The results showed that Protransf-CoQ10 could transform into transfersomal vesicles with particle sizes of approximately 201.5 ± 6.1 nm and a zeta potential of − 11.26 ± 5.14 mV. The dispersion of Protransf-CoQ10 into emulgel base resulted in stable Protransf-CoQ10 Emulgel during 28 days of observation at low temperatures. Moreover, the in vivo study revealed that Protransf-CoQ10 Emulgel successfully increases the collagen density and number of fibroblast cells in UV radiation skin-aged induced-mice which reflects its potential for repairing the skin ageing process. In addition, the 24-h topical application of Protransf-CoQ10 Emulgel showed that no erythema or skin rash was observed during the study. In conclusion, loading CoQ10 into protransfersomal Emulgel successfully enhanced the stability and anti-ageing efficacy enabling its potential use as anti-ageing cosmetics.Coenzyme Q10 (CoQ10) is a naturally produced organic molecule which acts as an antioxidant agent, including in skin anti-ageing, and plays a major role in the social determinants of health. However, its level in the body will decrease during ageing. Therefore, an external supplement is required to repair damaged skin, especially the skin dermis layer. This study aims to evaluate the use of a protransfersomal emulgel to improve the skin delivery and stability of CoQ10 which demonstrates low water solubility, poor permeability and instability. CoQ10 was initially dissolved in oleic acid at a weight ratio of 1:56. Protransfersome was then loaded with CoQ10 (Protransf-CoQ10) and prepared using a composition of L-α-Phosphatidylcholine and Tween 80 at a molar ratio of 85:15. The Protransf-CoQ10 was dispersed in an emulgel base consisting of Tween 80 and Span 80 to produce Protransf-CoQ10 emulgel. The in vivo studies of anti-aging activity and irritability were further evaluated by applying daily 200 mg of emulgels twice a day to a 4 cm Both these internal and external factors cause impaired tissue function and structural changes2 culminating in skin ageing characterized by thinning of the epidermis and skin dermis and, ultimately, resulting in wrinkles, fine facial lines, and loss of elasticity4. Skin elasticity is largely dependent upon young collagen fibers and fibroblasts, collagen-producing cells in the dermis layer, whose numbers decrease during the ageing process5.Premature skin ageing occurs because the skin, as the outermost organ, is always directly exposured to oxidants in the environment and is frequently a determining factor in social life. In addition, with increasing age, the activity of mitochondria in the body as a producer of energy in regenerating cells and tissues decreases6. Coenzyme Q10 (CoQ10) is one of the natural compounds often employed as an antioxidant, which plays a key role in stabilizing plasma and other intracellular membranes that protect against membrane phospholipid peroxidation7. CoQ10 acts by maintaining skin quality against free radicals3 which have been known to activate the mitogen-activated protein kinase (MAPK) pathway that produces matrix metalloproteinases (MMPs) such as collagenase, thus damaging collagen fibers10. During ageing, the levels of CoQ10 in organs, including the skin, also decrease with the result that it is necessary to supply CoQ10 to achieve normal levels of between 0.50 and 1.65 μg/mL within the body. Topical administration of CoQ10 has been shown to be effective in reducing wrinkles in skin that has been exposed to UV rays3.Anti-ageing cosmetics have been widely used to promote skin regeneration, especially of the upper skin layers which protect the skin against dehydration, penetration by various microorganisms, allergens, irritants, reactive oxygen species (ROS) and radiation, thereby maintaining healthy skin12. Moreover, CoQ10 decomposes when exposed to light13. Loading CoQ10 into protransfersome, a vesicular carrier would probably constitute an effective strategy to enhance its biological activity within the skin in addition to increasing its stability.CoQ10 demonstrates low solubility in water (0.193 μg/mL) with a large molecular weight of 863.36 g/mol and high lipophilicity with a log P value of 21. This limits its penetration of the skin and explains it tendancy to be deposited in the stratum corneum14. It possesses a flattened liquid crystal structure which is converted into an ultraflexible vesicle known as transfersome through the absorption of water from the skin during in situ hydration17. Transfersome is known to be an ultradeformable vesicle which is highly flexible and deformable, rendering it capable of passing through three skin penetration pathways18. Transfersome can rapidly penetrate the stratum corneum and enter the deeper skin layers via the intercellular lipid of the stratum corneum. It can fuse with the cell membrane, enabling it to enter the transcellular pathway, and is able to penetrate intact through the hair follicle pathway to penetrate the deeper layers of the skin21. Protransfersome is composed of amphiphatic lipid components such as phosphatidylcholine which, significantly, form double-layer membrane of vesicles, and surfactant as an edge activator that increases the vesicle flexibility or deformability22. In general, protransfersome contains a larger number of phospholipids than that present in transfersomes. During the manufacturing process, the protransfersome does not undergo an extrusion process to produce unilamellar vesicles as observed in the transfersome. This is because the protransfersome is a provesicular carrier system which will be converted into transfersome after it comes into contact with water in situ23. Therefore, under a light microscope, the protransfersome can be seen to possess a palisade crystalline liquid form, whereas transfersomes are vesicular when in liquid media24.Protransfersome, one of the provesicular nanocarriers that provides superior skin penetration and high stability, is widely used in transdermal delivery25. Previous reports have evaluated the use of nanocarriers for CoQ10 delivery such as a self-emulsifying drug delivery system (SEDDS)26, ethosomes27, transethosomes28, and microemulsion29. The use of transethosomes successfully encapsulated CoQ10 up to 97% in vesicles and produced > 95% drug deposition in different skin layers resulting in high efficacy for androgenic alopecia28. The low water solubility of CoQ10 frequently limits drug encapsulation efficiency in nanocarriers, thus the use of large amounts of lipid phase or ethanol may improve its loading.The use of ultradeformable vesicles has successfully improved the skin penetration of drugs and efficacy of anti-ageing properties of certain antioxidant molecules such as tocopherol which, when prepared in transfersome, possess good characteristics with a particle size < 100 nm and entrapment efficiency of up to 90%. Moreover, it is well distributed within the skin layer and in vitro tests have proved it biocompatible with keratinocytes and fibroblasts, indicating its protective effect against oxidative damage and the potential for wound healingIn this study, a protransfersome containing CoQ10 will be prepared for anti-ageing emulgel. The high level of phospholipids contained in protransfersome is intended to improve drug loading. The use of protransfersome in the anti-ageing activity and irritation level of Protransf-CoQ10 emulgel was evaluated in vivo using UV-induced aged mice models. This study could represent an attempt to improve CoQ10 anti-ageing activity with the result that is effective, safe and non-irritating.. There were improvements in stability and potential efficacy to inhibit premature ageing of the skin in UV-radiation skin aged-induced mice models as demonstrated in this study.This study aims to evaluate the potential use of protransfersome for topical delivery of CoQ10 as an anti-ageing agent. This study provides a scientific approach to successfully delivering low water solubility and poor permeable lipophilic substances and nanovesicular carriers specifically designed for anti-ageing cosmetics. The CoQ10 was loaded into protransfersomal emulgel composed of oleic acid containing soluble CoQ10, phospholipids as bilayer-forming lipids, and Tween 80 which acts as the edge activator of bilayer membrane after the protransfersome has been hydrated with skin water in situ, before being loaded into an emulgel baseAfter dissolving the CoQ10 in oleic acid and encapsulated it into protransfersomes composed of phospholipids and Tween 80, the protransfersome-loaded CoQ10 (Protransf-CoQ10) forms a bright orange, viscous, oily liquid, with a distinctive phospholipid smell, and viscous consistency. After hydration with saline, lamellar vesicular structures rapidly formed and were ultimately transformed into transfersome vesicles, as shown in Fig. 30 and easily oxidized when it is exposed to air in for lengthy periods32.The dispersion of Protransf-CoQ10 into the emulgel base Fig. A, B at a17, thus ensuring that a larger number of vesicles penetrate the skin. From the results, it is evident that the entrapment efficiency value of the CoQ10 in Protransf-CoQ10 is comparatively high at 45.64 ± 7.52% with particle size of 201.5 ± 6.1 nm , polydispersity index value of 0.229 ± 0.047, and ζ-potential of − 11.26 ± 5.14 mV, as presented in Table The particle size and polydispersity index value were further evaluated since they determine the ability of the vesicles to penetrate the deeper layers of the skin. The smaller the particle size of the vesicles, the easier the vesicles are to penetrate. In addition, the smaller the polydispersity index value, the more homogeneous the particle size of the vesicles35.In order to evaluate any interaction between CoQ10 and protransfersomal matrix, a Fourier Transform Infra Red (FTIR) analysis was further observed. As presented in Fig. Moreover, according to the result of differential thermal analysis, the CoQ10 encapsulation into protransfersome produced changes in the structure of cristallinity. CoQ10 and L-α-Phosphatidylcholine showed sharp endothermic peaks at 53.3 and 112.3 °C, respectively; however, protransfersomal CoQ10 showed weak endothermic peak at 143.9 °C indicating that less ordered crystalline structures were observed as presented in Fig. P < 0.05). On the other hand, a