In spite of intensively conducted research allowing for the development of more and more advanced wound dressing materials, there is still a need for dressings that stimulate not only reparative and regenerative processes, but also have a positive effect on infected and/or difficult-to-heal wounds. Porous dressing materials based on butyric-acetic chitin co-polyester containing 90% of butyryl and 10% of acetyl groups (BAC 90/10) can also be included in the group mentioned above. Two types of dressings were obtained by the salt leaching method, i.e. a porous sponge Medisorb R and Medisorb Ag with an antibacterial additive. The aim of the study was to evaluate biological effects of porous Medisorb R and Medisorb Ag dressings under in vitro and in vivo conditions. In an in vitro biodegradation test, no mass loss of Medisorb R dressing was observed within 14 days of incubation in physiological fluids at 37 °C. However, on the basis of the FTIR (Fourier Transform Infrared Spectroscopy) tests, surface degradation of Medisorb R dressing was observed. Additionally, the antibacterial activity of the porous Medisorb Ag dressing containing microsilver as an antibacterial additive was confirmed. The in vivo studies included inflammatory activity, skin irritation and sensitisation tests, as well an assessment of local effect after contact with subcutaneous tissue up to 6 months and skin wounds up to 21 days. In the in vivo tests, the dressings exhibited neither effects of skin irritation nor sensitisation. Under macroscopic examination, in full thickness defects of subcutaneous tissue and skin, the dressings caused wound healing with no inflammation, undergoing the most gradual biodegradation between weeks 4 and 8, and the observed differences were statistically significant. In the histological assessment, a weakened, limited inflammatory process associated with degradation of the material has been observed. The process of skin wound healing under Medisorb R dressing in the early period was accelerated compared to that observed in the control group with a gauze dressing. Wound healing is a spontaneous process that can be hampered in the case of large, long-lasting and difficult-to-treat chronic wounds. In such cases, if the autologous skin transplants are not available, biopolymers are used, which show the ability to initiate and stimulate the appropriate process of skin and epidermis healing . Skin woThe aim of the presented research was the biological evaluation of the effect of Medisorb R and Medisorb Ag dressings in vitro and in vivo. As part of the in vitro study, it was planned to check the degradation of the Medisorb R dressing under the treatment with plasma/serum enzymes and bacteriostatic/bactericidal activity of the Medisorb Ag dressing. Safety and effectiveness of Medisorb R and Medisorb Ag dressings was planned to be checked in vivo in studies involving: the tests concerned toxic and irritation activity after multiple application on the skin, allergic activity, healing process in the subcutaneous tissue in the period of up to 6 months and dressing impact on the healing process of the full thickness skin loss in the period of 21 days.Medisorb R and Medisorb Ag dressings ,43 manuf3 of water and then sonication of the blend was carried out using the ultrasound probe with 150 W power, frequency 40 kHz for 5–10 min, using a 2 cm diameter probe which was immersed in the solution 20 cm deep. Water excess was removed from the dressing in the drying process at 40 °C (at reduced pressure). The dressing materials, prepared in such a manner, were packed and submitted to the radiation sterilisation process in which the radiation dose was 25 kGy. The sterilisation process was validated according to the PN-EN ISO 11137-2 norm. It enabled obtaining materials which were characterised by high porosity up to 95–99% and chemical purity confirmed using organoleptic analysis, pH tests, permanganate oxidisability, absorbance in the ultraviolet radiation and the accurate conductivity of water extracts.Porous dressing enriched by the bactericide factor: The manufacturing process was analogical to the one described above with an additional phase of incorporation of the metallic silver on the surface in the form of micromolecules (microsilver) with bactericide characteristics confirmed. Silver was spread on the dressing material through spraying the silver suspension on the surface using a spray nozzle. The silver suspension was prepared by adding the metallic silver in powdered form of 1 g on 1 dm® rotator , CLW 53 STD  incubator, Christ/Alpha 2-4 LSC freeze-drier , and the following materials: sterile centrifuge tubes 15 mL (Axygen/28612055); sterile test tubes 1.5 mL (SARSTEDT/0000/2146101); test tubes for serum isolation (BD Vacutainer®/3119102) purchased by Sigma-Aldrich . The tests concerned the assessment of mass loss and possible products of decomposition of Medisorb R in plasma. Nine Medisorb R dressings of 10 cm diameter each were used. The dressing materials were divided into portions of the average mass of 20–60 mg. Next, the samples were placed in test tubes and were subjected to selected physiological fluid activity or water (control) and incubated with constant stirring (3 turns (360°)/min) for a given time at a temperature of 37 °C. After the elapsed time, the dressing samples were washed with water /min). After water removal, the samples were dried in a freeze-drier  and the mass was measured. The average from three independent tests constituted the result. During the tests, the following devices were used: Metler Toledo  XS105DU/M model analytical balance, SB3/StuartChemical composition of Medisorb materials were proved using IR spectra tests. After each time of treatment, Medisorb samples were recorded using FTIR spectrometer System 2000 Perkin Elmer Cracow, Poland in form of thin porous films using an ATR  adapter.Staphylococcus aureus ATCC 6538 and Klebsiella pneumoniae ATCC 4352. For each of the species, a suspension with a density from 1.0 × 105 to 3.0 × 105 CFU/mL was prepared. Tests with the use of each bacterial strain were conducted separately for each material. The test samples were treated with a bacterial suspension, where a 0.2 mL suspension was used for each sample. The suspension was pipetted on the sample on a couple of places. Directly after the application of bacterial suspension, 20 mL of neutraliser was added to the samples of control material and samples of Medisorb Ag. Six samples (three control samples of Medisorb R and tested samples of Medisorb Ag) were placed on a TSA  surface in dilution from 100 to 10−4 in order to establish the starting bacteria quantity per sample. Six further samples (three control ones of Medisorb R and three test ones of Medisorb Ag) were incubated at a temperature of 37 ± 2 °C for 24 h. Twenty millilitres of neutraliser was added to the samples after incubation. Then, the samples were placed on a TSA surface in dilution from 100 to 10−4 in order to establish the final bacterial quantity.For tests, two types of dressings were used: Medisorb R (control) and Medisorb Ag, which in the final stage of production, was modified with microsilver in order to achieve the antibacterial activity. Tests on antibacterial activity were performed in compliance with the PN-EN ISO 20743:2013 norm. Six control samples and six samples with an antibacterial additive were used. The tested samples were placed in a separate sterile container. The samples were previously subjected to radiation sterilisation, so additional sterilisation was not necessary. The following bacterial strains were used in accordance with the PN-EN ISO 20743:20163 norm: • Skin irritation (repeated exposure)For the test, three New Zealand White rabbits weighing 3.4–3.6 kg were used. The entire back and the flanks of the rabbits were depilated mechanically and cleaned with 70% ethanol. On the next day, two Medisorb R dressing samples with the dimensions of 2.5 × 2.5 cm, moistened with water to improve the contact with the skin, were applied to both flanks of each animal. The corresponding absorbent sterile gauze samples were used as the negative control. The samples were fixed using an occlusive compression dressing and remained in contact with the skin surface for 6 h. The same procedure was repeated over five consecutive days. After the last exposure, the skin reactions were evaluated at 1 h, 24 h, 48 h and 72 h based on the macroscopic assessment of erythema, eschar and oedema using a 0–4 grading scale.• Skin sensitisationThe testing procedure for skin sensitisation using the Buehler method was carried out on male albino guinea pigs. Prior to starting the procedure, the back and flanks of the animals were depilated mechanically. Then, the Medisorb R dressing samples of 2.5 cm × 2.5 cm, moistened with water, were applied to the left flanks of ten animals, covered with an occlusive dressing and secured using elastic bandage wrapped around the trunks of the animals. The negative control samples of sterile absorbent gauze saturated with water were applied to the skin of five additional animals. Both the test samples and control samples remained in contact with the skin surface for over 6 h. This procedure was repeated for 3 days a week, for three consecutive weeks (induction phase). After a break of 14 days, the flanks of all animals were shaved and the samples were applied to the contralateral untreated flank of each animal . As in the induction phase, the samples were held in contact with the skin for 6 h under occlusive dressing. After 24 h and 48 h from the removal of the challenge samples, the skin reactions were evaluated based on the appearance of skin sensitisation symptoms, such as erythema or oedema.