Background: While performing surgeries in the OR, surgeons and assistants often need to access several information regarding surgical planning and/or procedures related to the surgery itself, or the accessory equipment to perform certain operations. The accessibility of this information often relies on the physical presence of technical and medical specialists in the OR, which is increasingly difficult due to the number of limitations imposed by the COVID emergency to avoid overcrowded environments or external personnel. Here, we analyze several scenarios where we equipped OR personnel with augmented reality (AR) glasses, allowing a remote specialist to guide OR operations through voice and ad-hoc visuals, superimposed to the field of view of the operator wearing them.Methods: This study is a preliminary case series of prospective collected data about the use of AR-assistance in spine surgery from January to July 2020. The technology has been used on a cohort of 12 patients affected by degenerative lumbar spine disease with lumbar sciatica co-morbidities. Surgeons and OR specialists were equipped with AR devices, customized with P2P videoconference commercial apps, or customized holographic apps. The devices were tested during surgeries for lumbar arthrodesis in a multicenter experience involving author's Institutions.Findings: A total number of 12 lumbar arthrodesis have been performed while using the described AR technology, with application spanning from telementoring (3), teaching (2), surgical planning superimposition and interaction with the hologram using a custom application for Microsoft hololens (1). Surgeons wearing the AR goggles reported a positive feedback as for the ergonomy, wearability and comfort during the procedure; being able to visualize a 3D reconstruction during surgery was perceived as a straightforward benefit, allowing to speed-up procedures, thus limiting post-operational complications. The possibility of remotely interacting with a specialist on the glasses was a potent added value during COVID emergency, due to limited access of non-resident personnel in the OR.Interpretation: By allowing surgeons to overlay digital medical content on actual surroundings, augmented reality surgery can be exploited easily in multiple scenarios by adapting commercially available or custom-made apps to several use cases. The possibility to observe directly the operatory theater through the eyes of the surgeon might be a game-changer, giving the chance to unexperienced surgeons to be virtually at the site of the operation, or allowing a remote experienced operator to guide wisely the unexperienced surgeon during a procedure. The challenges of learning, planning and performing procedures in spine surgery have been enriched by the recent development of new technological tools and instrumentations, able to assist surgeons and reducing surgical invasiveness  but maintaining a valuable profile of safety –5. One oWhile performing surgeries in the operating room (OR), surgeons and assistants often need to access several information regarding surgical planning and/or procedures related to the surgery itself, or the accessory equipment to perform a wide spectrum of operations . FurtherInteractions with such digital objects were something considered science fiction, as seen in movies projecting us in the future; nevertheless, recently Microsoft implemented this technology with the “hololens,” an AR visor with a tracking system able to recognize hand motion thus allowing interactions with holograms. Most likely this technology will access the general consumer market within the next 10 years. Our group has already large experience with mixed reality, having worked on one of the first large-scale setups for AR interactions “CAVE” , which wGenerating 3D models from medical images does not imply similar challenges compared to electron micrographs –12 , we took advantage of four different state of the art AR goggles: the Epson BT-300 and BT-350, both allowing HD projection, with a 5 MPx camera on board, and the Vuzix Blade, also allowing HD projection . The latTeamViewer pilot is a cross platform remote assistance software that was developed and enhanced for the purpose of exploiting AR and AI features combined . Key perAnother in-house Unity-based software tool customized specifically for the HoloLens goggles is the Holosurgery app. This piece of software holds features that enable more convenient input methods such as hand gestures and voice commands. Summoning optimized pre-processed imaging data such as 3D models of a patient's spine is achieved with simple key vocal inputs, e.g., “Show 3D Model.” In addition, the 3D model is manipulated using hands and fingers motions and that achieves re-scaling, movement, and rotating of the 3D model. There is also more complex geometry operations such arbitrarily clipping planes which neatly visualizes a clipped region of interest within the displayed 3D model.All three state of the art software tools along with the implemented AR goggles empowers the OR staff to perform normally in non-normal and challenging times similarly in the case of global pandemics.n = 5 expert who have used to devices in the OR.System Usability Scale is an industry standard used to give a gross but reliable evaluation of the usability of a product. It is a questionnaire that can be customized to a certain extent, based on individual needs. Each answer requires an answer on a scale from 1 (strongly disagree) to 5 (strongly agree). A 9 questions questionnaire reported in AR goggles allowed to stream videos and transmit still images from the surgical field to different specialists . ProcessSurgeons had the possibility to get a live visualization of the CT reconstruction and of the planned trajectories  7) of t of t7) oThe ability to obtain an ergonomic live-sharing of surgeons view, together with the possibility to overlay images or videos offered the opportunity to involve a group of young residents and medical students for a remote step-by-step interactive learning of the surgical procedure , 3.A total number of 12 lumbar arthrodesis have been performed while using the described AR technology. Five cases of Lateral Lumbar Interbody Fusion (LLIF) and 7 Transforaminal Lumbar Interbody Fusion (TLIF) were performed with posteriore screwing through Standard  or Cortical Bone Trajectory . Intraoperative neuromonitoring was used in all the cases. Neuronavigation was used in two TLIF procedures.In three cases  surgical procedures have been shared through enhanced videoconferences among three experienced surgeons. The surgeon in the OR discussed the case while showing the screw entry-point and the trajectory, with the aid of the fluoroscopy and neuromonitoring. In the LLIF case, the discussion about the procedure involved the lateral positioning, the trans-psoas approach and the cage placement. In seven cases (5 LLIF and 2 TLIF procedures) the positioning of neuromonitoring electrodes on patients skin and the wires connection to the central monitoring platform and display was made by surgeons wearing AR goggles with the remote assistance from specialized technicians. Similarly, the remote assistance allowed the surgeon to set neuronavigation in two cases. In all the procedures, companies ensured a live support for nurses assisting surgeons with regard to the devices and surgical instrumentations needed.In three cases AR goggles allowed the surgeon to access to the surgical planning of patients that underwent CBT fixation in real time while maintaining the view on the operator field.In two cases  a group of four residents belonging to their first year of the Residency program and two medical students got access to the procedure with a remote connection, with the possibility to interact with the surgeons. Surgeries were performed in a step-by-step manner.No complications potentially linked to the use of AR were registered, such as malfunction of the neuromonitoring and of the neuronavigation system, or infections. Surgeons reported a positive feedback as for the ergonomy, wearability and comfort during the procedure, as confirmed by the results shown on the graphs in AR represents the possibility to create a useful and real-time interaction between multiple environments and/or images/videos of interest . AR systIn this case series a simple, ergonomic and successful use of AR goggles is presented. In addition, the unfortunate conjunction with the COVID-19 pandemic has led to the chance of facing physical restrictions adding further applications of this technology.These tools, indeed, allow surgeons to view images and use apps anywhere and anytime they like overlaying digital content on their real field of view. Moreover, images, videos or screens shared by other devices could be watched on these see-through lenses, through an enhanced videoconference app . ConsideAnother important advantage offered by this AR system, is the real-time visual feedback of the operative field that allows the mentor to provide a better coaching, as reported in other previous papers , 20. DavThese examples could represent a starting point to better investigate the potential development of AR for the teaching/supervision of surgical techniques, reducing the need for physical presence of experienced surgeon and consequently its related constraints on time and budgets.The remote mentoring could also be considered to coordinate the setting up of the operative room for newer procedures or to help surgeons with the use of new instrumentations, even when specialist consultants could not physically enter the operative room. It is well-known that many traditional neurosurgical procedures often required the use of intraoperative neuromonitoring (IONM) in order to guarantee the best result, both in terms of extent of resection and neurological safeguarding. With the advent of new emerging minimal invasive techniques for spinal degenerative disease , this need has spread further. Consequently, the great spread of the use of these techniques has increased the need for IONM , 22, witFinally, and as already mentioned, although the remote mentoring and specialist counseling with the AR seemed, until few months ago, only a window on the future of the operative rooms and surgical activities, the recent dramatic experience of lockdown due to COVID-19 pandemic spread has changed the perspective, making it an everyday tool for the OR.Another important application of AR for surgery is represented by its role in surgical planning. In this series CBT planning was visualized by the surgeon while maintaining the view on the surgical field and obtaining a real time feedback of the planned screw entry points. During the past few decades, several tools have been developed to improve pre-operative surgical planning both for spine and cranial surgery , 23.The 3D printing era brought most surgical fields to an advanced new level, where even minimal differences from standard anatomy are detected, helping surgeons during the pre-operative planning and during the procedure, and then, leading to a customized surgical management. Nowadays, the so-called image guidance surgery is widely used in different surgical specialties  but recently, due to reached high accuracy, have been widely implemented in neurosurgery for cranial, spinal and skull base procedures . Penner To this end, systems incorporating haptic feedback for realistic rendering of contact forces experienced during the interaction with tissues are considered of fundamental importance for speeding up