significant difference was observed in the pH during the same period, although the pH value remained within the pH range of the skin. No significant difference existed in the particle size or particle size distribution of the preparation after 28 days of storage.A physical stability test was subsequently carried out to determine the physical resistance of the system when stored at different temperatures, namely; room temperature and a lower temperature for 28 days. During the study, the parameters of particle size, polydispersity index, and pH were observed. As seen from Fig. To evaluate the ability of protransfersomes to topically deliver CoQ10 and produce an effective anti-ageing activity, the Protransf-CoQ10 Emulgel was topically applied for 14 days to the back skin of UV-rays-induced subjects who were subsequently observed for skin histopathology. The control group subjects which received UV rays had the lowest collagen density of 52.30 ± 7.87%, indicating that UV rays damage the collagen in the skin dermis. The administration of both Protransf-CoQ10 Emulgel and CoQ10-Ole Emulgel significantly improved the collagen density of UV-ray radiated subjects’ skin as indicated in Fig. The anti-ageing activity test result was further analyzed by observing the number of fibroblast cells capable of producing collagen. Therefore, the higher the number of fibroblasts, the more collagen was formed. In this study, the assessed fibroblasts were young and light purple in appearance. The results showed that the CoQ10 Emulgel had a significantly different number of fibroblasts compared to the control group, with pro-CoQ10 Emulgel producing the highest number of fibroblasts, which was 31.50 ± 9.48 cells per field view, as indicated in Fig. The safe use of Protransfersome-loaded emulgels in this study was also evaluated by conducting an in vivo irritation test. Epidermis liquefaction, subepidermis edema, collagen fiber swelling, inflammatory cells infiltration, dan appendages degeneration were observed for determining irritation in model’s skin. As presented in Fig. The results of the histopathological scoring of the models’ back skin after 24 h of application showed that CoQ10 Emulgel had an irritation score of 0.52, while CoQ10-Ole Emulgel had one of 1.36, and Protransf-CoQ10 Emulgel one of 0.92 as presented in Fig. 37. The addition of lipid vesicles to gel is beneficial for increasing vesicle stability, prolonging drug release, improving dermal permeability, and enhancing drug deposition in the skin38.In this study, the Protransfersomes and Protransfersomal emulgel preparations for CoQ10 delivery as the active cosmetic ingredient have the potential to inhibit premature ageing of the skin. The main purpose of protransfersome formulation is to significantly encapsulate CoQ10 in order to modify the physicochemical characteristics of CoQ10, rendering it more water dispersible and able to penetrate the skin since high lipophilic CoQ10 demonstrates low water solubility and poor skin penetration. However, the high content of oleic acid, which accounted for approximately 37% of the final weight of protransfersomal emulgel, would render it unacceptable for daily use as a skin cosmetic. Therefore, it was added to emulgel to increase its appropriateness for use. As far as the functional aspects of vesicles are concerned, the formation of transfersome due to hydration of protransfersome by water content in the emulgel base produces ultra-deformable vesicles which allow them to easily penetrate the skin. In addition, previous reports showed that the presence of a gelling agent would act as a steric hindrance which would be adsorbed onto the vesicle surface preventing fusion or aggregation, thus increasing physical stability during storage24. Protransfersomes are able to carry active ingredients through the skin pores into the deeper layer. The protransfersome system analyzed in this study has positive characteristics including nanometer size, and thick consistency resulting from its large phospholipid content. When the protransfersome is observed using a light microscope, a palisade lamellar structure appears in the form of liquid crystals. This is due to differences in the degree of hydration of surfactants and phospholipid molecules triggered by solvent limitations. The protransfersome forms as a mixture of flat liquid crystals resembling palisade and vesicular lamellae linked together39. The percentage of entrapment efficiency (EE%) of the protransfersome system is a parameter used to predict the stability of the dispersion40 describing the amount of drug present in the vesicle41. In this study, the EE% value was comparatively large because it corresponded to the phospholipid content in the formula42 and the tendency of CoQ10 to be retained in the phospholipid membrane due to its lipophilic properties39.Protransfersomes have been developed as the nanometer-sized carrier form of transfersome provesicles and have a higher phospholipid content compared to transfersomes. This enables the protransfersome system to demonstrate greater entrapment efficiency due to a higher number of vesicles formed that are subsequently available for encapsulating drugs, thus providing high stability when compared to the transfersome systemTo improve acceptability, the protransfersome was formulated as an emulgel preparation incorporating the use of an emulgel as the gel base. In this study, three types of emulgels were developed and evaluated for their anti-ageing and irritability activity, namely; Protransf-CoQ10 emulgel, emulgel loaded CoQ10 which was previously dissolved in oleic acid (CoQ10-Ole emulgel) and CoQ10 dispersed in an emulgel base (CoQ10 emulgel).37, while spontaneous hydration of protransfersome produces larger vesicles than those resulting from dispersion into emulgel. When compared to the particle sizes of CoQ10-Ole Emulgel and Co-Q10 Emulgel, those of all three emulgels-loaded CoQ10s can be measured in nanometers. The order of particle size from the smallest to the largest is Protransf-CoQ10 Emulgel < CoQ10-Ole Emulgel < Co-Q10 Emulgel. Co-Q10 Emulgel is the largest in size because CoQ10 is only dispersed in the emulgel base, while Protransf-CoQ10 Emulgel and CoQ10-Ole Emulgel had similar particle size and PDI probably due to CoQ10 solubility in Oleic Acid for both formulas43. From the results of the polydispersity index, it is evident that all particles have a uniform size distribution. This indicates that the preparation will be stable during storage because it reduces the tendency for particle aggregation which causes the system to become unstable.During the homogenization method for preparing necessary samples the particle size test involves manual shaking which is considered to closely replicate real-life conditions. The particle size of the emulgel loaded Co-Q10 remained in the nanometer range, indicating that adding emulgel base to the particle size of Protransf-CoQ10 had no effect. The particle size of Protransf-CoQ10 Emulgel is smaller than that of Protransf-CoQ10 itself. This indicates that the particles have turned into transfersome vesicles because they have been partially hydrated by the presence of water in the emulgel base. The decreased vesicle size of protransfersomal CoQ10 after dispersion into the emulgel base is probably due to the shearing stress that occurs during the incorporation of Protransf-CoQ10 into hydrated Carbopol-based emulgel. This causes the small vesicles formed and the emulgel matrix to be adsorbed onto the vesicle surface, preventing vesicle fusion or aggregationA test was carried out to determine the physical stability of Protransf-CoQ10 emulgel when stored at different temperatures, namely; room temperature and colder temperatures for 28 days and whether differences in particle size, polydispersity index, and pH existed. There was no significant difference in particle size, polydispersity index, and pH of Protransf-CoQ10 emulgel during the study period.P > 0.05). These two groups demonstrated significant improvement in collagen density compared with the control group whose subjects had been exposed to UV and who recorded the lowest density value. This is probably due to soluble CoQ10 in Oleic Acid loaded into emulgel had been easily released from emulgel than that of Protransf-CoQ10 Emulgel, which the formation of vesicle during hydration results in semipermeable bilayer membrane as water diffusion-limiting barriers for CoQ10 release. The low collagen density has been known caused by imbalance between collagen synthesis by fibroblasts and collagen degradation of UV irradiation, while collagen synthesis is proportionally relate to fibroblasts resident44. Moreover, collagen synthesis by fibroblast will actively occur on the 4th day of 21 days45. The faster CoQ10 release from CoQ10 Ole Emulgel will stimulates fibroblast proliferation which increase expression of collagen matrix46, while the late CoQ10 release from Protransf-CoQ10 Emulgel will result in delayed effects on fibroblast-stimulated collagen synthesis.The results of the anti-ageing activity of CoQ10 loaded in emulgel and evaluated for skin collagen density confirmed CoQ10-Ole Emulgel as having the highest percentage of collagen density, followed by Protransf-CoQ10 Emulgel. However, no significant difference existed between these groups  was obtained from Kangcare Bioindustry Co. Ltd. . L-α-Phosphatidylcholine is a product of Sigma-Aldrich . Tween 80 and Span 80 were both purchased from Enviro Prima Co. Ltd. . The oleic acid used in this study was acquired from Brataco Co. Ltd. . All other reagents were of the available pharmaceutical and analytical grades.16. Initially, CoQ10 was stirred until completely dissolved in a mixture of oleic acid and Tween 80. Finally, L-α-Phosphatidylcholine was added and stirred until dissolved to produce Protransf-CoQ10.The protransfersome was composed of L-α-Phosphatidylcholine, Oleic Acid, and Tween 80 as shown in Table A CoQ10-loaded protransfersome emulgel was prepared by adding the Protransf-CoQ10 to the emulgel base with a final CoQ10 content of 1%. The emulgel base was produced using Carbopol 940 added to a combination of Tween 80 and Span 80 (1:1) to form a homogenous emulgel base with the addition of Triethylamine (TEA) to adjust the pH to 6.0 ± 