• Test on the healing process in the subcutaneous tissue up to 6 months in the presence of Medisorb R dressingMedisorb R sterile dressings of size 1.5 × 1.5 cm were used for the implantation research. The local effect of subcutaneous tissue was checked at week 1, 2, 4, 8, 16, 20 and 28 after dressing implantation in the subcutaneous tissue in the abdominal cavity of the rats. Tests at all examination points were made in the same manner.Animal pre-operation stage: The research was done on 35 rats of the same inbreeding, Wistar, female, approximately 4–6 months old and 200 g in weight. The rats were kept under laboratory conditions and were given standard LSM (Lab Service Manager) feed and water ad libitum. The abdominal skin of the rats was depilated mechanically the day before the operation, and the size of the depilated surface was 7 × 5 cm. The animals were not fed 24 h before the treatment.Operation: The process of dressing implantation was done under aseptic conditions. Rats were under the influence of general intraperitoneal anaesthetisation of Ketamine  at a 25 mg/kg dose and Xylazine  at a 5 mg/kg dose. The abdominal operational field was disinfected twice with SkinSept . The 4 cm incision was made along the linea alba. After exposition of subcutaneous tissues and fascia of the abdominal wall, the pocket preparation was made in the subcutaneous tissue. Four pockets were made in each animal, two pockets on each side of the linea alba. Four implants  in 10% water formaldehyde solution  in a phosphate buffer. After preliminary formalin maintenance, the tissue excess was removed from the samples. Next, the samples were dehydrated in acetone (at 56 °C), overexposed in xylene at room temperature and were put in paraffin blocks. The scraps of 4 µm width were cut on a rotation microtome Leica 2025 . Prepared samples were coloured with haematoxylin and eosin (HE) using the Van Gieson method (VG) differentiating the connective tissue stroma, and then they were put in medium for submersion . Histopathological samples were assessed under an optical microscope  using various magnifications. Histopathological images were documented with the use of a software for picture analysis and acquisition . The inflammatory cells , necrosis presence, connective tissue growth, fibrous degeneration and fatty streak were taken into consideration in the tissue assessment.• Test on healing process in full thickness skin loss in the period of 21 days in the presence of Medisorb dressingMedisorb R dressings (size 2.5 × 2.5 cm) were used to evaluate their influence on the wound healing process. The gauze dressings of the same size were used as a control material. The research was run on New Zealand rabbits of both sexes and approximate weight of 2.7 kg (±200 g), which came from inbreeding at the Nofer Institute of Occupational Medicine in Lodz. Experimental animals were brought to the lab 1 month before the tests. The animals were kept in single cages and under controlled conditions regarding humidity (28–37%) and temperature (16–20 °C). The animals had unlimited access to water and were fed with standard granulated feed for rabbits . The average use of feed per rabbit was 50–70 g. The test was run on 18 rabbits, divided into groups of three rabbits for each planned research date.Animal preparation for operation: Rabbits were on a starvation diet 24 h before the planned procedure, though they had water access.Operation: The rabbits were anaesthetised via intramuscular injection of Xylazine  at a dose of 0.5 mg/kg and Ketamine  at a dose of 5 mg/kg. The condition of full analgesia was obtained 10–15 min after addition of pharmaceuticals and lasted for 60–80 min. The full recovery lasted 120–140 min. After total analgesia was reached, the skin on both sides of the spine was disinfected with SkinSept Pur or SkinSept Color . Afterwards, four operational, oval-shaped, full thickness skin wounds reaching fascia were made with a diameter of 2 cm. Two wounds on the right side of the spine were treated with Medisorb R, and two wounds on the left side were treated with the sterile control gauze dressing ; the maximum dose was up to 80 mg/kg. The drug was given in fraction doses till the breathing and heartbeat stopped. Before the specimen was given, the