the learning curve . On the For these tasks, haptic rendering is required to provide realistic forces and torques created by the complex interactions between the surgical tools and tissues involving tool penetration, tissue removal, rotational speed and vibrations . The accApart of these considerations, the proposed system can be used for gathering data related to surgical tool trajectories that can be used for fitting haptic models describing the tool-tissue dynamics that can be derived through contact models  or more Masciatelli et al. and Cabrilo et al. firstly described the application of AR in neurovascular surgery showing optimized workflow by providing essential anatomical information , 36. In Resident training in surgical specialties is based on the apprenticeship model developed by Dr. William Halsted in 1980s and the training paradigm of “see one, do one, teach one” have been the pivotal concept until nowadays . DiffereThe operating theater has been the main classroom for many surgeons and is well-known that acquisition of surgical skills requires repeated occasions for hands-on practice. However, the limited number of people that can access to the OR and the large number of residents that need to learn surgical procedures often represent an issue, especially for small surgical centers and less developed countries.Thus, the application of new technologies to increase residents' exposure to surgical procedures could play a key role for the learning curve. Thanks to its integrated camera and the previous described videoconference function, the EPSON glasses gives to the surgeon the possibility to record all the procedure and to create a live streaming that could be shared with residents and medical students, reducing the need for physical presence in the operative room. Moreover, the possibility to watch the pre-operative planning and reconstructed 3D models superimposed on the surgical field through the EPSON glasses, provide a double advantage; on one hand, indeed, surgeon has the possibility to see the model without taking eyes off of the operative field, while on the other hand, the simultaneous view of the real surgical field and of the 3D model could improve and speed up the residents' learning process.Henssen et al. reported interesting results with their experience with AR comparing two different methods to study neuroanatomy; the classic method of studying cross sections of the brain and the one based on an AR-based neuroanatomy learning app . Hence, Understanding the true usability of the system, in order to assess whether it is not merely a technical exercise but rather a potential “everyday use tool” was key to us. In order to quantify how specialists perceived the use of the devices in the OR, they filled a SUS questionnaire  and rateTherefore, providing a precise and reliable 3D virtual and interactive environment, AR may become an extremely valuable tool for education of neurosurgical procedures, due to their intricate and complex nature.fabio.cofano@gmail.com.The data analyzed in this study is subject to the following licenses/restrictions: Ethical. Requests to access these datasets should be directed to Fabio Cofano, Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.CC wrote the first proof of the paper, with inputs from LD and MC. FC retrieved all the relevant literature, put together all qualitative/quantitative contributions from all authors and other users, and wrote the whole manuscript. MA contributed with a critical review of the manuscript and significant inputs during the rebuttal phase. GD, AL, MB, NM, FZ, and NZ tested the AR setup on field and gave their feedbacks to the technical team. DB was responsible for the technical support of the system and coordinated the work on the Unity code for the Microsoft hololens setup, together with MC. LD and DG coordinated the operations in OR with the AR setup. DG and CC coordinated the study. All authors contributed to the article and approved the submitted version.LD is the owner of the company LD Consulting. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Streptococcus pyogenes (group A Streptococcus (GAS) is an important human pathogen that can cause severe invasive infection, such as necrotizing fasciitis and streptococcal toxic shock syndrome. The mortality rate of streptococcal toxic shock syndrome ranges from 20% to 50% in spite of antibiotics administration. AR-12, a pyrazole derivative, has been reported to inhibit the infection of viruses, intracellular bacteria, and fungi. In this report, we evaluated the bactericidal activities and mechanisms of AR-12 on GAS infection. Our in vitro results showed that AR-12 dose-dependently reduced the GAS growth, and 2.5 μg/mL of AR-12 significantly killed GAS within 2 h. AR-12 caused a remarkable reduction in nucleic acid and protein content of GAS. The expression of heat shock protein DnaK and streptococcal exotoxins was also inhibited by AR-12. Surveys of the GAS architecture by scanning electron microscopy revealed that AR-12-treated GAS displayed incomplete septa and micro-spherical structures protruding out of cell walls. Moreover, the combination of AR-12 and gentamicin had a synergistic antibacterial activity against GAS replication for both in vitro and in vivo infection. Taken together, these novel findings obtained in this study may provide a new therapeutic strategy for invasive GAS infection. Streptococcus (GAS), Streptococcus pyogenes, is an important human pathogen, which induces a wide spectrum of symptoms, from superficial skin and throat infections to life-threatening streptococcal toxic shock syndrome and necrotizing soft tissue infections. The mortality rate of the streptococcal toxic shock syndrome may exceed 50% in spite of aggressive treatment [Group A reatment ,2,3. GASreatment . The increatment ,7.AR-12 (OSU-03012), a pyrazole-based compound derived from celecoxib, has completed the first phase of clinical trials as an anticancer agent . AR-12 iCryptococcus neoformans [Salmonella enterica, Francisella tularensis, and Leishmania donovani [Mycobacterium abscessus in cells but also reduces the bacterial levels in the lungs of M. abscessus-infected mice [Staphylococcus aureus, Streptococcus pneumonia, Listeria monocytogenes, Bacillus anthracis, and Mycobacterium smegmatis [Staphylococcus aureus [Streptococcus infection.Reports also indicate that AR-12 can inhibit fungal acetyl CoA synthase and PDK1 activity, and improve the antifungal activity of fluconazole to inhibit the growth of oformans ,18. In aoformans ,20,21. Adonovani ,23,24. Zted mice . Celecoxmegmatis . Severals aureus . The cyts aureus . We cons6 CFU/mL and 3.8 × 107 CFU/mL to 2.3 × 103 CFU/mL and 1.1 × 103 CFU/mL by 2.5 μg/mL of AR-12 treatment for 2 h incubation, respectively. After 24 h incubation with 2.5 μg/mL of AR-12, the remaining bacteria of A20 and NZ131 strains amounted to 5.9 × 105 CFU/mL and 1.2 × 105 CFU/mL, respectively, which were two log folds lower than those of the medium control, 0.25 μg/mL, or 0.5 μg/mL of AR-12-treated groups. These data suggest that AR-12, at a concentration ≥2.5 μg/mL rapidly killed both GAS strains, and 90% of the A20 and NZ131 GAS cells were inhibited by 2.5 μg/mL of AR-12. This indicates that the MIC at 90% inhibition level (MIC90) of AR-12 against both GAS strains was 2.5 μg/mL. In addition, compared with the medium-only treatment of A20 cells (7.4 × 106 CFU/mL) and NZ131 cells (3.8 × 107 CFU/mL) at 2 h incubation time, there were 1.0 × 106 CFU/mL of A20 cells and 1.1 × 107 CFU/mL of NZ131 cells after 2 h treatment by 0.5 μg/mL of AR-12, respectively, and this indicates that AR-12 temporary inhibited A20 and NZ131 cell growth at a concentration of 0.5 μg/mL  was further used to analyze the surface morphological change of the AR-12-treated NZ131 strain. After treatment with 2.5 μg/mL of AR-12 for 2 h, the SEM images revealed the remarkable change in GAS architecture. Untreated NZ131 cells showed a smooth surface pattern of cell walls A,B, wherReports have indicated that AR-12 inhibits the activity of several protein kinases, including AKT, JAK2, PDK1, and STAT3 of different mammalian cell lines and induces cell death ,12,13,14Acinetobacter baumannii and Klebsiella pneumoniae [Streptococcus A20 strain and NZ131 strain was evaluated. Our previous data indicated that the MIC90 of AR-12 against both GAS strains was 2.5 μg/mL  or the AR-12-only group (5.2 × 108 CFU/mL) (8 CFU/mL (the gentamicin-only group) or 5.3 × 108 CFU/mL (the AR-12-only group) to 9.7 × 107 CFU/mL at the same incubation time  B. Howeveion time D. Even tBased on the equation used to figure out the antibacterial mechanism of compounds ,30, we c8 CFU of NZ131, different doses (10 μg/kg to 400 μg/kg) of AR-12 were injected into the air-pouch immediately, and then the survival rates of mice were examined. The results showed that while all of the PBS-treated control mice, the 10 μg/kg and the 100 μg/kg AR-12-treated mice died within 10 days post infection, 10% of the 400 μg/kg AR-12-treated mice survived over the 14-day experimental period. However, there was no statistically significant difference between the AR-12 (400 μg/kg)-treated mice and the GAS-infected control mice. Subsequently, the protective efficacy of the combination of gentamicin and AR-12 was examined. As shown in 8 CFU/mouse) without treatment caused 100% of mice death within 9 days; treatment of AR-12 (10 μg/kg) still caused 100% of mice death within 12 days. Treatment of gentamicin (2 μg/kg) with GAS-infected mice caused 25% of mice survival over 14 days, even though there was no statistical difference compared with the GAS-infected group. However, combination treatment of 2 μg/kg of gentamicin and 10 μg/kg of AR-12 significantly elevated the GAS-infected mice survival rate to 50%  with a spectrophotometer . The bacterial suspension was diluted with phosphate-buffered saline (PBS) to 2 × 109 CFU/mL for in vivo experiment. To quantitate the exact bacterial concentration, the bacterial suspension was serially diluted with sterile PBS, then 0.1 mL was poured on THY agar plates and incubated at 37 °C overnight.GAS strain A20 (M1 serotype) was isolated from a patient with necrotizing fasciitis . GAS str600. The MIC of AR-12 or gentamicin was determined by the agar dilution method. For exact quantification of bacterial counts at different time points, the bacterial suspensions were serially diluted, plated on THY agar, and cultured at 37 °C overnight. The results of one of three experiments are reported.A fresh GAS colony was grown in THY broth (Difco Laboratories) for 12 h at 37 °C and then subcultured into the fresh broth. At the time of subculture, varying concentrations of AR-12 or gentamicin were added to the bacterial suspension, and bacterial growth at different time points was determined with a spectrophotometer by measuring A2 in an incubator .A549 and RAW264.7 cell lines were grown in DMEM  supplemented with 5% fetal bovine serum, 100 μg/mL of penicillin, and 100 μg/mL of streptomycin (Gibco). U937 cells were grown in RPMI 1640 (Life Technologies) supplemented with 5% fetal bovine serum, 100 μg/mL of penicillin, and 100 μg/mL of streptomycin (Gibco). HMEC-1 cells were grown in M200 (Life Technologies) supplemented with low serum growth factor, 100 μg/mL of penicillin, and 100 μg/mL of streptomycin . The cells were cultured at 37 °C and with 5% CO+ or SYPRO+ cells were calculated and expressed as the mean ± standard deviation.To examine the mechanisms of the bacteriostatic and bactericidal effects of AR-12, GAS NZ131 