0.2. Protransf-CoQ10, CoQ10 solution in oleic acid, and CoQ10 powder were subsequently added to this emulgel base and mixed homogenously to produce Protransf-CoQ10 emulgel, CoQ10-Ole emulgel, and CoQ10 emulgel, respectively.The evaluation of physical characteristics includes particle size, polydispersity index, ζ-potential, microscopic observation, entrapment efficiency, and physical stability during storage.The dispersion of Protransf-CoQ10 into an emulgel base at a weight ratio of 2:1 produced Protransf-CoQ10 emulgel whose color changes to brownish orange and the reduction on its pungent odor. Meanwhile, the CoQ10 dissolved in oleic acid (CoQ10-Ole) emulgel had an odorless, jelly-like consistency and was bright orange in color. These characteristics were identical to those of CoQ10 emulgel, although the latter had a more transparent appearance due to the absence of oleic acid from the formula.24. The suspension was further diluted by pipetting 150 μL of sample and added with 2 mL of deionized water  for sample measurement.Evaluation of particle size and ζ-potential were respectively carried out using a Delsa™ Nano Submicron Particle Size Analyzer  and light scattering and electron scattering methods. Approximately 50 mg of CoQ10-loaded protransfersome and emulgels were resuspended in 5 mL of 0.9% NaCl. The samples were then prepared using the manual shaking method for 5 min50. The evaluation was conducted using an optical microscope before, during, and after addition of 0.9% NaCl at 400 × magnification.The Protransf-CoQ10 was observed microscopically to evaluate its transformation ability in relation to transfersome vesicles by placing a small amount of sample on a glass slide and covering it with a cover glass. A drop of 0.9% NaCl saline solution was added to the other side of the cover slip’s cavity17. Approximately 100 mg of Protransf-CoQ10 was weighed, and then hydrated with 2 mL phosphate buffered saline (PBS) pH 7.4 and sonicated for 30 min until homogeneous. The suspension formed was then centrifuged at 3000 rpm for 30 min to obtain supernatant and sediment in a 10 mL glass tube. The sample was prepared by taking 1.5 mL of supernatant and then dissolved in 2 mL methanol, added to 2 mL PBS pH 7.4 and, finally, sonicated for 15 min. The sediment was dissolved in 1.5 mL methanol, added to 2 mL of PBS and sonicated for 15 min. The absorbance of each sample was measured by UV–Vis spectrophotometry at a wavelength of 275 nm. The EE (%) of CoQ10-loaded in protransfersome was calculated by means of the following equation:The EE% was measured for CoQ10 loaded in protransfersome by means of UV–Vis spectrophotometry−1. While, the thermal analysis was evaluated using Differential Thermal Analysis (DTA) instrument . About 3–5 mg samples was put into crucible sample pan. The sample was then subsequently heated from 30 to 300 °C at a heating rate of 10 °C per minutes.In order to evaluate whether any chemical or physical changes occurred in samples, spectroscopical and thermal analysis were further investigated. The spectroscopical analysis was evaluated using a Fourier Transformed Infra-Red analysis by using Spectrophotometer ECO ATRS Bruker Alpha II (Germany). About 1 mg sample was analyzed at wavenumbers of 450–4000 cm53. The emulgel was evaluated for physical characteristics, i.e., pH and particle size, on the 28th day after preparation.Moreover, a stability test of the Protransf-CoQ10 emulgel was carried out by storing the samples at in the dark at room temperature (24 ± 2 °C) and, subsequently, a cold temperature (4 ± 2 °C) for 28 daysMus musculus) within the terms of a study protocol approved by The Ethics Commission of Faculty of Veterinary Medicine, Universitas Airlangga . All methods were performed in accordance with ARRIVE guidelines and relevant regulations54. Within this research, two types of study involving the uses of experimental models were evaluated, firstly, anti-ageing activity as indicated by collagen density and number of fibroblasts, and, secondly, a safety test incorporating irritancy scoring of skin tissue. The effect of the Protransf-CoQ10 emulgel was compared with those of CoQ10-Ole and CoQ10 emugels. Each group comprises of 4 mice as the study model. Prior to the study, the hair on the models’ backs was trimmed with mechanical hair clippers, ensuring that their skin was not injured during this process. Each model was housed in a separate cage to prevent their touching the part to be smeared with the sample.The in vivo anti-ageing activity was evaluated using Balb/c mice  using Cellsen Standard Software. Collagen density was measured by histochemical scoring, while the number of fibroblasts was calculated by digital analysis using Adobe Photoshop and Image J software. Density measurement involved measuring the area of collagen coir and comparing it with the field of view. The denser the coir collagen, the higher the density value, and vice versa. Calculation of the density value was completed by means of calculating the area of the field of view and the black colored area using Image J software calibrated fin advance or each degree of magnification. The comparison of the black stained area with the field of view produced the density value.The anti-ageing activity test was evaluated to establish the parameters of collagen fiber density and the number of fibroblasts. The study was carried out by applying 200 mg of the emulgels twice a day every day to a 4 cm2 area of skin on their backs. Twenty-four hours after application, the models were sacrificed by dislocation. Skin was excised with a microtome before being immersed in a formalin solution and stained with hematoxylin–eosin. The preparations were observed with a light microscope to assess the degree of skin irritation by means of histopathological scoring. Histological change data is semi-qualitative and features five variables, namely; epidermis liquefaction, subepidermal edema, collagen fiber swelling, inflammatory cell infiltration, and degeneration of the appendages in hair vesicles. The scoring method comprised a score of 0 = normal skin, 1 = mild irritation, 2 = moderate irritation, and 3 = severe irritation56. The data from each sample consisted of the mean value of the variable score for each of the five different fields of view at 100 × and 400 × magnification. All examinations involved the use of an ordinary light microscope .In order to observe the irritant effects of CoQ10-loaded in proransfersome and emulgels, histopathological changes in the skin tissues of each model after a 24-h period of exposure were observed. Firstly, the back hair of the models had been shaved. Approximately 200 mg of the sample was then applied to a 2 × 2 cmP value < 0.05, then a significant difference between the results of the tests performed existed. However, if the data was not normally distributed and homogeneous, the data would be analyzed using non-parametric statistics by means of the Kruskall Wallis method and, subsequently, a Post Hoc Mann Whitney U test. If the P value < 0.05; then a significant difference existed.The data in this study consisted of three replicates. In order to test the significance of differences in the data relating to Protransf-CoQ10 emulgel, CoQ10-Ole emulgel, and CoQ10 emulgel, a statistical analysis was performed using the one-way variant analysis (ANOVA) method. After the normality and homogeneity of the data had been tested, a Post Hoc Tukey HSD test was administered. If 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 .


We found that TMEM206 is frequently overexpressed and that high levels of TMEM206 correlated with clinical stage and pulmonary metastasis in patients with OS. We provided evidence that TMEM206-silenced OS cancer cells exhibit decreased proliferation, migration, and invasion in vitro. Mechanistically, we identified β-catenin, a key member of Wnt/β-catenin signaling, as a downstream effector of TMEM206. TMEM206 silencing inhibits the Wnt/β-catenin signaling pathway in expression rescue experiments, confirming that TMEM206 silencing attenuates OS cell tumorigenic behavior, at least in part, via the β-catenin mediated downregulation of Wnt/β-catenin signaling. More importantly, TMEM206 knockdown-related phenotype changes were replicated in a xenograft nude mouse model where pulmonary metastases of OS cells were suppressed. Together, our results demonstrate that silencing TMEM206 negatively modulates the Wnt/β-catenin signaling pathway via β-catenin to suppress proliferation, migration, invasion, and metastasis in OS carcinogenesis, suggesting TMEM206 as a potential oncogenic biomarker and a potential target for OS treatment.Transmembrane protein 206 (TMEM206), a proton-activated chloride channel, has been implicated in various biochemical processes, including bone metabolism, and has emerged as a novel cancer-related protein in multiple tumor types. However, its role in primary malignant bone tumors, particularly in osteosarcoma (OS), remains unclear. This study is aimed at exploring the effects of TMEM206 gene silencing on the proliferation, migration, invasion, and metastasis of human OS cells  Osteosarcoma (OS) is the most common primary aggressive and malignant tumor of the bone, with high morbidity and metastatic potential. Approximately 75% of OS patients are between 15 and 25 years of age, which heavily threatens the health of adolescents . The pooProliferation, invasion, and metastasis are the essential biological characteristics of OS  and are Ion-activated chloride channels (IACC) are members of the transmembrane (TMEM) chloride channel family and are widely distributed in human tissues, including bone , 9. The in vitro and tumor growth in vivo to confirm clinical observations. Our findings provide novel insights into the role of TMEM206 in OS cell proliferation, migration, invasion, and metastasis while shedding light on the potential molecular mechanisms.In this study, we collected OS patient tissue samples to investigate whether TMEM206 was dysregulated in OS patients and correlated with prognosis. TMEM206 loss-of-function experiments in well characterized OS cell lines were then performed Human OS tissue samples were collected from Renmin Hospital of Wuhan University, from November 2017 to June 2019, from patients aged 7-33 years . There were 33 adjacent nontumor tissues and 48 OS samples. Grade of OS tissues were distinguished on the basis of Ennecking staging system during pathological examination. All participants have signed an informed consent form and been informed of all the surgery and experiment details in advance. Histologic sections were reviewed by two expert pathologists to verify the histologic diagnosis. We obtained written informed consent from all patients. The Ethics Committee of Renmin Hospital of Wuhan University approved the study protocol. Eighteen male BABL/c nude mice (6-8 weeks old) were purchased from the Wuhan University experimental animal center . All experimental procedures involving animals were strictly followed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by Animal Care and Use Committee of Renmin Hospital of Wuhan University. The human fetal osteoblastic cell line hFOB1.19 and human OS cell lines SAOS2, U2OS, and MG63 were purchased from the Cell Bank of the Chinese Academy of Sciences . All cell lines were cultured in Dulbecco's Modified Eagle Medium DMEM , and 10% fetal bovine serum , 100 U/mL penicillin, and 100 mg/mL streptomycin  were added. Cells were cultured at 37°C with 5% CO2 in an incubator .