrocytes, and microglia. A comprehensive understanding of Wnt signaling among different cells may help to evaluate its potential value for the development of novel therapeutic strategies based on Wnt activation that can ameliorate complications and improve functional rehabilitation after ischemic stroke. Stroke is the leading cause of disability and mortality worldwide and is classified as ischemic or hemorrhagic. Ischemic stroke accounts for 87% of all stroke incidences and is defined as the interruption of blood flow to the brain due to blockage of the cerebral artery, causing severe damage to focal brain tissue . PatientVascular recanalization remains the primary therapeutic option . To dateThe above studies suggest that the ischemic penumbra still has great therapeutic potential for neuroprotection. The Wnt pathway is part of an evolutionarily conserved intracellular signal transduction cascade that regulates multiple processes crucial for cell proliferation, differentiation, migration, and fate decision during development . RecentlSeveral studies suggest that a series of biochemical reactions occur within a few minutes after ischemia/reperfusion, causing strong oxidative stress and excitotoxic damage to the brain tissue . MeanwhiOwing to the complexity of the physiological and pathological mechanisms of the human brain, drug development to ameliorate ischemic stroke is acknowledged as a very challenging task. Thousands of lead compounds that show promising therapeutic effects in preclinical trials rarely show sufficient efficacy during the trial phases . In 2020Previous studies in drug development have shown that a single protective mechanism cannot completely block the cascading damage after ischemic stroke; therefore, it is difficult to achieve adequate therapeutic effects . The treWnt signaling is a complex regulatory network consisting of two branches: canonical and non-canonical pathways . The canDkk1 gene in mouse neural stem cells in the sub granular zone increased with age and inhibited Wnt signaling activity, while neural stem cell-specific Dkk1 knockout significantly increased Wnt signaling activity and adult neurogenesis in aged mice  . The BBBWnt7a/7b (which shows the highest expression in the brain tissue) or receptor Fzd4,2,7, LRP5/6, receptor activator GPR124 in endothelial cells, or Ctnnb1 (β-catenin) can lead to abnormal cerebrovascular development and BBB function  deficiency of the endothelial Wnt/β-catenin signaling pathway affects the development of cerebrovascular and BBB, but does not affect the development and function of other organs and tissues ; 2) knocfunction ; and 3) function .Lrp6 may be correlated with the risk of ischemic stroke , Wnt signaling directly regulate the transcription of an endothelium-specific transcytosis inhibitor called major facilitator superfamily domain-containing protein 2a (MFSD2A), in a Wnt/β-catenin-dependent manner. Mice lacking either the Lrp5 or the Wnt ligand Norrin exhibit increased retinal vascular leakage and enhanced endothelial transcytosis . TherefoWnt signaling pathway has been well-established to play a critical role in neural development, axonal outgrowth, synaptogenesis, fate decision, and survival . DysreguAlthough Wnt3a protein-mediated Wnt/β-catenin signaling activation showed a decent neuronal effect, due to its hydrophobicity, Wnt3a can barely exert any biological function through systematic administration without a cosolvent, such as detergents  or solubilizers , which makes it almost impossible to conduct clinical studies. Therefore, genetic engineering-based Wnt surrogates may be a promising strategy for the development of BBB protective drugs in the future.The white matter consists of axons, oligodendrocytes, and astrocytes, which are the most common injury sites for ischemic stroke . NeuronaTo achieve remyelination after brain injury, oligodendrocytes must develop from oligodendrocyte precursor cells (OPCs). Wnt/β-catenin appears to play a crucial role in spatiotemporal regulation of oligodendrocyte differentiation . A recenAs the dominant immune cells in the CNS, microglia are well-characterized for their secretory and phagocytic properties. More importantly, microglia express various immunological receptors, which endow them with a Janus face to function in both positive and negative manner towards neurons. For instance, triggering receptor expressed in myeloid cells 2 (TREM2) is a pattern recognition receptor expressed in myeloid cells, including microglia. TREM2 was found to prohibit β-catenin degradation, thereby activating the canonical Wnt pathway . GeneticFrom the perspective of neurogenesis, microglia were found to selectively engulf synapses based on specific chemokine signals such as CR3/CX3CL1. CX3CL1 interacts with its receptor, fractalkine, specifically expressed on neurons, and thus activates microglia by phagocytosis . This prDkk1 and further inactivates the Wnt/β-catenin signaling pathway , and agonists towards the co-receptor of Wnt receptors . However