cells were incubated with different concentrations of AR-12 for 2 h and fixed with 4% paraformaldehyde. The fixed bacteria were reacted with SYPRO dye (Thermo Fisher Scientific) in 0.04% SDS for 0.5 h. Then, the fixed GAS was reacted with propidium iodide (PI) dye  for 1 h to analyze the amounts of nucleic acids of GAS. After washing, the bacteria were suspended in 0.5 mL of PBS and analyzed by the flow cytometer FACSCalibur . The total protein and nucleic acid contents in the GAS were detected by the flow cytometer and determined by the mean fluorescence intensity (MFI) of each group. The percentages of PIWST-1 is a sensitive colorimetric assay for determining cell viability in cell proliferation and cytotoxicity assays. An appropriate number of cells were plated in 96-well microtiter plates and then treated with 0.25, 0.5, and 2.5 μg/mL of AR-12 for various time courses. The WST-1 reagent solution (Roche) was added to each well of the 96-well microplate containing 100 μL of cells in the culture medium, and the plate was then incubated for 2 h at 37 °C. Absorbance was measured at 450 nm by using a microplate reader (Life Technologies). The results of the three experiments are represented and expressed as the mean ± standard deviation.2 for 24 h, and then the culture supernatants were collected. Cytotoxicity of the AR-12-treated cells was determined using the lactate dehydrogenase (LDH) assay and detected by a microplate reader (Life Technologies). The levels of LDH released into the cell culture supernatants were measured using the LDH Cytotoxicity Detection Kit  according to the manufacturer’s instructions. The cytotoxicity percentage was calculated as follows: cytotoxicity % = /(Triton X − 100 − treated group − medium background) × 100%. The results of the three experiments are represented and expressed as the mean ± standard deviation.Cell lines were cultivated in the absence or presence of different concentrations of AR-12 at 37 °C and 5% CO600 = 0.2~0.3) and late-log (A600 = 0.4–0.5) phases of the GAS culture supernatants, respectively, and these culture supernatants were incubated with different concentrations of AR-12. Subsequently, these supernatants were confirmed by Western blot analysis using anti-SPE B and anti-SLO monoclonal antibodies (Sigma-Aldrich). The horseradish peroxidase-conjugated goat anti-mouse IgG  was used as the secondary antibody, and the blots were developed by ECL, as previously described.The NZ131 strain was incubated with different concentrations of AR-12 at 37 °C for 2 h and then harvested by centrifugation (6000 rpm) at 4 °C. After centrifugation, the bacteria were washed twice with ice-cold sterile water and suspended in Tris-EDTA buffer  containing 200 μL of lysozyme  and 50 μL of mutanolysin . Total protein contents of each group were isolated using the Bacterial Total Protein Extraction Kit . Then, 20 μg of the protein samples were separated using SDS–PAGE and then transferred to the PVDF membrane. After blocking, the blots were hybridized with anti-DnaK or anti-GroEL antibodies as the primary antibody, and horseradish peroxidase-conjugated goat anti-rabbit IgG  was used as the secondary antibody. Finally, blots were developed using an ECL Western Blot Detection Kit  according to the manufacturer’s instructions. In addition, streptolysin O (SLO) and streptococcal pyrogenic exotoxin B (SPE B) were collected from the mid-log  and fixed with 1% aqueous osmium tetroxide in 0.1 M PBS solution at 4 °C for 24 h. The fixed GAS was washed three times in 0.1 M PBS, serially dehydrated from 10 to 100% ethanol, followed by 33%, 66%, and absolute acetone, and dried in a Hitachi HCP-2 critical point dryer. The specimens were mounted and coated with gold by Hitachi E-1045 ion sputter and investigated with a Hitachi S-4700 scanning electron microscope at 1 kV.8 CFU in 0.1 mL of sterile PBS was inoculated into the air pouch. After the GAS inoculation, 0.1 mL of different doses of AR-12  was given immediately into the air pouch. To determine the effects of the combination of AR-12 and gentamicin on GAS infection, 2 μg/kg of gentamicin, 10 μg/kg of AR-12, and a mixture of both agents were administered immediately post GAS infection. The animals were observed every day, and the mice survival rate was examined for a total of 14 days.BALB/c mice were purchased from the National Laboratory Animal Center in Taiwan. Eight-week-old male mice (body weight 25 ± 1.5 g per mouse) were used in all experiments. The animals were raised and cared for in accordance with guidelines established by the Ministry of Science and Technology in Taiwan. All procedures, care, and handling of the animals were reviewed and approved by the Institutional Animal Care and Use Committee at I-Shou University (ISU108022). Groups of eight to ten mice were anesthetized and then injected subcutaneously with 1 mL to form an air pouch. The NZ131 cell suspension containing 2 × 10p < 0.05, ** p < 0.01, and *** p < 0.001.The statistical analysis was conducted using Prism 5.0 software . The data shown in In this study, we demonstrated that AR-12 has an anti-GAS effect in vitro and in vivo. Moreover, the mechanisms for the antibacterial effect of AR-12 on the GAS strain NZ131 cells were evaluated, leading to the indication that AR-12 impeded the synthesis of proteins and nucleic acids, and damaged the bacterial surface structures by weakening the cell wall of GAS. Furthermore, there was a synergistic effect of the AR-12 and gentamicin to act against GAS. The sub-MIC dose of AR-12 potentiated the GAS killing efficacy of a sub-MIC dose of gentamicin in vitro and in vivo. Thus, these results suggest the possible pharmacological use of AR-12 as an effective anti-GAS agent and may provide a new therapeutic strategy for invasive GAS infection.


The associations between hypertension, diabetes, and dyslipidemia with Bell’s palsy have been controversial and only a few studies have assessed risk factors for Bell’s palsy based on population-based data. The aim of the present study was to evaluate whether sociodemographic factors such as sex, age, residence, household income, and metabolic diseases such as hypertension, diabetes, and dyslipidemia were risk factors for Bell’s palsy using the National Health Insurance Service National Sample Cohort data of Korea. Patients who visited an outpatient clinic twice or more or had one or more admission and received steroid medication under the International Classification of Diseases diagnostic codes for Bell’s palsy from 2006 to 2015 were defined as patients with Bell’s palsy in this study. The associations between sociodemographic factors and metabolic diseases to Bell’s palsy were analyzed with univariate and multivariate Cox proportional hazard regression models. There were 2708 patients with Bell’s palsy recorded from 2006 to 2015. Male sex, advanced age, residence in a location other than the capital and metropolitan cities, hypertension, and diabetes were significant risk factors for Bell’s palsy. This study is significant for patients and providers because we analyzed the relationships using a population-based database over a long-term follow-up period. It is the most common cause of peripheral facial palsy9. The annual incidence of Bell’s palsy has been reported to range from 11.5 to 53.3 per 100,00011. The etiology of Bell’s palsy remains unclear14; however, it has been attributed to the reactivation of latent herpes simplex virus type 1 (HSV-1) infection in the geniculate ganglion14. Ischemia and autoimmune conditions have also been proposed as causes14.Bell’s palsy is an acute idiopathic paresis or paralysis of the peripheral facial nerve that contracts muscles of the face13. These are characteristic of metabolic syndrome, which is a chronic metabolic disorder15. Metabolic syndrome has been reported to be associated with a worse recovery rate in Bell’s palsy10. Although several reports have indicated that there is an association between hypertension, diabetes, and dyslipidemia and Bell’s palsy, to date the reports remain controversial16.Several factors associated with Bell’s palsy have been reported, including hypertension, diabetes, and dyslipidemia, which are associated with microangiopathy and can be risk and prognostic factors for Bell’s palsyA few studies have assessed risk factors for Bell’s palsy based on population-based data. Considering the controversial and uncertain relationship, we used population-based national sample cohort data from Korea to evaluate whether sociodemographic factors such as sex, age, residence, household income, and metabolic diseases such as hypertension, diabetes, and dyslipidemia were risk factors for Bell’s palsy.This retrospective study included public data from the National Health Insurance Service National Sample Cohort data of Korea, which is a 2% representative subsample of the entire National Health Insurance Service data. It was established with systematic stratified random sampling with proportional allocation within each stratum, resulting in the representativeness of the whole data. All Korean citizens are required to join the National Health Insurance Service; thus, the results of this study are based on the whole population in Korea.Patients who visited the outpatient clinic twice or more or had one or more admission from 2006 to 2015 and received steroid medication under the diagnostic codes for Bell’s palsy in the International Classification of Diseases (ICD) (G51.0) were defined as patients with Bell’s palsy. Thus, steroid treatment was performed for all members of the patient group. The control group was established using the rest of the patients from the database. Considering the easy accessibility to medical services due to the mandatory National Health Insurance Service in Korea, as well as the large size of the control group, patients with Bell’s palsy who had not visited the hospital in the control group were thought to be negligible. The patients with Bell’s palsy and control groups were divided according to sociodemographic factors such as sex, age, residence, and household income, and metabolic diseases such as hypertension, diabetes, and dyslipidemia, that were diagnosed during 2003 to 2015, prior to the onset of Bell’s palsy. Patients with hypertension, diabetes, and dyslipidemia were defined using diagnostic codes in the ICD  and relevant medication claim codes including beta blockers, alpha blockers, calcium channel blockers, angiotensin converting enzyme inhibitors, angiotensin II receptor blockers, diuretics for hypertension, insulin, biguanide, alpha-glucosidase inhibitors, sodium glucose co-transporter-2 inhibitors, sulfonylurea, meglitinide, thiazolidinedione, dipeptidyl peptidase-4 inhibitor for diabetes, statin, ezetimibe, niacin, fibrate, cholestyramine, and omega-3 fatty acid for dyslipidemia.p < 0.05 was considered statistically significant.First, the incidence and sociodemographic distribution of patients with Bell’s palsy were investigated because they were fundamental to the analysis of risk factors. Associations between sociodemographic factors such as sex, age, residence, household income, and metabolic diseases such as hypertension, diabetes, and dyslipidemia with Bell’s palsy were analyzed. Data from 2006 to 2015 were included in the univariate and multivariate Cox proportional hazard regression models. Statistical analyses were performed using SAS 9.4 . In all analyses, This study was in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The Institutional Review Board of the National Health