β-catenin , c-Myc , CyclinD1 , MMP7 , MMP9 , and GAPDH  were purchased as primary antibodies. HRP-predicted sheep anti-rabbit second antisubstances (1 : 5000) were acquired as well. Eventually, the visualization of the protein bands were realized via an ECL system, and grayscale results were determined via Image J soft.RIPA lysis buffer  was adopted to abstract the overall proteins as per supplier's guideline. Immunoblotting was used to identify the protein expressing levels herein as per the guideline provided in the previous study. Primary antibodies against TMEM206 , 6 cells were transfected with 2.5 μg of plasmid using 3.75 μL based on manufacturer's protocol.For knockdown of TMEM206, lentiviruses containing TMEM206 short hairpin (sh) RNAs/scramble controls (SC) were produced in HEK-293T cells with packaging vectors from ObiO . The medium supernatants were collected and concentrated by PEG-it virus precipitation solution . The obtained viruses were then used to infect U2OS and MG63 cells. Transient transfections of plasmids TMEM206-shRNA and SC-shRNA  were performed with Lipofectamine 3000 . Approximately 2.5 × 10The transfected U2OS cells were fixed with 4% paraformaldehyde for 30 min and rinsed thrice with PBS and. The cells were then permeabilized with PBS containing 0.5% Triton X-100 for 15 min. The TMEM206 antibody  was applied to the coverslip and incubated in a wet box overnight at 4°C. After washing three times with PBS, the fluorescent secondary antibody  was added and incubated for 1 h in the wet box at 25°C. Following three additional washes with PBS, the DAPI mounting solution  was added to stain the nuclear compartment. The immunoreactive cultured cells were observed using a fluorescence microscope .CCK-8 detection  uses a similar method, as described earlier. Transfected cells were inoculated into 96-well plates and incubated overnight. Add CCK-8 reagent 10 mL to each well and incubate at 37°C for 2 hours. The absorption value was measured at 450 nm with a microplate analyzer .5) were inoculated into 96-well plates, and cell proliferation was assayed using an EdU kit  according to manufacturer's instructions. Hoechst 33342 was added to stain the nuclei. After a 30-minute incubation in the dark, the cells were observed under a fluorescence microscope .U2OS and MG63 cells  as previously described . The migβ-catenin binding site and cotransfected with the control (pcDNA-Con) or TMEM206 overexpression (pcDNA-TMEM206) plasmid. The resulting Firefly and Renilla luciferase activity were measured 48 h posttransfection using the Dual-Glo™ Luciferase Assay System, according to manufacturer's instructions .Reporter assays were performed with U2OS and MG63 cells, which were transfected with either wild type or mutant reporter plasmids containing the putative 6 cells) were inoculated subcutaneously into the nude mice. Tumor volumes were measured every 7 days. After 21 days, mice were sacrificed, and tissues were collected for further analyses.MG63 cells stably transfected with TMEM206-shRNA or SC-shRNA  and GraphPad Prism Software version 7.01 , respectively. All data were expressed as mean ± standard deviation (SD) or mean ± standard error means (SEM). Comparison between two groups was determined by unpaired P < 0.001). The IHC results indicated that TMEM206 was aberrantly expressed in the advanced Enneckin stage (P < 0.05). We then analyzed the endogenous expression of TMEM206 in a panel of human OS cell lines . As shown in P < 0.01).We first set to explore whether TMEM206 was aberrantly expressed in OS tissues. Compared to paired adjacent normal tissues, the protein expression of TMEM206 was dramatically higher in tumor tissues . Next, we investigated whether TMEM206 regulates cell proliferation. EDU-staining and CCK-8 assays were used to evaluate the effects of silencing TMEM206 on cell proliferation. When TMEM206 was downregulated in OS cells, a significant decrease in proliferation rates was observed compared with SC-shRNA transfection . The colony formation assay showed that TMEM206-shRNA-transfection displayed significantly fewer colonies than SC-shRNA-transfected U2OS and MG63 cells (P < 0.001). Collectively, these data suggest that TMEM206 plays an important role in OS cell growth in vitro.To elucidate the role of TMEM206 in OS, U2OS and MG63 cells were transfected with control scrambled shRNA or TMEM206-shRNA. Immunofluorescence staining indicated that TMEM206 expression was reduced after TMEM206-shRNA transfection , and thiP < 0.01). We then examined the role of TMEM206 in U2OS and MG63 cells on cell migration using a wound scratch assay. As illustrated in P < 0.001). Collectively, these data demonstrate that TMEM206 is involved in OS tumor metastasis in vivo and cell migration and invasion in vitro.Given that TMEM206 expression correlates with tumor stage, we investigated whether TMEM206 regulates OS cell migration and invasion, two critical features of malignant cell behavior. First, we analyzed the human OS tissues and found that the OS patients with metastasis showed much higher TMEM206 expression than the primary tumors without metastasis . The correlation between the TMEM206 and β-catenin reminds us that there is a interaction mechanism among them.Considering that Wnt/in stage , and WB β-catenin. The results showed that overexpression of TMEM206 significantly promoted the activity of firefly luciferase that carried wild-type but not mutant TCF-binding sites both in U2OS and MG63 cells (P < 0.01), which indicated that β-catenin is indeed a downstream target of TMEM206. In addition, by using immunofluorescence staining, we also observed that upregulation of TMEM206 expression noticeably promoted the β-catenin accumulation in MG63 cells  were further confirmed by Western blotting. As shown in β-catenin, c-Myc, cyclinD1, MMP7, and MMP9, which are all key signaling intermediates of the Wnt/β-catenin signaling pathway, were all significantly decreased in both U2OS and MG63 cells .To further confirm the underlying mechanisms by which TMEM206 interacted with 63 cells . Wnt/β-cβ-catenin is required for the observed phenotypes induced by TMEM206 knockdown in OS cells, we then conducted the following rescue experiments: first, the Wnt/β-catenin signaling activator lithium chloride (LiCl) was added to the TMEM206-shRNA-transfected U2OS and MG63 cells to rescue the function of Wnt/β-catenin signaling. As expected, the decreased migration and invasion of MG63 cells transfected with TMEM206-shRNA was reupregulated after LiCl administration (P < 0.001). To further confirm our hypothesis, we constructed TMEM206 overexpressing MG63 cells, and the β-catenin inhibitor IWP-2 was added to observe its effect. Transfection of the plasmid containing TMEM206 promoted the migration and invasion of MG63 cells, and this promotive effect was abolished by IWP-2 treatment (P < 0.001). Taken together, these data revealed that the defects in proliferation and migration induced by TMEM206 knockdown in OS cells were at least in part via the β-catenin-mediated downregulation of the Wnt/β-catenin signaling pathway.To determine whether in vitro. In this study, a xenograft model in nude mice injected with TMEM206-shRNA-transfected MG63 was used to determine the effect of silencing TMEM206 on OS tumor growth in vivo. Decreasing the expression of TMEM206 remarkably suppressed tumor growth, as reflected by tumor volume, compared with the SC-shRNA vector-transfected control cells . The decrease in cell proliferation in the xenograft model was also validated by immunohistochemical staining of TMEM206 and β-catenin . Taken together, silencing TMEM206 negatively regulated the proliferation and migration of OS cells in vivo and in vitro , the most common malignant bone tumor in children and adolescents, is characterized by a high degree of malignancy and high occurrence of lung metastasis . The lacTaking advantage of clinical specimens and well-characterized OS cell lines, we sought to establish whether TMEM206 is associated with OS pathogenesis. TMEM206 was consistently found to be upregulated in clinical tumor samples and OS cell lines, and the levels of TMEM206 positively correlated with clinical stages in OS patients, which demonstrates that TMEM206 overexpression might drive OS progression. Similar to our results, the expression of TMEM206 positively correlated with the prognosis of HCC  and promβ-catenin signaling pathway was regulated by TMEM206 in OS cells. TMEM206 knockdown significantly altered the expression levels of Wnt/β-catenin signaling pathway markers in OS cells dual-luciferase reporter analysis showing that TMEM206 could directly interact with β-catenin. The results of rescue experiments further proved that the defects in proliferation and migration induced by TMEM206 knockdown in OS cells were, at least partly, due to the downregulation of the Wnt/β-catenin signaling pathway. Along the same line of arguments, Jiang et al. reported that TMEM48 could promote cervical cancer invasion and metastasis in vitro and in vivo via activating the Wnt/β-catenin pathway [β-catenin pathway [β-catenin pathway is crucial for bone morphogenesis and bone regeneration [in vitro and OS tumor growth in vivo [Tumor biological functional experiments in OS cells showed that TMEM206 downregulation influences OS cell proliferation, colony formation, migration, and invasive capacity, the most important features of malignant cell behavior. Consistent with our present data, Zhao et al. found that knockdown or upregulation silencing of TMEM206 inhibited or promoted the proliferation of colorectal cancer cells , which s pathway . Moreove pathway . The canneration , and var in vivo . TMEM206in vivo. Therefore, we utilized a xenograft model to determine the effect of silencing TMEM206 on OS tumor growth in vivo. Inhibition expression of TMEM206 remarkably suppressed tumor growth, as reflected by the tumor volume accompanied by decreased immunohistochemical reactivity of Ki-67 and CD31. The lung is the most common site of distant metastasis of OS [β-catenin pathways, MMP7 and MMP9, have been regarded as metastasis-related genes, which play a critical role in OS invasion, metastasis, and tumor cell angiogenesis [in vivo could be explained by the inhibition of the Wnt/β-catenin pathway. Taken together, our results collectively indicate that blocking the TMEM206 could inhibit OS. Nevertheless, although the results are inspiring, the antitumor effects of the TMEM206 target should be investigated in patients in future clinical studies.Based on the above evidence, we speculate that targeting TMEM206 might be a useful approach to repress the proliferation, migration, and metastasis of OS is of OS . Our patogenesis , 28. Thein vitro and in vivo by downregulating Wnt/β-catenin signaling. These findings provide sufficient in vitro and in vivo evidence of the anticancer effects and tumor inhibitory role of TMEM206 inhibition in OS and confirm TMEM206 as a key regulator of Wnt/β-catenin signaling. Our study highlights a critical role for the TMEM206/Wnt/β-catenin signaling axis in OS progression and suggests that TMEM206 is a potential therapeutic target for OS diagnosis and therapy.In summary, our findings are the first to show that TMEM206 is increased in human OS tissues and may represent a novel biomarker of OS. Silencing TMEM206 impedes the proliferation and invasion of OS cells


The pangolin is the only scaly mammal in the world. Among the eight extant pangolin species, the Chinese pangolin (CP) is most threatened with extinction. Thus, this species is categorized as “Critically Endangered” by the International Union for Conservation of Nature (IUCN) and listed under “Appendix I” of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Taiwan is a single Formosa Island where the population of CP has been increasing for the last decade due to continuous conservation initiatives. The presence of sufficient samples of CP that are adapted to distinct environmental conditions could provide valid morphometric results. However, the morphometric relationships, growth type, and condition factors have not yet been enumerated for any pangolin species including Chinese pangolin. The study included 282 rescued and rehabilitated individuals from central and southern Taiwan. Adult male and female pangolins measure between 75.2–103 cm and 66–114.9 cm from the snout to the tip of the tail and weigh between 4–7.6 kg and 3–5.8 kg, respectively. Adults also showed negative allometric growth (b < 3) as their length increased. However, the average relative condition factor (KR) was 1.02 ± 0.16 and showed the majority of rescued adults in normal (59.7%) and good (39%) health conditions.b was used to estimate the length-weight relationships where R2 = 0.70, a = 0.61, and b = 1.98. The ratio of total body length and tail length was between 1.1 and 2.7. Pangolins exhibited negative allometric growth (b < 3) as their length increased. The average relative condition factor (KR) was 1.04, indicating a state of good health for rescued individuals. However, Fulton condition factor (KF) and KR fluctuated in different months but was significantly heavier during the wet seasons. This study recommended to release healthy animals with KF of 0.8 or higher back into the wild in order to increase their survival rate. The study, thus will be helpful to promote standardized guidelines for conducting physical measurements and understanding health status. Additionally, it will support the recovery of this threatened species by husbandry and diet management in ex-situ and in-situ conservation.Morphometric relationships and condition factors are crucial to quickly understanding the fitness and well-being of animals. Total length (cm) and bodyweight (g) of 282  pangolins were accounted for in this study which was received and rehabilitated in Pingtung Rescued Center, Pingtung and Endemic Species Research Institute, Nantou, Taiwan. The allometric equation; W = aL Manis pentadactyla) is one of the most illegally traded mammals for meat consumption, traditional medicine, and souvenirs. Wildlife authorities are constantly confiscating and seizing hundreds of live pangolins from illegal traders each year around the world [Manis javanica) [Manis crassicaudata) [Chinese pangolin (he world . The illhe world . The reshe world . The Chiavanica)  and Indicaudata) . This spcaudata) . Pangolicaudata) . Globallcaudata) . It is aF) and relative condition factor (KR) are key determinants of an individual animal’s provide overall fitness, and its implications regarding health in terms of biomass fitness of a population in a habitat [Pangolin is an idiosyncratic mammal with a typical morphological characteristic. The morphological parameters including length-weight relationships (LWRs), growth and condition factors (K) of Chinese pangolin are lacking ,9. Previ habitat ,13.The body condition of an animal refers to its energetic state, so an animal in good condition has more energy reserves  than an animal in poor condition. In mammals, the amount of fat that an individual carries can have significant fitness consequences. For instance, individuals with larger fat reserves may have better fasting endurance and better survival than individuals with smaller reserves . ConditiThe information on rescued Chinese pangolins received in the Pingtung Rescue Center for Endangered Wild Animals (PTRC), Pingtung (n = 178), and the Endemic Species Research Institute (ESRI), Nantou (n = 104), Taiwan, was managed by excluding missing data and injured individuals. These rescued pangolins were in temporary care and released back into a safe natural habitat. Length and weight measurement was conducted during the first aid treatment following the wild mammals handling procedure  under thb, was used to estimate the relationship between the weight  of the pangolins with their total length . Logarithmic transformation of LWRs parameters and estimation by using the linear regression of data transformed to base 10 logarithms; LogW = Loga + b LogL [The allometric equation for length-weight relationships (LWRs), W= aL+ b LogL . Based o+ b LogL . To esta+ b LogL , gender F) and relative condition factor (KR) were developed for quick evaluating the physical status or well-being of the confiscated or rescued pangolins, based on the assumption that heavier pangolins of a given length are in better conditions. The factor is calculated using the Fulton 1904 suggested formula for the Condition Factor (KF) = weight/length3 × 100 [R) was calculated by the ratio of observed weight (Wo) and calculated weight (Wc). KR = 1 or near to 1 indicates a “normal” health condition whereas KR greater than 1 (KR > 1) is good and fat, however, less than 1(KR < 1) indicates poor health status with a skinny body [The Fulton condition factor . The coefficient of determination (r2) is a measure of the quality of the linear regression’s model prediction; a value close to 1 means a better model and correlation is significant at the 0.01 level (p < 0.01). Pearson correlations were analyzed at the p < 0.05 level of significance. In addition, Kendall’s tau-b and Spearman’s rho tests were used to analyze the corrrelation between length and weight. The correlation of bodyweight with Fulton’s condition factor (KF), relative condition factor (KR) and calculated weight (WC) were tested by correlation tests.All the data were managed in Excel and analyzed using IBM SPSS statistics 26. Descriptive analysis was used to summarize the morphometric measurement of length and weight as well condition factors of rescued pangolins and conducted the correlation and regression analysis . Regressp = 0.000) . Pangolins older than one year  were rescued and rehabilitated, followed by 6 month old pangolins , while the adult pangolins were 2 years old , and 3 years old and above . The total length increase was statistically significant different between ages  .The correlation between total length (TL) and bodyweight (BW) of the Chinese pangolin (CP) was highly positive (around 80%). The bodyweight grows faster relative to increasing length; however, the growth type was negative allometric growth (b < 3) . The regF) values of the Chinese pangolin fluctuated between 0.23 and 2.12 whereas the average value of the relative condition factor (KR) was 1.04 ± 0.24 (p = 0.002), calculated weight , and relative condition of ages were significantly different  (Fulton condition factor (K4 ± 0.24 . Nearly 4 ± 0.24 . Althoug= 0.006) .R ≥ 1) which showed that most rescued adults were in normal (59.7%) and good (39%) health conditions. However, the mean values of the Fulton condition factor (KF) and relative condition factor (KR) fluctuated in different months . The adult pangolins both male and female were found to be slightly fatter, and their wellbeing was higher in the wet season; however, KF and KR were not statistically significantly different between dry and wet seasons (p > 0.005) but they were positively correlated with each other  as their length increased but positive allometric growth (b = 3.6) in June as the bodyweight was proportional with respect to length increase . The relt months a but KR ch other .The population of Chinese pangolin in Taiwan was comparatively increased due to conservation initiatives and strict implementation of rules and regulations after 1970 . Many liMale pangolins were found