Insurance Service Ilsan Hospital approved this study (NHIMC 2020-10-009). All methods were performed in accordance with the relevant guidelines and regulations. Written informed consent was waived by the Institutional Review Board of the National Health Insurance Service Ilsan Hospital because of the retrospective nature of the study.There were 2,708 patients with Bell’s palsy from 2006 to 2015. The average annual incidence of Bell’s palsy was 25.9 per 100,000 for 10 years , advanced age , residence in regions other than the capital and metropolitan cities , hypertension , diabetes , and dyslipidemia .Multivariate Cox proportional hazard regression analysis indicated that significant risk factors for Bell’s palsy were male sex , advanced age , residence in regions other than the capital and metropolitan cities , hypertension , and diabetes   than males (47.6%)5. However, there were more male patients with Bell’s palsy (52.5%) than females (47.5%) in our population-based study, and male sex was a significant risk factor for Bell’s palsy. Recently, Lee and Kim also reported that there were more male patients with Bell’s palsy than females and that male sex was also a risk factor for Bell’s palsy based on population-based data in Korea11.Several reports have indicated that the distribution of Bell’s palsy is equal among men and women12. However, Lee and Kim’s study, which was based on National Health Insurance Service data in Korea, showed a peak incidence of Bell’s palsy between 60 and 69 years of age11. In the study based on the Korean National Health and Nutrition Examination Survey, another population-based data set in Korea, advanced age was significantly associated with facial palsy, including Bell’s palsy17. Another epidemiologic study in Italy showed that advanced age was a significant risk factor for Bell’s palsy, and correlated with a linear trend when age increased12. In our study, the risk for Bell’s palsy increased when age increased and peaked at 60–69 years of age. The age of the general population is increasing, which can impact reactivation of latent herpes simplex virus infection in the geniculate ganglion and also the incidence of ischemia, which are more likely with old age, thus resulting in a high risk for Bell’s palsy for older patients compared with a peak incidence in a previous report.Bell’s palsy has demonstrated a peak incidence in patients between 15 and 45 years of ageTo date, no studies have been conducted to verify the relationship between socioeconomic factors such as household income and residence with Bell’s palsy. In the present study, residence in a location other than the capital and metropolitan cities, which indicates small-sized cities or rural areas, was associated with significant risk factors for Bell’s palsy. It is hypothesized that the risk was higher due to the aging of the population in rural areas.2. In their systematic review, Jörg et al. reported that there might be an association between uncontrolled severe hypertension and peripheral facial palsy in children18. Hung et al. reported that hypertension was significantly higher in the Bell’s palsy group than in the control group in a population-based study in Taiwan5.The association between hypertension and Bell’s palsy has been described in many studies. Savadi-Oskouei et al. reported that hypertension may increase the risk of Bell’s palsy in patients over 40 years of age1. Monini et al. reported that hypertension was not a significant risk factor for Bell’s palsy12. Riga et al. reported that hypertension was not associated with the severity of Bell’s palsy at initial diagnosis, nor was it associated with the recovery from Bell’s palsy after six months13. Additionally, Chang et al. reported that hypertension was not associated with facial palsy, including Bell’s palsy with House-Brackmann grade 3–6, whereas cardiovascular disease was associated with those clinical indicators17.In contrast, several studies reported that there were no associations between hypertension and Bell’s palsy. Peitersen reported that Bell’s palsy is not more frequent in patients with hypertension than in those without hypertension16. Hematoma or thrombus caused by hypertension in the facial canal could compress the facial nerve. Thickened vessel and perineural edema due to hypertension could also compress the facial nerve10. Of note, uncontrolled hypertension is particularly associated with Bell’s palsy7. Patients with uncontrolled hypertension have a higher possibility of hemorrhage into the facial canal and necrosis in the facial nerve than those with controlled hypertension, which likely contributes to these findings.In our study, hypertension was confirmed to be a risk factor for Bell’s palsy; Bell’s palsy and hypertension was defined by the diagnostic and medication claim codes. Furthermore, our study analyzed population-based data over a long-term period from 2006 to 2015. Thus, based on the long-term data, hypertension has been confirmed as a risk factor for Bell’s palsy. However, the etiology is unclear, as small hemorrhages in the facial canal, vascular lesions, and partial necrosis in the nerve may cause facial palsy2. Riga et al. reported that diabetes with abnormal glycosylated hemoglobin (Hb1A1c) was associated with the severity of Bell’s palsy at initial diagnosis13. Hung et al. reported that diabetes was significantly higher in the Bell’s palsy group than in the control group in a population-based study in Taiwan5.Many studies have reported the association between diabetes and Bell’s palsy. Savadi-Oskouei et al. reported that diabetes was a predictor of Bell’s palsy12. Sevik Elicora and Erdem reported that diabetes was not associated with the severity and recovery rate in Bell’s palsy9. Chang et al. reported that diabetes was not associated with facial palsy, including Bell’s palsy with House-Brackmann grade 3–617. However, our study revealed that diabetes was an associated risk factor for Bell’s palsy.In contrast, Monini et al. reported that diabetes was not a significant risk factor for Bell’s palsy14. A vascular rather than metabolic mechanism has been suggested for facial palsy in diabetes2. Chronic nerve ischemia can occur due to the reduction in endoneurial oxygen, reduced blood flow in the nerve, and epineurial arteriovenous shunt13. Consequently, microangiopathy may be a compensatory response to endoneurial ischemia or hypoxia. In addition, hyperglycemia induces direct nerve injury due to increased oxidative stress, accumulation of advanced glycation end products, impaired axonal transport, and impaired flow through the polyol pathway13.Microangiopathy such as vascular insufficiency and diabetic polyneuropathy were associated with a poor prognosis5.Several studies have described an association between dyslipidemia and Bell’s palsy. Hung et al. reported that hyperlipidemia was significantly higher in the Bell’s palsy group than in the control group in their population-based study in Taiwan. Regular statin (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor) use (≥ 60 days within 6 months) was significantly associated with Bell’s palsy13. Chang et al. found that hyperlipidemia was not associated with facial palsy, including Bell’s palsy with House-Brackmann grade 3–617.In contrast, Riga et al. reported that hypercholesterolemia was not associated with the severity of Bell’s palsy at initial diagnosis nor was it associated with the recovery from Bell’s palsy after six months6. Because we defined patients with dyslipidemia using diagnostic codes and relevant medication claim codes such as statins, our results indicated that dyslipidemia with statin use may not be a risk factor for Bell’s palsy, which is in agreement with the report by Kim et al.Kim et al. reported no relationship between previous statin use over the course of 2 years and the incidence of Bell’s palsy in patients 40 years of age or older, based on population-based data in Korea, although there were significantly more patients with dyslipidemia in the Bell’s palsy group than in control group10.Triglyceride and high-density lipoprotein-C are associated with atherosclerosis. Microcirculatory disorders caused by atherosclerosis increase the accumulation of lipids and cholesterol in the vessel, which induces endothelial dysfunction, plaque formation, vascular inflammation and obstruction, microvascular ischemia, and neuropathy5. Statins can induce a disturbance in neurite outgrowth and maintenance and the neurotoxic effects of statins could increase the risk of Bell’s palsy6. Statins inhibit the synthesis of ubiquinone, which is essential for the mitochondrial respiratory chain, resulting in disruption to the energy supply to neurons6. Changes in neuronal cell membrane composition could influence protein interactions and neuronal functions. Axonal degeneration and segmental demyelination could be associated with neuropathy in statin use6. In addition, statins could stimulate pro-inflammatory responses and induce autoimmunity5.There are several possible etiologies for the association between statin use and Bell’s palsy. The potential risk of peripheral neuropathy due to statin use has previously been reported6. Anti-inflammatory and anti-oxidative effects of statins have also been reported. These neuroprotective, anti-inflammatory, and anti-oxidative effects could reduce the risk of Bell’s palsy6. However, the neuroprotective effect of statins may be restricted to neuron bodies, not to nerve sheaths, and statins might negatively impact oligodendrocytes and myelin formation5. In this respect, Bell’s palsy, which is a peripheral neuropathy, could increase due to statin use. However, considering previous studies that reported an association between peripheral neuropathy and statin use, these adverse effects of statins might be minimal6.In contrast, there is also a neuroprotective effect with statin useIn the present study, metabolic diseases were defined using both diagnostic and medication claim codes, which was more accurate than a definition using only diagnostic codes. Based on the results of the present study, hypertension and diabetes are associated with Bell’s palsy, whereas dyslipidemia is still controversial regarding its association with Bell’s palsy when considering the administration of statin.There were several limitations to our study. First, the risk factors for Bell’s palsy were analyzed based on the diagnostic codes and medication claim codes, which do not reflect the actual incidence of the disease. Second, only the predicting factors for Bell’s palsy were analyzed and the prognostic factors were not evaluated because the degree and prognosis of Bell’s palsy could not be verified in the population-based database that was based on diagnostic and medication claim codes. Nonetheless, this study analyzed the relationship between sociodemographic factors and major chronic metabolic diseases with Bell’s palsy comprehensively using population-based database over a long-term period, which contributes to clinical and socio-economic understanding of this condition. Although novel risk factors were not revealed in the present study, this study was performed using population-based data and suggests that patients with risk factors for Bell’s palsy should be carefully evaluated for prevention and clinical practice of Bell’s palsy.In summary, male sex, advanced age, residence in a location other than the capital and metropolitan cities, hypertension, and diabetes were significant risk factors for Bell’s palsy based on population-based data in Korea.