to be bigger and longer than females in different age groups, like in other mammalian species . Body sib) to understand the LWRs which have been used widely to understand the biometric length-weight relationships in mammalian species [Phataginustricuspis) in Southwestern Nigeria [This is the first length-weight relationships and condition factors study on Chinese pangolin. We used the allometric equation  were found in normal and good health condition. It could be due to sufficient food supply and minimized threats along with facilities and knowledge on handling of species during rescue and transformation in Taiwan. Therefore, veterinary training including species handling for the government authorities and stakeholders is of utmost need to minimize the unwanted effects on the species’ health for in-situ and ex-situ pangolin conservation. Female individuals of these ages; sub-adults (1-year old) and adults (2 years old and those 3 years and older) were slightly fatter and healthier than males. This might be due to sex-specific consequences of variation in fat reserves . In femaF) for the quick assessment of the health status of rescued individuals. This is because it can be calculated easily with a simple formula, and KF values do not differ significantly across sexes and ages  and relative condition factor (KR) for the majority of rescued pangolins (around 90%) were close to 1 and greater and equal to 1, thus showing an overall state of wellbeing of the rescued pangolins. It is only recommended to release healthy animals with K-values of 0.8 or higher back into the wild in order to increase their survival rate. Similarly, KF and KR fluctuated in different months, but it is not statistically different between dry and wet seasons in adults. However, overall pangolins were found in better condition in the wet season compared to the dry season which might be due to the increase in feeding activity and food availability when temperatures are higher. Thus, this study fulfilled the aims set for it, and the data presented might constitute a valuable guideline for establishing future biometric studies for live pangolins in other range countries.This study provided the first data on length-weight relationships (LWRs) and condition factors for the Chinese pangolin. Almost all LWRs showed a negative allometric growth of species. The Fulton condition factor (K


A series of functional truncations of different regions further identified that the C-terminal domains of MRAP2 protein were required for the pharmacological modulation of intracellular Ca2+ coupled cascades and membrane transport. These findings elucidated the broad regulatory profile of MRAP2 protein in the central nervous system and may provide implications for the modulation of central MCHR1 function in vivo.Melanin concentrating hormone (MCH), an orexigenic neuropeptide, is primarily secreted by the hypothalamus and acts on its receptor, the melanin-concentrating hormone receptor 1 (MCHR1), to regulate appetite and energy homeostasis. The Melanocortin Receptor Accessory Protein 2 (MRAP2), a small single transmembrane protein broadly expressed in multiple tissues, has been defined as a vital endocrine modulator of five melanocortin receptors (MC1R–MC5R) and several other GPCRs in the regulation of central neuronal activities and peripheral energy balance. Here, we demonstrated the interaction between MRAP2 and MCHR1 by immunoprecipitation and bimolecular fluorescent assay and found that MRAP2 could inhibit MCHR1 signaling  Deletio2+ level , and ind2+ level .Melanocortin receptor accessory proteins (MRAPs) include two members, the melanocortin receptor accessory protein 1 (MRAP1) and melanocortin receptor accessory protein 2 (MRAP2). MRAP2 is highly expressed in the hypothalamus, especially the paraventricular nucleus (PVN), and has been linked to mammalian obesity syndromes , 7. It hin vitro and the pharmacological regulation of MCHR1 signaling required distinct functional regions of MRAP2 protein. Collectively, we observed that the C-terminus of MRAP2 was mainly responsible for the inhibition of MCHR1 signaling and cell surface translocation. Our data not only identified MCHR1 as a novel metabolic-associated GPCR target of the MRAP2 protein, but also elucidated the complex endocrine network of GPCR signaling, which may explain the composite metabolic phenotypes of MRAP2 deficient murine models.In this study, we investigated the regulatory profile of the MRAP2 protein on the trafficking and signaling of MCHR1. MRAP2 interacted with MCHR1 https://www.gtexportal.org/) and mouse RNA-seq datasets were downloaded from all RNA-seq and ChIP-seq sample and signature search (ARCHS4) database (https://amp.pharm.mssm.edu/archs4/). Datasets of mouse cerebral and hypothalamic single-cell RNA-seq including GSE74672, GSE87544, GSE130597, and GSE125065 were downloaded from Gene Expression Omnibus (GEO) datebase.Human bulk RNA-seq datasets were downloaded from the Genotype-Tissue Expression (GTEx) project  as the normalized value. Next, we analyzed the samples of whole brain and hypothalamus of humans and mice, respectively. We calculated Pearson’s correlation coefficient between MCHR1 and MRAP1 or MRAP2 expression value across all samples. For single-cell RNA-seq analysis, all datasets were filtered and low-quality cells with unique feature counts less than 200 were excluded. The co-expression of cell numbers of MRAP2 and MCHR1 positive neuronal populations was calculated. Next, Pearson’s correlation coefficient was calculated between the expression value of MRAP2 and MCHR1 across all cells.To calculate the changes in MRAP2 and MCHR1 expression under high-fat diet condition, we counted the proportion of cells expressing these transcripts in the high-fat diet and normal chow condition, respectively. We also tested the significance of the differences using Fisher’s exact test. In order to explore the pathways in which these genes are involved, we performed GO pathway enrichment analysis using clusterProfiler R package.MCHR1, MRAP1, and MRAP2 were amplified from wild-type C57/BL6 mice cDNA library. All PCR products were ligated into pcDNA3.1(+) vector and the constructs were verified by Sanger sequencing. Melanin concentrating hormone (MCH) was purchased from BACHEM. SNAP-94847 (MCHR1 antagonist) was purchased from MCE (MedChemExpress). In the following assays, we purchased anti-mouse HA monoclonal antibody , anti-mouse Flag monoclonal antibody , anti-mouse IgG antibody , and HRP-conjugated antibodies against mouse antigens .2. Transfection was conducted using P-PEI reagent according to the manufacturer’s protocols. The total amount of transfected plasmids was kept identical in each group by adding empty pcDNA3.1 vector. The concentration of cells seeded for each single assay was 105/ml.HEK293T cells were cultured in DMEM medium (high glucose) supplied with 10% FBS and 1% penicillin–streptomycin (P/S). Cells were incubated in a 37°C incubator consisting of 5% CORT-PCR was performed as previously described . BrieflyProteins were extracted 24–36 h upon transfection and then incubated with mouse anti-HA or mouse anti-Flag antibody at 1:5,000 dilution overnight at 4°C. The next day, protein A+G beads  were added and rotated at 4°C for 4 h. Finally, beads were resuspended in protein loading buffer after three washes and boiled for 15 min. Samples were loaded on 12% SDS-PAGE gels and mouse anti-FLAG antibody was used for detecting MRAP1/MRAP2 in MCHR1 co-immunoprecipitation experiments. Images were captured by ImageQuant 4000.HEK293T cells were seeded on poly-L-lysine pretreated coverslips of 12-well plates and transfected with MCHR1-F1 and MRAP1-Flag-F2 or MRAP2-Flag-F2. Each well was transfected with 1 μg of plasmids in total. The next day, cells were fixed with 4% PFA Fix Solution for 20 min. Cells were incubated with anti-FLAG antibody  at 1:5,000 for 2 h at room temperature for the detection of MRAP1 and MRAP2 proteins. Then, samples were washed 3 times and incubated with 1:5,000 secondary antibody Alexa Fluor594 (Abcam) for 2 h at room temperature. To detect membrane translocation of MCHR1 in the presence of MRAP2, we transfected GFP-MCHR1 and MRAP2 or RAMP3 at a ratio of 1: 9 without antibody treatment. In order to observe co-fluorescence of MCHR1 and MRAP2 chimeras, 3HA-MCHR1 and each 2xFLAG MRAP2 chimera were transiently transfected. Cells were incubated with both anti-HA and anti-FLAG antibody  at 1:5,000 for 2 h at room temperature. Samples were then washed 3 times and incubated with both 1:5,000 secondary antibody Alexa Fluor488 (Abcam) and Alexa Fluor594 (Abcam) for 2 h at room temperature.Coverslips were fixed with nail polish on the glass slide containing ProLong (R) Gold Antifade with DAPI Molecular Probes . Imaging was collected using a 63× oil objective with laser-scanning Zeiss confocal microscopy (LSM880).HEK293T cells were seeded in 12-well plates and transfected with MCHR1 and MRAP2 (1:0 to 1:9 ratio receptor to MRAP2). Each well was transfected with 1 μg of plasmids in total. Upon 24–36 h transfection, ELISA was performed as previously described . Cells wvia P-PEI according to the manufacturer’s instructions. A total of 1 μg of plasmid was transfected in each group of three wells. After 24–36 h transfection, medium was removed and different concentrations of MCH (from 10-6 to 10-11 M) in DMEM supplemented with 0.1% BSA were added and incubated for 9 h at 37°C. For competitive inhibition assay, a concentration of SNAP-94847 (MCHR1 antagonist) ranging from 10-6 to 10-11 M was added in HEK293T cells supplemented with 2 ×10-9M (EC80) MCH.HEK293T cells were cultured in 24-well plates and different MRAP2 mutants were co-transfected along with MCHR1, NFAT (firefly luciferase), and pRL-TK (Renilla luciferase) reporter vectors Dual-luciferase reporter assays were conducted using Dual-Glo kits  according to the manufacturer’s instructions. Luciferase activities were measured by a Spectramax M5 multimode plate reader. Firefly luciferase values were normalized to Renilla luciferase values for relative quantification.DNAMAN software was utilized to compare the protein sequence similarity of human and mouse MRAP2. Distinct colors highlighted the similarity score of various amino acids, in which red represented 100% consistency