Nurses are faced with varying job stressors depending on their positions and duties. Few previous studies have compared job stress and related chronic conditions among different nursing positions. The objectives were to compare job stressors among clinical registered nurses, nurse practitioners, and head nurses and explore the impact of job stressors and stress level on hyperlipidemia, hyperglycemia, and hypertension.Secondary data extracted from a survey of health-care workers conducted from May to July 2014 across 113 hospitals in Taiwan was used. This analysis included 17,152 clinical registered nurses, 1438 nurse practitioners, and 2406 head nurses. Socio-demographic characteristics, job stressors, stress levels, and hyperlipidemia, hyperglycemia, and hypertension variables were extracted.Perceived stressors differed among clinical registered nurses, nurse practitioners, and head nurses, but overall stress level did not. Nurse practitioners and head nurses showed significantly higher prevalence of hyperlipidemia, hyperglycemia, and hypertension than clinical registered nurses. Higher stress levels, age, body mass index, work hours, and caring for family members were positively associated with hyperlipidemia, hyperglycemia, and hypertension. After adjustment for these variables, risk of hyperlipidemia, hyperglycemia, and hypertension did not differ across the nursing positions.Although stressors vary by different nursing positions, overall stress level does not. Hyperlipidemia, hyperglycemia, and hypertension are related to stress level, age, body mass index, weekly working hours, and caring for family members. Hence, alleviating job stress and avoiding long working hours are likely to reduce the risk of hyperlipidemia, hyperglycemia, and hypertension in nurses. In many countries, nursing is considered as a highly stressful occupation , and higEncountering stress from various sources at work may cause distress symptoms, and inadequate treatment of these symptoms could result in the development of physiological and/or mental diseases . Over thSeveral cross-sectional studies have reported that work stress is a risk factor for hyperlipidemia , 9, 10. Nurses account for a large proportion of the personnel in the health-care industry. They play a crucial role in clinical care, and have a wide variety of responsibilities . HoweverThe United States (US)’ health-care system features four types of advanced-practice registered nurses: certified nurse practitioners (NPs), certified registered nurse anesthetists, clinical nurse specialists, and certified nurse midwives . NPs’ prPrevious studies on work stress among nurses have compared job stress across hospital levels, work units, and qualifications , 28; howThis secondary data analysis was performed using data sourced from a national cross-sectional study of full-time health-care workers in Taiwan that was conducted from July to September 2014. This study invited those health facilities that ever jointed the Taiwan Health Promoting Hospital Network, ever participated in the previous similar survey in 2011, or ever applied for certification of health promoting hospital and health-care institution to participate. Of the 157 health facilities invited, 113 agreed to participate, including 13 medical centers, 59 district hospitals, 32 regional hospitals, eight public-health centers, and one nursing home. After obtaining approval from the hospitals, envelopes with consent forms and questionnaires were distributed to each of the hospital. Health-care workers were asked to return the signed consent forms and filled questionnaires using the provided two separated and sealed envelopes to the data collection site at each hospital. Overall, 111,110 questionnaires were distributed, 89,014 filled questionnaires were returned, and 75,841 valid questionnaires were collected . The dataset with valid records were released to the research team by the Taiwan Health Promotion Administration. For the present study, questionnaires answered by non-nursing staff  and non-clinical staff  were excluded, remaining 20,996 anonymous questionnaires completed by full-time clinical nurses, which formed the analytical sample for this secondary data analysis. Among these nurses, 2406 (11.5%) were HNs, 1438 (6.8%) were NPs, and 17,152 (81.7%) were CNs. Details of the study design are presented elsewhere .Data were collected through a structured questionnaire that focused on the physical and mental health, and safety of the participants. Prior to the data collection, six experts reviewed and revised the questionnaire content over two meetings, and a pilot test was performed on 10 health-care workers to ensure its clarity and readability.Hyperlipidemia, hyperglycemia, and hypertension were measured using yes/no questions regarding the presence of any of those chronic conditions. The study variables related to hyperlipidemia, hyperglycemia, and hypertension included socio-demographics , work experience , medical history and chronic diseases, self-reported weight and height, and family-care needs. Family-care needs was measured by a question enquiring “current family-care needs” with four items -- (1) self or spouse being pregnant, (2) having children at preschool age, (3) having children in elementary school and/or high school, (4) having other family member who is in need of care. Any positive answer to the four items was coded as yes, otherwise it was coded as no.Sources of work stress and level of perceived stress were measured by a scale of 23 items. A five-point Likert scale (1–5) was used to measure level of perceived stress; higher scores indicated greater stress, given a summary score ranged from 23 to 115 points. The reliability and validity of the scale have been described previously . The scaStatistical analysis was performed using SPSS 23.0 . Means and standard deviations were used to describe continuous variables, and frequencies and percentages were used to describe categorical variables and the overall distribution of the sample. Analysis of variance (ANOVA) was used to compare the level of stress among the three different nurse positions. If a significant difference was found, the Scheffé post-hoc test was conducted for pairwise comparisons. Further, logistic regression models were applied to examine the factors associated with the presence of any of the chronic conditions including hyperlipidemia, hyperglycemia, and hypertension.SD]: 7.09), followed by NPs , and CNs . Most participants (98%) were women. Over one quarter (26.2%) were overweight or obese (body mass index [BMI] > 24). Over half (60%) of the NPs and HNs were married; most CNs (> 60%) were never married. HNs had the highest mean number of years of work experience, at 20.35 (SD: 9.75), followed by NPs at 15.14 (SD: 9.72) and CNs at 11.96 (SD: 10.44). The general ward was the most-represented work unit (42.7%). HNs had the highest mean number of work hours per week , followed by NPs  and CNs . The CN group had the highest proportion of members who had family-care needs (49.6%).Table n = 931, 4.8%), followed by hyperlipidemia  and hyperglycemia . The prevalence of hyperlipidemia, hyperglycemia, and hypertension was highest among HNs (14.8%), followed by NPs (12.2%) and CNs (8.5%).The prevalence among the sample of any of the constituents of hyperlipidemia, hyperglycemia, and hypertension was 9.5% Table . Of thesAn ANOVA Table  showed nFactors associated with hyperlipidemia, hyperglycemia, and hypertension among nurses were examined using logistic regression Table . Crude o> 55, 46–54, 36–45, and 26–35 years were 16.361- (95% CI: 9.303–28.773), 9.856- (95% CI: 6.964–13.950), 4.125- (95% CI: 3.007–5.659), and 1.673-times (95% CI: 1.251–2.239), respectively, more likely to have hyperlipidemia, hyperglycemia, and hypertension than those aged under 26 years. Nurses with a BMI of > 24 kg/m2 were 1.237-times (95% CI: 1.041–1.471) more likely to have hyperlipidemia, hyperglycemia, and hypertension than those with a BMI of < 24 kg/m2. Nurses who worked over 50 h a week were 1.237-times (95% CI: 1.041–1.471) more likely to have hyperlipidemia, hyperglycemia, and hypertension than those who worked 40–49 h a week. Finally, nurses who had family-care needs were 1.179-times (95% CI: 1.017–1.366) more likely to have hyperlipidemia, hyperglycemia, and hypertension than those without family-care needs.After controlling for other variables in the model, no significant differences were found among the nursing positions regarding the ORs for hyperlipidemia, hyperglycemia, and hypertension . After adding level of work stress, both weekly work hours and family-care needs lost their significance. These results suggested that the effect of extended work hours (≥ 50 h) and family-care needs on hyperlipidemia, hyperglycemia, and hypertension can be explained by perceived level of work stress. After adding level of work stress to the model, we found that women were 1.669-times (95% CI: 1.080–2.577) more likely to have hyperlipidemia, hyperglycemia, and hypertension than men. Age and BMI remained risk factors for hyperlipidemia, hyperglycemia, and hypertension. The results of adjusted model 3 showed that there was no significant interaction between job position and level of work stress in regard to hyperlipidemia, hyperglycemia, and hypertension, meaning the association between work stress and hyperlipidemia, hyperglycemia, and hypertension did not differ among HNs, NPs, and CNs.We further used logistic regression to analyze whether the association between work stress and hyperlipidemia, hyperglycemia, and hypertension differed across sources of work stress Table . The adjThe present research found significant differences among HNs, NPs, and CNs regarding sources of perceived work stress; these differences in stressors were determined to be associated with the different work contents of the respective job positions. Our results showed that evaluation, hospital management, and communication with others is the primary stressor for HNs, that work complexity, uncertainty regarding medical results, urgency of work, and lack of job security and support is the primary stressor for NPs, and that promotion is the primary stressor for CNs. Hospitals in Taiwan must undergo evaluations every three years to certify/retain their status as medical centers, regional hospitals, district hospitals, and/or teaching hospitals. In addition, there are several other types of external evaluations/certifications that hospitals can seek to underline their high standards and quality of care . HNs areAlthough the significance of each source of stress varied across the different nursing positions, the overall work-stress perception did not significantly differ across the sample. The prevalence of hyperlipidemia, hyperglycemia, and hypertension differed across job positions, being highest among HNs, followed by NPs and CNs. Through univariate analysis, we observed distributional differences among the three nursing positions in terms of age, gender, BMI, marital status, years of work experience, work unit, work hours per week, and family-care needs. In sum, nurses in all three job positions had distinct characteristics, and these attributes, rather than job position, influenced prevalence of hyperlipidemia, hyperglycemia, and hypertension.> 24 in accordance with the criteria in Taiwan; among the participants, 33.0% of HNs were obese, followed by NPs (26.7%) and CNs (25.2%). The HNs’ average age was higher than those of the other groups, and they mainly performed administrative work. Further, most NPs and CNs worked on the front line, which entails more physically strenuous tasks. Regarding the correlation of hyperlipidemia, hyperglycemia, and hypertension with working hours, the longer the working hours, the higher the risk of developing hyperlipidemia, hyperglycemia, and hypertension [Our finding regarding factors related to hyperlipidemia, hyperglycemia, and hypertension agree with previous findings. Older individuals have a higher risk of suffering from hyperlipidemia, hyperglycemia, and hypertension , 37. Excrtension . Finallyrtension , which wrtension .Our study revealed that an increased level of perceived stress is associated with an increased OR for hyperlipidemia, hyperglycemia, and hypertension, which is consistent with the literature –5. StresThis study used self-report data, and we were unable to verify the accuracy of the participants’ reports. No objective measurement of hyperlipidemia, hyperglycemia, and hypertension was available, and under-reporting was possible; however, the rate of under-reporting across the three groups could be non-differential. Finally, causal relationships could not be inferred as a result of the use of a cross-sectional design.Medium to high job stress was observed in nurses. Although stressors varied by nursing position, the overall stress level was similar across HNs, NPs, and CNs. Higher job stress levels, increased age, higher BMI, long work hours, and caring for family members were positively associated with hyperlipidemia, hyperglycemia, and hypertension. Further studies are needed to identify and verify effective strategies for reducing work stress in nursing staff.