between sequences; blue indicated that the amino acids at this position were not conserved with a similarity score between 0 and 33%.vs. response equation (Y = Bottom+(Top-Bottom)/(1 + 10^((LogEC50–X))) (X: log(agonist); Y: response values) method. One-way ANOVA with Tukey post-test was applied to measure significance between groups, and results were shown as mean ± SEM. The tests were performed with a nominal significant level of *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.All experiments in this study were repeated at least three times. Data were analyzed by the GraphPad Prism6 software. Pharmacological curves were carried out by the log(agonist) First we developed a custom script to interrogate the co-expression correlation of MCHR1 and MRAP1 or MRAP2 in 1,698 human or mouse bulk RNA-seq samples of central nervous system  from the Genotype-Tissue Expression (GTEx) project databases and all RNA-seq and ChIP-seq sample and signature search (ARCHS4) database. As shown in MCHR1 and MRAP2 both played a critical role in the mammalian energy homeostasis. Therefore, we analyzed the cell number ratio change of MCHR1 and MRAP2 under different metabolic conditions. The results showed that MCHR1 and MRAP2 exhibited consistent cell ratio shift in the High-Fat Diet induced group compared to controls, which were both significantly upregulated  assays in HEK293T cells transfected with 3HA-MCHR1 and 2Flag-MRAP1 or 2Flag-MRAP2. As shown in These findings provided actual evidence for the co-expression of these proteins Next, to further assess the ability of MCHR1 to form a functional protein complex with MRAP2 but not MRAP1 in live cells, we performed bimolecular fluorescence complementation (BiFC) assays. To achieve this goal, we generated MCHR1 constructs fused to the YFP-F1 fragment in the C-terminus, while the C-terminal of MRAP1 or MRAP2 was fused to the complementary YFP-F2 fragment and Flag tag  were co-transfected with 3HA-MCHR1. The results showed that the nonfunctional MRAP2 mutants were all properly expressed Figure 52+ influx of MCHR1 by CRE-luciferase reporter assay. The results showed that MRAP2 strongly decreased the agonistic efficacy of MCH  to observe the effect of MRAP2 mutants on MCHR1. We found that consistent with the results observed in In addition, we also performed competitive inhibition analysis  is the first reported energy homeostasis-related GPCR that was regulated by MRAP2. MRAP2 potentiates MC4R signaling  in vivo , 21. MRA in vivo , pro-ore in vivo , and gro in vivo , 14. In via a different mechanism of action. As previously reported, a glycosylated asparagine at position 9, which was inactivated or deleted, could result into the functional loss of MRAP2. Also, we showed that the interaction between MCHR1 and MRAP2 still occurred upon deletion or mutation of the C-terminal and N-terminal, respectively. These results make sense, because it has been reported that the TM region of MRAP2 is the smallest region for MRAP2 to form homodimers  regions, whereas the C-terminal portion is variable . Unlike modimers , suggestin vivo. Based on some recent work demonstrating that MCHR1 is abundantly expressed in nerve cilia and in which it regulates cilia length and signaling pathways , which activates downstream MC4R to transmit satiety signals , and MRApathways , 27, we The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.The animal study was reviewed and approved by the Department of Plastic and Reconstructive Surgery, Shanghai Institute of Precision Medicine, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.ChZ conceived and designed the study. MW, JX, XL, and BJ performed the experiments. YZ, CoZ, SL, and ZK performed data analysis. TZ, L-NJ, QL, and ChZ are responsible for writing and revising manuscript. All authors contributed to the article and approved the submitted version.This work was supported by grants from the National Key Research and Development Program of China (Grant Nos. 2017YFA0103902 and 2019YFA0111400), Shanghai Municipal Key Clinical Specialty (Grant No. shslczdzk00901), the Innovative Research Team of High-level Local Universities in Shanghai (Grant No. SSMU-ZDCX20180700), and the Key Laboratory Program of the Education Commission of Shanghai Municipality (ZDSYS14005).The 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The synthesis conditions are carefully optimized in order to yield monodisperse and uniform nanomedicines, which will ensure reliable and steady therapeutic properties. Compared with the strategies aiming at a single carbon source, improved starvation therapy efficacy is observed. Besides, more than boosting the energy shortage, CHC/GOx@ZIF‐8 can block the lactate‐fueled respiration and relieve solid tumor hypoxia, which will enhance GOx catalysis activity, depleting extra glucose, and producing more cytotoxic H2O2. By the synergistically enhanced anti‐tumor effect, both in vitro and in vivo cancer‐killing efficacies of CHC/GOx@ZIF‐8 show twice enhancements than the GOx mediated therapy. The results demonstrate that the dual‐depriving of lactate and glucose is a more advanced strategy for strengthening cancer starvation therapy.Recent investigations reveal that lactate is not a waste product but a major energy source for cells, especially in the mitochondria, which can support cellular survival under glucose shortage. Accordingly, the new understanding of lactate prompts to target it together with glucose to pursue a more efficient cancer starvation therapy. Herein, zeolitic imidazolate framework‐8 (ZIF‐8) nanoplatforms are used to co‐deliver  Metal‐organic‐framework nanomedicines encapsulating an inhibitor of monocarboxylate transporters 1 and glucose oxidase are developed for efficient glucose depletion while inhibiting the lactate influx. At meanwhile, inhibiting the lactate influx can improve the intracellular oxygen pressure, which subsequently promotes the catalytic activity of glucose oxidase. Cancer cells are substantially killed from ATP shortage and high ROS levels. The overproduced H2O2 will induce systemic toxicity, which will enhance the cancer‐killing efficiency in addition to glucose‐blocking induced energy shortage. However, this process will consume O2 and elevate the local hypoxia in vivo. And considering the inherent hypoxic conditions in the solid tumor, the efficiency of GOx‐mediated cancer starvation therapy was restrained by the insufficient O2 supply. Therefore, many efforts have been made to improve the efficacy of GOx‐mediated starvation efficacy. For example, catalysts such as MnO2 and catalase were used to convert H2O2 to H2O and O2 to improve the local oxygen pressure, which could subsequently promote the catalytic activity of GOx. Unfortunately, for this strategy, the improved oxygen pressure was at the price of sacrificing the toxic H2O2, which will make the treatment less effective. On the other hand, substrates such as lactate, glutamine, and aspartate may compensate for the starvation of glucose, subsequently pulling down the efficacy of starvation therapy.Cancer cells are greedy for glucose as energy to drive their rapid proliferation. Accordingly, cancer starvation therapy based on glucose‐deprivation is emerging as an effective method for suppressing tumor growth and survival.1 In m Lactate was previously considered a waste product that must be eliminated by cancer cells despite the fact that blood lactate is a prominent fuel for mitochondrial metabolism  in both normal and cancer tissues. Oxidative cancer cells can directly use lactate in the tumor microenvironment (TME) to support the aggressive development of tumors and preserve cellular survival under glucose shortage. Some recent findings revealed that lactate would support cancer angiogenesis and metastasis, acts as a catalyst to activate mutated genes into cancer, and interferes with the immune response to cancer. The essential roles of lactate, such as the major signaling molecule and the regulator of cancer development, have been realized gradually and, very recently, applied in nanomedicine design. For instance, direct lactate removal using lactate oxidase was used to regulate the lactate abundance in TME, which subsequently generated toxic H2O2 to kill cells. Also, approaches targeting the dominating enzyme of pyruvate/lactate shuttle, lactate dehydrogenase, were used to inhibit the lactate generation in TME. Monocarboxylate transporters (MCTs) are the guards of lactate flux between cells and the microenvironments. MCT1 inhibitory strategies, including using inhibitors and gene regulations, were reported to interfere with lactate‐fueled respiration in mitochondria. Blocking the lactate influx of cancer cells could improve the local oxygen pressure, which subsequently improves the therapeutic efficacy of oxygen‐consuming therapeutics, including photodynamic therapy and radiotherapy.The glucose consumed by cancer cells further metabolizes predominantly through glycolysis, thus producing high levels of lactate even in oxygen‐rich conditions (Warburg Effect).9 Lactα‐cyano‐4‐hydroxycinnamate (CHC). To co‐deliver CHC and GOx, the porous and biocompatible zeolitic imidazolate framework‐8 (ZIF‐8) was selected as the carrying‐nanoplatform, which has been widely applied in the delivery of biomacromolecules, such as protein and DNA, as well as small‐molecular drugs. Moreover, ZIF‐8 can sensitively decompose and release the cargos under weak acidic environments, which will promise the targeting delivery of drugs toward cancers. Benefiting from the strong interaction between Zn2+ and carboxyl groups in CHC and GOx, we successfully realized the in situ co‐loading of CHC and GOx during the crystallization process of ZIF‐8 to obtain the final product, CHC/GOx@ZIF. In this structure, both GOx and CHC were entrapped in the crystal structures of ZIF‐8 to rescue from the burst release of the cargos to normal tissues. Moreover, we demonstrated that N,N‐dimethylformamide (DMF) played important roles in CHC/GOx@ZIF‐8 synthesis, including weak basic property, structure‐directing, and excellent dissolving ability toward CHC and Zn2+. The as‐obtained monodisperse and uniform nanomedicines will ensure reliable and steady therapeutic properties.Besides these essential roles, lactate is also the major energy source for the