Podospora anserina demonstrated pronounced age-dependent changes of mitochondrial morphology and ultrastructure, as well as alterations of transcript and protein levels, including individual proteins of the oxidative phosphorylation (OXPHOS). The identified protein changes do not reflect the level of the whole protein complexes as they function in-vivo. In the present study, we investigated in detail the age-dependent changes of assembled mitochondrial protein complexes, using complexome profiling. We observed pronounced age-depen-dent alterations of the OXPHOS complexes, including the loss of mitochondrial respiratory supercomplexes (mtRSCs) and a reduction in the abundance of complex I and complex IV. Additionally, we identified a switch from the standard complex IV-dependent respiration to an alternative respiration during the aging of the P. anserina wild type. Interestingly, we identified proteasome components, as well as endoplasmic reticulum (ER) proteins, for which the recruitment to mitochondria appeared to be increased in the mitochondria of older cultures. Overall, our data demonstrate pronounced age-dependent alterations of the protein complexes involved in energy transduction and suggest the induction of different non-mitochondrial salvage pathways, to counteract the age-dependent mitochondrial impairments which occur during aging.The accumulation of functionally impaired mitochondria is a key event in aging. Previous works with the fungal aging model  Biological aging is commonly described as a process associated with the time-dependent decline of physiological functions and the accumulation of cellular and molecular damage ,2,3. OvePodospora anserina [P. anserina the mitochondria undergo pronounced morphological and ultrastructural changes. In juvenile hyphae, the mitochondria occur as a network of filamentous organelles. Due to an altered balance of the mitochondrial fusion and fission, the network disintegrates during aging, leading to punctuated mitochondria [1Fo-ATP synthase dimers [1Fo-ATP-synthase dimerization process, via the ablation of the F1Fo-ATP-synthase assembly factor PaATPE, experimentally supported this scenario. The PaAtpe deletion mutant displays a vesicular mitochondrial ultrastructure and impairments in mitochondrial function and is short-lived, due to a detrimental induction of mitophagy [One model organism with a strong mitochondrial etiology of aging is the ascomycete anserina ,13,14. Tanserina ,16. Durichondria . At the chondria ,19. Thise dimers . Recentlitophagy ,22. Age-itophagy ,26,27,28P. anserina, the age-related changes in mitochondrial morphology and ultrastructure go along with a pronounced reorganization of the mitochondrial DNA (mtDNA), which encodes a number of essential proteins of the respiratory chain [P. anserina and this was found to be evolutionary conserved [P. anserina further supported the impact of respiration on aging and lifespan. For instance, impairments of the standard complex IV -dependent respiration in different P. anserina mutants led to the induction of an alternative oxidase (AOX)-dependent respiration and an increased lifespan [P. anserina were discussed in a recent review [P. anserina, such a structure was previously demonstrated [P. anserina F1Fo-ATP synthase revealed hints at an age-dependent alteration of the respiratory chain composition and loss of mtRSCs [In ry chain ,31,32,33ry chain ,35,36,37onserved ,39,40. Aonserved ,42,43,44lifespan ,46. Thist review . A genert review ,47,48. At review . Individt review ,51. In anstrated . The mannstrated . Recentlf mtRSCs . Moreovef mtRSCs .P. anserina. A longitudinal transcriptome analysis revealed that the transcript profiles associated with mitochondrial function fluctuate during the aging of P. anserina [P. anserina, but mainly with the soluble protein part of mitochondria and a focus on protein modifications [In the past, a number of studies focused on age-dependent changes in mitochondrial transcript and protein levels in different biological systems, including anserina . This flications ,57,58. Oications . HoweverP. anserina wild type, uncovered detailed age-dependent alterations of the mitochondrial complexome. We also observed an age-associated switch from a standard complex IV-dependent, to an AOX-dependent, respiration in the P. anserina wild type. Most strikingly, we identified that components of the proteasome, as well as the endoplasmic reticulum (ER) proteins, had an increased abundance of enriched mitochondrial fractions of older strains, suggesting the induction of compensatory salvage pathways occurs during aging.Here we report results from a complexome profiling analysis, in which high molecular weight protein complexes were separated using BN-PAGE, subsequently sliced into even fractions, and analyzed by quantitative mass spectrometry ,61,62. TP. anserina wild-type strain ‘s’ [In this study, the rain ‘s’  was usedrain ‘s’ . For allrain ‘s’ . These sP. anserina wild-type strains were grown on cellophane foil covered M2 agar plates under constant light at 27 °C. After two days of growth, mycelia were transferred to CM-liquid medium and incubated for an additional two days at 27 °C, in constant light and shaking. The mitochondria of young (6 d) and old (18 d) P. anserina wild-type cultures were isolated, according to a published protocol [g) was used [protocol . For purwas used .g. The obtained supernatants were immediately loaded on a linear gradient gel (4–13%) overlaid with 3.5% stacking gel. Electrophoresis was started at 100 V and 10 mA. When samples had completely migrated into the stacking gel, limits were set to 500 V and 15 mA. Electrophoresis was stopped when the Coomassie front approached the gel front. After electrophoresis, the gel was incubated for 30 min in fixing solution , 10% acetic acid , 100 mM ammonium acetate , and subsequently proteins were visualized after 1-h Coomassie staining  in 10% acetic acid . Afterwards the gel was destained with water and 10% acetic acid.BN-PAGE was performed, to separate native protein complexes according to size, as described ,67. Brie® Turbo™ transfer system , according to the manufacturer’s specifications. Afterwards, blocking, antibody incubation, and washing steps were performed according to the Odyssey ‘Western Blot Analysis’ handbook . As primary antibody, an anti-PaAOX1  was used. Subsequently, a conjugated IR Dye CW 800  was used as secondary antibody. For detection, an Odyssey® Fc imaging system  was used, and densitometric quantification was performed with the manufacturer’s software, Image Studio 5.2.Mitochondrial protein extracts (50 µg) were separated by 2-phase SDS-PAGE (12% separating gels) according to the protocol published in Brust et al. . BrieflyDetermination of COX- and AOX-dependent oxygen consumption was performed using strains cultivated on M2 medium for 2 days and in CM liquid medium for 2 days, as described above. Small pieces of mycelium were subsequently transferred into the high-resolution respirometer  and oxygen consumption was measured in liquid CM medium according to the manufacturer’s instructions. Then, 1 mM potassium cyanide  was added to inhibit respiration via COX, and 4 mM salicylhydroxamic acid  was added to inhibit respiration via AOX. Data were analyzed using the manufacturer’s software DatLab 6.v/v) methanol , 10% (v/v) acetic acid , and 10 mM ammonium acetate  for 30 min and stained with Coomassie  and 10% (v/v) acetic acid ). Afterwards, each lane of the gel was cut from the bottom to the top into 48 almost equal fractions. Fractions were collected in 96 filter well plates . The gel pieces were destained in 60% Methanol, 50 mM ammonium bicarbonate (ABC) . Solutions were removed by centrifugation for 2 min at 600× g. Proteins were reduced in 10 mM DTT  and 50 mM ABC  for one hour at 56 °C and alkylated for 45 min in 30 mM iodoacetamid . Samples were digested for 16 h with trypsin , at 37 °C in 50 mM ABC , 0.01% Protease Max , and 1 mM CaCl2 . Peptides were eluted in 30% acetonitrile  and 3% formic acid , centrifuged into a fresh 96 well plate, dried in a speed vac, and resolved in 1% acetonitrile and 0.5% formic acid.Complexome profiling was used to identify native protein complexes after BN-PAGE and their alterations during aging. It was performed as published previously . In briem/z, with a resolution of 70,000 and an automatic gain control (AGC) value of 3E6 total ion counts, with a maximal ion injection time of 160 ms. Only higher charged ions (2+) were selected for MS/MS scans with a resolution of 17,500, an isolation window of 2 m/z, and an automatic gain control value set to E5 ions, with a maximal ion injection time of 150 ms. MS1 data were acquired in profile mode.Liquid chromatography/mass spectrometry (LC/MS) was performed on a Thermo Scientific™ Q Exactive Plus equipped with an ultra-high-performance liquid chromatography unit (Thermo Scientific Dionex Ultimate 3000) and a Nanospray Flex Ion-Source (Thermo Scientific). Peptides were loaded on a C18 reversed-phase precolumn (Thermo Scientific), followed by separation with a 2.4 µm Reprosil C18 resin (Dr. Maisch GmbH) in-house packed picotip emitter tip , separated using a gradient from 4% acetonitrile, 0.1% formic acid, to 30% eluent B  for 30 min and an additional gradient to 60% for 5 min with a flow rate 400 nL/min and washout with 99% B for 5 min. MS data were recorded by data-dependent acquisition. The full MS scan range was 300 to 2000 P. anserina proteome database UniProtKB with 10657 entries, released in 8/2021. The enzyme specificity was set to trypsin. Acetylation (+42.01) at the n-terminus and oxidation of methionine (+15.99) were selected as variable modifications, and carbamidomethylation (+57.02) as fixed modification on cysteines. The false discovery rate (FDR) for the identification protein and peptides was 1%. Intensity-based absolute quantification (IBAQ) values were recorded. The sum of all IBAQ values of data sets were normalized to data of the 18-day-old sample. Protein abundance within native lanes were normalized to the maximum appearance, to enable comparison of mitochondrial complexes between samples. Slice number, according to a native mass ladder, was used for native mass calibration. The software NOVA (v.0.5.7) was used for hierarchical clustering of complexomics data [MS data were analyzed using MaxQuant (v 2.0.1.0)  and the