cell in addition to glucose; therefore, the influence of lactate blocking on GOx mediated cancer starvation therapy is attractive to be explored. Herein, a new strategy of starvation therapy by dual‐blocking the main energy sources, glucose and lactate, simultaneously, has been designed, in which glucose will be depleted by GOx catalysis, and the cellular influx of exogenous lactate is blocked by MCT1 inhibitor, s protein18 and2O2 production  were cultured in the media supplied with different concentrations of lactate but free of glucose, glutamine, and pyruvate  and lactate (5 mm) were exogenously added to the cell culture media under GOx@ZIF‐8 treatment  by water, and crystals were formed with the injection of Znd Figure . The aci  CHC e Figure  and stru2.3Figure −1, corresponding to the cyano groups stretching vibration in CHC and the vibration of peptide‐bond in GOx were observed in CHC/GOx@ZIF‐8. Powder X‐ray diffraction (PXRD) analysis disclosed that the incorporation of GOx and CHC would not change the crystallinity of ZIF‐8 , as CHC was filled in the pores due to its small size. Differently, CHC/GOx@ZIF‐8 showed comparable BET surface and pore volume (1345.0 m2 g−1 and 0.80 cm3 g−1) to CHC@ZIF‐8, which indicated that GOx, a biomacromolecule, was directly encapsulated into the crystal of ZIF‐8. Further characterized by TGA under N2 atmosphere, all the three samples showed good thermal stabilities with small weight loss (<10%) up to 500 °C   and lactate (10 mm) were analyzed by DCFH‐DA. According to the LSCM analysis  in tumor tissues  the starvation effect by depriving of both lactate and glucose and 2) cytotoxicity induced by elevated ROS levels. Finally, the main organs of mice treated with PBS and CHC/GOx@ZIF‐8 were subjected to H&E staining analysis 2∙6H2O dissolved by 0.4 g of water was quickly injected into the 2‐MIM solution under vigorous stirring at room temperature. Ten minutes later, the formed ZIF‐8 crystals were separated from the reagents by centrifugation at a speed of 13 200 rpm for 10 min, washed with water twice, and re‐dispersed in water.1.135 g of 2‐MIM was first dissolved by 4.0 g of water, then 58.5 mg of Zn(NO3)2∙6H2O dissolved by 0.4 g of water was quickly injected into the 2‐MIM solution under vigorous stirring at room temperature. After another stirring for 10 min, the formed crystals were separated by centrifugation , washed twice with water, and finally redispersed in water.1.135 g of 2‐MIM was first dissolved by 4.0 g of water, then 6 mg of GOx was added into the 2‐MIM solution. After stirring for 5 min, 58.5 mg of Zn(NOm) in DMEM cell culture media. Cell proliferation and lactate concentrations in the supernatants were measured by CCK‐8, LA assay kit according to the manufacturer's instructions respectively. To test the lactate‐mediated cellular survival while glucose was depleted by GOx@ZIF‐8, different concentrations of GOx@ZIF‐8 were dispersed in the DMEM media supplied with glucose (10 mm) or media supplied with glucose (10 mm) and lactate (5 mm) and incubated with the cells for different periods. Then, cell proliferation was measured by CCK‐8 assay.Cells were seeded at a density of 10 000 cells per well and incubated 24 h before the tests. To characterize the lactate mediated cell survival without other nutrition supply and incubated with different concentrations of lactate 2∙6H2O dissolved by 0.4 g of water was quickly injected into the above solution under vigorous stirring at room temperature. After another stirring for 10 min, the formed crystals were separated by centrifugation , washed with ethanol twice, washed with water twice, and finally redispersed in water.1.784 g of 2‐MIM was first dissolved by 4.0 g of water, then 16 mg of CHC and 720.8 mg of DMF were added. After stirring for 5 min, 58.5 mg of Zn(NO3)2∙6H2O dissolved by 0.4 g of water was quickly injected into the above solution under vigorous stirring at room temperature. After another stirring for 10 min, the formed crystals were separated by centrifugation , washed with 10% ethanol v/v twice, washed with water twice, and finally redispersed in water.1.784 g of 2‐MIM was first dissolved by 4.0 g of water, then 16 mg of CHC, 720.8 mg of DMF, and 6 mg of GOx were added. After stirring for 5 min, 58.5 mg of Znwt%Ws represents the weight of GOx determined by the SDS‐PAGE analysis, and W represents the weight of the final product.The loading capacity of GOx in ZIF‐8 was calculated according to the SDS‐PAGE analysis. The final volume of the synthesized products was adjusted to 4 mL, and 10 µL of the final product was subjected to SDS‐PAGE analysis. The intensity of the GOx band was quantified by using the Image J 2X software, and the amount of GOx was calculated according to the calibration curve of GOx based on SDS‐PAGE. The loading capacity of GOx in GOx@ZIF‐8 or CHC/GOx@ZIF‐8 was calculated according to Equation .−1 of CHC/GOx@ZIF‐8 was dispersed in PBS with different pH values  and incubated at 37 °C. The supernatants were collected by centrifugation every one hour by centrifugation. The fluorescence intensities of AF‐647 were measured to monitor the released GOx. To test the catalytic activity of GOx in CHC/GOx@ZIF‐8, the synthesized CHC/GOx@ZIF‐8 was dispersed in PBS (pH = 5.2) and incubated at 37 °C for 6 h. The amount of GOx was quantified by measuring the fluorescence of AF 647. Then GOx (0.2 µg mL−1), HRP (0.1 µg mL−1), TMB (200 nm), and glucose (2 mm) were mixed in PBS (pH = 5.2) and incubated at 37 °C. The generation of OxTMB was monitored by measuring the absorbance at 649 nm.AF‐647 labeled GOx was used to synthesis CHC/GOx@ZIF‐8. To test the GOx release under acidic conditions, 50 µg mL−1) of CHC@ZIF‐8 and corresponding CHC were incubated with cells for different periods in MEM media containing 10 mm of lactate. Then, cell proliferation and lactate concentrations in the supernatants were measured by the CCK‐8 assay and LA assay, respectively. To test the oxygen consumption rate, cells were first incubated with 20 µg mL−1 of CHC@ZIF‐8 and the corresponding amount of CHC for 12 h. Then the cells were subjected to the OCR assay according to the manufacturer's instructions.Cells were seeded at a density of 10 000 cells per well in 96‐well plates and incubated 24 h before the tests. Different concentrations  of ZIF‐8, CHC@ZIF‐8, GOx@ZIF‐8, and CHC/GOx@ZIF‐8 were incubated with cells in the DMEM cell culture medium with 10 mm of glucose and 10 mm of lactate. Then, cell proliferation was measured every 24 h by CCK‐8 assay for the period of 96 h.Cells were seeded at a density of 10 000 cells per well in 96‐well plates and incubated 24 h before the tests. Different concentrations  and lactate (10 mm) for 12 h. Then cells were washed with PBS and stained with DCFH‐DA (1 µm) in PBS for 30 min. For LSCM analysis, the fluorescence of DCF was excited by 488 nm laser, and emission was collected from 510 to 550 nm. For FCM analysis, cells were dissociated by trypsin and dispersed in PBS. The fluorescence of DCF was collected from 10 000 cells and analyzed by the FloJo‐V10.Cells were seeded at a density of 10 000 cells per well in a 96‐well plate for LSCM analysis and 200 000 cells per well in a 6‐well plate for FCM analysis. Cells were incubated at 37 °C for 24 h to allow complete adherence. Then 20 µg mL−1 of CHC@ZIF‐8, GOx@ZIF‐8, and CHC/GOx@ZIF‐8 were incubated with cells in DMEM media supplied with glucose (10 mm) and lactate (10 mm) for 12 h. Then cells were dissociated and co‐stained by Annexin V‐FITC and PI according to the manufacturer's instructions. Then fluorescence of FITC and PI were collected from 10 000 cells and analyzed.Cells were seeded at density of 200 000 cells per well in 6‐well plate and incubated for 24 h. Then 20 µg mL−1 of CHC@ZIF‐8, GOx@ZIF‐8, and CHC/GOx@ZIF‐8 were incubated with cells in DMEM media supplied with glucose (10 mm) and lactate (10 mm) for 12 h. Then cells were stained with CAM and PI according to the manufacturer's instructions. CAM and PI were excited by the 488 and 552 nm laser, respectively. Emission of CAM and PI were collected at 500—540 nm and 615—645 nm.Cells were seeded at a density of 10 000 cells per well in a 96‐well plate and incubated for 24 h. Then 20 µg mL6 cells were injected into the posterior flank regions of the mice. The tumors were measured by a vernier caliper and calculated according to the formula: Volume = (Length × Width × Width)/2.All the animal experiments were conducted according to the Institutional Animal Use and Care Regulations (protocol no. SUSTC‐JY2019068) approved by the Laboratory Animal Ethics Committee of SUSTech. All the animals were acclimated and tested for infectious diseases for one week since they were received. To fabricate the subcutaneous tumor, 3 × 103, the mice were randomly divided into 4 groups. PBS, CHC@ZIF‐8, GOx@ZIF‐8, and CHC/GOx@ZIF‐8 were intratumorally injected into the tumors. Histological analysis of the tumor tissues was conducted 24 h after the injection of drugs. The tumor size and bodyweight of the mice were recorded every two days after the injection. 16 days after the drug administration, the mice were sacrificed, and major organs  were used for the tissue slides and histopathological assessment. The isolated tumors were weighted and imaged.When the tumor size reached 200 mmTo measure the intra tumoral lactate level, tumors were isolated and ground 24 h after the drug administration. Then, the lactate concentration was measured by using the LA assay kit. Histopathological assessment, TUNEL analysis, and immunofluorescence analysis were conducted according to previous studies.27p‐values were calculated according to the analysis of variance (ANOVA) with Bonferroni correction by using the SPSS 19.0 software, *p < 0.05, **p < 0.01, ***p < 0.001. Diameters of nanoparticles were calculated using the Nano Measure 2.0 software. Fluorescence intensity analysis of the images was conducted using the Image J 2X software. The data of FCM was analyzed by FloJo‐V10.The cell proliferation (%) was calculated by normalizing average OD450 of the control group to 100%. All the values are presented as mean ± SD, and the data were obtained based on three independent tests. The authors declare no conflict of interest.Supporting InformationClick here for additional data file.