ics data .P. anserina, resulting in the deletion of large parts of the mtDNA, suggested that severe changes of the mitochondrial transcriptome and proteome occur during aging [The pronounced age-associated reorganization of the mtDNA of ng aging ,31,32,33ng aging ,57,58,59ng aging .P. anserina wild-type cultures. The older age-stage lies in the range were the first cultures of a population died (1Fo-ATP-synthase). Furthermore, all three known mtRSCs, S0, S1, and S2, which consist of CI1CIII2CIV0, CI1CIII2CIV1, and CI1CIII2CIV2, respectively, were visible (1) and monomeric complex IV (IV1) occurred, suggesting a reduced activity of these complexes in aged cultures. Such a reduced activity of complex I and complex IV was found in aged rodents [In the current study, we aimed to focus on a comprehensive analysis of the age-related mitochondrial complexome ,61,62. Fion died A. After  visible B. In agr rodents ,74,75.Next, 48 equal pieces C of the 1Fo-ATP-synthase dimers in the aging of P. anserina [1), as well as in a dimeric (V2), state [1 domain of complex V, indicating an imbalanced assembly of F1 and Fo domains , state . An age-), state ,21, was  domains F.P. anserina, which ultimately must lead to impaired mitochondrial function.Overall, these data identified clear age-dependent alterations of OXPHOS in P. anserina wild-type cultures , PaNDE1 , and PaNDE2 , are active [P. anserina mutants. Interestingly, both external NADH dehydrogenases are not able to compensate complex I deficiency in P. anserina [S. cerevisiae partially restores complex I deficiencies in other organisms, such as worms and mice, and in human cell lines [+/NADH pool in the mitochondrial matrix. Altogether, this is the first hint at a role of PaNDI1 in the aging process of P. anserina.Under standard growth conditions, complex I-dependent NADH oxidation is coupled with proton-pumping. In plants and fungi, this oxidation can also occur through another family of proteins. These so-called alternative NADH dehydrogenases oxidize NADH from the cytosol  or from the mitochondrial matrix  without proton pumping . In the cterized ,90. Howee active ,91. A prll lines ,94,95,96PaCox1 gene codes for the plDNA, which becomes liberated during aging, leading to the mtDNA reorganization [PaCoxI gene might be downregulated. Indeed, a reduced Pa Cox1 transcript level was found in a previous transcriptome analysis [In the next set of analyses, we investigated the reduction of complex IV in more detail. Complexome profiling identified ten subunits of complex IV, whose abundance was reduced during aging A. Among nization ,31,32. Tanalysis . Other aP. anserina, the standard complex IV-dependent respiration is known to be bypassed under certain conditions by respiration via an alternative oxidase (AOX), e.g., under the oxidative stress induced by paraquat [P. anserina mutant strains grisea, PaCox17::ble, and PaCox5::ble all are devoid of intact complex IV and respire via the AOX. In these mutants AOX induction leads to lifespan extension [P. anserina has hardly been analyzed. An initial study analyzing PaAOX in the aged P. anserina wild type revealed that induction of alternative respiration does not occur during wild-type aging [PaAox transcript in middle-aged cultures, suggesting that the amount of AOX in aged cultures may also be increased [Ciona intestinalis AOX [In paraquat . Anotherxtension ,100,101.ncreased . Both stncreased  and thosncreased . Consistncreased ,103,104 alis AOX .P. anserina wild-type cultures (6-days-old) respire predominantly (75%) via complex IV, and only moderately (25%) via AOX. In older cultures, this is almost turned around. We determined only 40% complex IV-dependent respiration and 60% alternative respiration  and in fractions between 40 (1.5 MDa) and 46 at 2.4 MDa . The 20 rganisms . This derganisms ,31,32,33Neurospora crassa, which is closely related to P. anserina, but also in humans, the eIF-3 consists of 13 subunits, eight of them form the core structure [A further striking alteration is observed in fraction 27 (550 kDa). The seven corresponding proteins were more abundant in the 18-day-old sample than in the 6-day-old sample  and PaPRE2 (β5) we detected two proteolytic active subunits, which were more abundant in the 18-day-old sample than in the 6-day-old sample  compared to the young (6-day-old) sample (lower panel of S. cerevisiae [The increased abundance of the proteasomal proteins suggests an increased recruitment of the proteasome to the mitochondria in the aged achinery ,122,123.revisiae . The datrevisiae ,125. It revisiae . The incP. anserina [Another possible non-mitochondrial salvage pathway may be the attachment of the ER to the mitochondrion. Indeed, we found four ER proteins in fraction 22 as part of the other prominent uncharacterized BN-PAGE band . Additioanserina . Such a anserina ,136,137.P. anserina, pronounced changes in the mitochondrial complexome profile occur. The fundamental age-dependent reorganization of the respiratory chain via the induction of AOX, together with the decline in the amount of mtRSC, clearly affect the ability of the mitochondria to generate ATP. Moreover, the loss of mitochondrial ribosome subunits impairs mitochondrial translation; thereby, further compromising mitochondrial function. Interestingly and hitherto unknown, in P. anserina these alterations appear to induce the recruitment of components of compensatory pathways, such as the proteasome and the ER to mitochondria, to counteract these impairments. Future studies need to address the regulation of these salvage pathways in more detail, to provide a better understanding of their role in the prevention of age-dependent impairments.Overall, during the aging of


Allergic rhinitis (AR) is considered a major nasal condition impacting a large number of people around the world, and it is now becoming a global health problem. Because the underlying mechanisms of AR are complex, the development of single-drug treatment might not be enough to treat a wide spectrum of the disease. Although the standard guidelines classify and provide suitable diagnosis and treatment, the vast majority of people with AR are still without any means of controlling it. Moreover, the benefits of AR drugs are sometimes accompanied by undesirable side effects. Thus, it is becoming a significant challenge to find effective therapies with limited undesirable side effects for a majority of patients suffering from uncontrolled AR. Aller-7/NR-A2, a polyherbal formulation, has revealed promising results in patients by reducing nasal symptoms and eosinophil counts without serious adverse effects. Interestingly, three out of seven of the herbals in the Aller-7/NR-A2 formulation are also found in an Ayurvedic polyherbal formulation known as “Triphala,” which is a potential candidate for the treatment of AR. However, there are no current studies that have examined the effects of Triphala on the disease. This review aims to describe the complexity of AR pathophysiology, currently available treatments, and the effects of Triphala on AR in order to help develop it as a promising alternative treatment in the future. Although allergic rhinitis (AR), commonly known as hay fever, is not considered a serious condition or one with the potential for mortality, it might become a clinical burden if it were to become uncontrollable in a majority of patients. Epidemiological studies have revealed that patients with AR have a high probability of subsequently developing asthma, and a large proportion of asthmatic patients also have AR as a concomitant condition . MoreoveSeveral studies have highlighted the beneficial effects of phytochemicals in attenuating allergic responses and the overall symptoms of AR. Curcumin, ginger, ginseng, resveratrol, and quercetin compounds have all shown effective results in relieving nasal symptoms and suppressing AR-related mediators in animal models by controlling the main anti-inflammatory mitogen-activated protein kinase/nuclear factor κB (MAPK/NF-κB) pathway . A majorTriphala possesses several activities that could be beneficial for respiratory and immune systems. However, there is no study directly evidencing the potential benefits of Triphala on AR, even though its constituent phytochemicals have been suggested to serve many aspects of AR therapeutic strategies. Therefore, it is important to review and clarify the possible mechanism of action of Triphala and its active constituents on AR. This review focuses mainly on the antioxidative, anti-inflammatory, immunomodulatory, and other effects of Triphala on the AR-affected respiratory system. The quality of the articles relevant to the scope of AR has been evaluated according to the guideline reported by AR is a condition of nasal hypersensitivity caused by an immunologically mediated inflammation by non-infectious stimulants or environmental allergens, such as pollen, dust, fungi, cockroaches, etc. AR is becoming a global health problem affecting approximately 10–40% of the global population . AlthougCurrently, AR is classified according to the duration (intermittent or persistent) and severity  of symptoms. Intermittent AR is diagnosed if the frequency of symptoms is fewer than four times per week or a month per year. A frequency greater than this is classified as persistent. Moreover, the impact of the disease on a patient’s QoL is also used to evaluate the severity of the symptoms. Symptoms are considered to be mild if normal daily activities, such as sleep, study, and work, can be undertaken without troublesome or interfering symptoms. On the other hand, if a patient presents at least one symptomatic difficulty, then the symptoms are defined as moderate/severe  Klimek . TherefoThe key elements for AR management are as follows: 1) avoidance of suspected allergens in sensitized individuals, 2) providing appropriate therapeutic agents to reduce nasal symptoms and improve patients’ QoL, and/or 3) considering allergen-specific immunotherapy (AIT) . The lisThe pathophysiology of AR is quite complex , beginniThe early phase occurs in sensitized individuals within minutes of exposure to allergens. Soluble IgE antibodies, which are produced by B cells in the sensitization phase, locally diffuse and systematically enter the body fluids via lymphatic vessels . IgE canThere are many signs and symptoms associated with the early phase of AR, such as nasal pruritus, sneezing, and coughing caused by the histamine-stimulated sensory nerve endings of the trigeminal nerves . The actThe late phase response is characterized by recruitment of T cells, eosinophils, basophils, and neutrophils following a release of their various mediators, which leads to a continuation of the symptoms and inflammatory induction. This phase usually begins around 4–5 h following exposure to allergens and can last for 24 h. Some cytokines, such as IL-4, IL-13, and tumor necrosis factor-α (TNF-α) released from mast cells, have the ability to upregulate two adhesion molecules, which are vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) . These aTerminalia chebula Retz (Family Combretaceae R. Br), Terminalia bellirica (Gaertn.) Roxb (Family Combretaceae R. Br), and Phyllanthus emblica L (Family Phyllanthaceae Martinov). The composition of Triphala is derived from a rational combination of T. chebula Retz., T. bellirica (Gaertn.) Roxb., and P. emblica L., which are combined to balance the three pillars of life or elemental substances, known in Sanskrit as the three doshas, namely, Vata (elements of space and air), Kappha (elements of earth and water), and Pitta (elements of fire and water)  Roxb., and three parts P. emblica L (1:2:3) (T. chebula Retz. and T. bellirica (Gaertn.) Roxb. and two parts P. emblica L (1:1:2)  . Normall (1:2:3)  or one p (1:1:2) . AccordiP. emblica L. methanolic extracts , flavonoids (quercetin and kaempferol), alkaloids (phyllantidine and phyllantin), ascorbic acid, carbohydrates, proteins, etc. . The amoextracts . Moreoveextracts . TherefoThe phytochemicals contained in Triphala may be beneficial for the treatment of AR. Gallic acid and quercetin are prominent phenolic compounds and are well-known antioxidants in AR-related studies. Gallic acid was shown to alleviate nasal inflammation by shifting the immune response toward Th1 in a mouse model of OVA-induced AR . The incin vitro and in vivo. In IL-4-induced human nasal epithelial cells (HNEpCs), nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) mRNA expression, and signal transducer and activator of transcription 6 (STAT6) activation are all attenuated (Perilla frutescens (L.) Britton, and vitamin D3] known as “Lertal®” was effective in relieving the overall symptoms of AR and reducing the use of anti-allergic drugs without additional adverse effects  and kaempferol. In particular, the plasma level of ascorbic acid has been found to be reduced in allergic-related diseases in which patients with allergy-related respiratory and cutaneous symptoms can derive benefits after receiving an exogenous vitamin C treatment . One stuConsidering the beneficial aspect of Triphala for manipulating the pathophysiology of AR, specific convincing compounds shown in . are lika-tocopherol, bilirubin, lipoic acid, transferrin, and albumin, in order to prevent overwhelming by and accumulation of oxidative stress  urban pollutants aggravate nasal inflammation and AR-related symptoms, 2) pollen grains that contain endogenous reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase damage airway epithelial cells and trigger granulocyte recruitment . Howevere stress . The imbe stress .Oxidative stress is now considered to be one of the markers of the pathogenesis of AR, asthma, and chronic obstructive pulmonary disease (COPD). Epidemiological and clinical studies of AR have found that eroallergen and pollutant co-exposure can induce immunological effects, leading to recruitment of inflammatory cells, cytokines, chemokines, and finally AR-related symptoms . Ozone ein vitro and in vivo models. For this aspect, several studies reported the activity of Triphala by using chemical assays in which the results do not directly represent the antioxidative properties of Triphala in the actual physiological condition and cannot guarantee its intracellular antioxidant activities  Roxb., and its scavenging ability for 2,2-diphenyl-1-picrylhydrazyl (DPPH) and peroxynitrite radicals seems to be superior. On the other hand, although T. bellirica (Gaertn.) Roxb. contains fewer polyphenolic compounds than the other two herbs, it still has a greater effect in reducing the number of superoxide, nitric oxide, and hydroxyl radicals  Roxb. and T. chebula Retz.) exhibited an ability to decrease NF-κB activation and nuclear translocation by suppressing the phosphorylation of p38, c-Jun N-terminal kinase (JNK), and ERK and activating the Akt/AMP-activated protein kinase/nuclear factor erythoid 2-related factor (Akt/AMPK/Nrf) signaling pathway  were relieved following topical application of T. chebula Retz. extract. As well as lesion improvement, the extract attenuated atopic markers (MMP-9 and IL-31) and eosinophil infiltration in affected and adjacent areas, respectively   , and altpy (AIT) . Moreovepy (AIT) .In theory, it is accepted that a balance of the numbers and ratios of Th cells and Tc cells is considered to be a homeostasis marker of the intrinsic immune system. Significant increments in Th cells, Tc cells, and natural killer cells following exposure to Triphala and its active constituents could confirm an additional improvement in the general cellular-mediated immunity of these agents in order to deal with harsh environments and exogenous microorganisms.Although intranasal corticosteroids are now considered to be the most effective treatment for AR, their mechanism of action acts mainly preferentially through immunosuppression rather than immunomodulation . It willFollowing exposure to allergens or the start of the pollen season, some AR patients may have symptoms of cough (dry cough or a cough containing phlegm that is loose in character) together with nasal symptoms . ChronicP. emblica L. and T. chebula Retz. extracts have the ability to alleviate coughs induced by mechanical stimulation and citric acid, respectively (T. bellirica (Gaertn.) Roxb. itself can dose dependently reduce BHR induced by carbachol  has been accumulated, and that increases the reliability and confidence of using this lineage of medicine. Recently, several plants and their constituents are approved to be safe, effective, and less expensive for managing the development and progression of various diseases, and many of them are already on the WHO’s list of essential medicines. Therefore, Triphala which contains three different plants may be one of the effective remedies to alleviate AR-related symptoms. Experimental data from previous Nigella sativa (black caraway or black cumin). However, there have not been any in vivo studies that utilize a direct model for AR to test the effects of Triphala. The ovalbumin-induced guinea pig model of AR could well be a proper assay to definitively evaluate if Triphala can reduce AR-associated inflammation. However, there is a limitation to these types of study models because it is not a direct AR model to give us enough reliable information specific to AR. It would provide strong evidence if in the future there are direct experimental designs specifically developed for animal AR models to verify the effectiveness of Triphala.The anti-inflammatory, immunomodulatory, antioxidant, and antibacterial activities of Triphala are similar to those of other herbal formulas clinically proven to treat AR effectively, such as Shi-Bi-Lin , butterbur, and During allergic inflammation, the nasal epithelial barriers of AR patients appear to become impaired. Consequently, this condition leads to the development of sinusitis and otitis media, with effusion caused by bacteria, viruses, and fungi. Although Triphala shows promise in being an effective bacteriostatic/bactericidal agent, most studies have focused only on oral streptococci. Thus, further studies are needed to address whether Triphala is effective in killing other types of bacteria, as well as viruses and fungi that are related to allergic rhinosinusitis. Additionally, clinical studies should be conducted to verify the efficacy of this herbal formula. As a step toward the clinical investigation of Triphala for its efficacy and safety, our research team has created the study design/clinical trial, and the study protocol has already been registered in Thai Clinical Trials Registry (TCTR20191008005). Moreover, study models for pharmacokinetics are crucial to fulfill all aspects of the pharmacology of Triphala and to obtain valuable information about the active compound profiles. If the accumulated data clearly show that Triphala can be used effectively to inhibit inflammation, modulate immunity, alleviate unwanted symptoms in patients with AR, and prevent AR-related infections, this will provide a novel alternative treatment for patients. For example, Triphala could be used as a monotherapy in patients with mild AR. For patients with moderate-to-severe AR  but who do not want to use steroidal drugs, Triphala may be used in combination with an antihistamine. For patients using steroids who have inadequate control of AR, Triphala may be added to the treatment plan to reduce the use of high-dose steroids. Similarly, for patients who require immunotherapy, a Triphala supplement may offer a faster and better response to immunotherapy.In conclusion, Triphala exhibits beneficial effects if used as an alternative treatment for AR. However, extensive investigations in both animals and humans are first needed to be certain about its definitive mechanisms of action and the efficacy/safety of treatments. Although most studies reported the pharmacological effects of Triphala in a reasonable range of concentrations, some studies evaluated the effects of Triphala at relatively high doses. This may create concerns about its low efficacy and toxicity. With this, specifying the efficacy and adverse effects of Triphala is a vital base to ensure its safe use. Triphala should be carefully examined further to gain adequate data about toxic target organs, safe dose range, safety window of effective dose, and minimum toxic dose. Considering the actual applications, appropriate dosage and course of treatment are required to be precisely adjusted to ensure that Triphala is safe and effective. The accumulated evidence obtained from such studies will be beneficial to properly prescribe Triphala to individual AR patients with different clinical symptoms and varying degrees of severity in order to achieve the highest efficacy with the fewest side effects.