general health condition of the rabbits was evaluated. During autopsy, the macroscopic wound image, presence and dressing was assessed. Skin fragments were taken for histopathological tests. The assessment of selected organs was also made.Microscopic tests: Skin fragments for microscopic examination were prepared in the same way as described above.Statistics: Data is presented as average. Also, the standard deviation of average quantities from measurements was determined on samples taken from n = 35 animals. The data was analysed with the ANOVA statistical test. Differences between averages were notified as significant with α = 0.05.frozen plasma (for checking the influence of freezing/de-freezing process and impact of EDTA for enzyme activity)fresh plasma (sole effect of the presence of EDTA for enzyme activity)fresh serum with full enzymatic activity.While planning the tests and selection of physiological fluids, the following factors were under consideration: (a) influence of the plasma freezing on enzyme activity, and (b) method of blood collection for plasma isolation (with the use of EDTA). Assuming that the dissolution of Medisorb R should be correlated with enzymatic activity, the following physiological fluids were used:Test results are presented in During the tests, it was found that even after 14 days of the treatment of Medisorb R dressing with all tested physiological fluids, no weight loss was observed. This can be connected with the fact that during incubation at a temperature of 37 °C, the enzymatic activity of the physiological fluid components was significantly reduced with time . The observed changes in the sample mass could be the result of plasma and/or serum protein absorption on the dressing surface. This finding is consistent with the fact that the observed changes of the mass loss of Medisorb R were not dependent on the incubation time.−1 in relation to the amide I signal at 1659 cm−1 was observed. It confirmed the sample surface degradation, which was connected to the hydrolysis of ester BAC 90/10 groups. The signals visible at 1445 cm−1 and 1002 cm−1 were connected with absorbed protein on the surface of dressing. An intense band at 667 cm−1 probably stemmed from the vibration of binding of phosphate derivatives.FTIR ATR analysis was made for samples of untreated Medisorb R dressings and material treated with fresh human plasma in order to test the dressing surface degradation and protein absorption on the dressing surface . CompariThe studies of skin irritation and sensitisation were conducted in accordance with the European Standard EN ISO 10993-10. Skin irritation in the form of erythema/eschar and oedema formation was scored according to the guideline-based scoring system depicted below .The scores were summed up and averaged independently for the test and control samples. The severity of the irritant reaction was calculated by subtracting the average score for the control from the average score for the test sample. The obtained value was the cumulative irritation index, allowing for classification of the skin irritation hazard .All animals appeared clinically normal throughout the study. There were no perceptible adverse local reactions following application of the tested product to the occluded rabbit skin. The calculated cumulative irritation index was “0”. Thus, the dressing can be classified as non-irritant. Since rabbit skin is more sensitive to irritant agents than human skin, the risk of local irritation from the Medisorb R dressing in humans should be regarded negligible.Skin sensitisation reactions were graded according to the Magnusson and Klingman scale . Grade 1Daily observation of the animals did not reveal any adverse clinical signs. There were no significant changes in body weight gains between the control and treatment group. Since after the challenge sample application, none of the animals presented erythematous or swelling responses, grade 0 was given to both animal groups. Based on this finding, it may be concluded that the Medisorb R dressing exhibited no sensitising potential in animal testing. Therefore, the risk of allergic reaction in humans can be considered negligible.All the animals survived till the moment of scheduled autopsy. During observation, all the animals had appropriate weight gain and appetite. Operational wounds healed through primary adhesion. During autopsy, no pathological changes in the peritoneum or organs were observed. The macroscopic evaluation was made at weeks 1, 2, 4, 8, 16, 20, 24 and 28 after Medisorb R dressing implantation in the subcutaneous tissue .At week 1 after the implantation of the Medisorb R dressing on the surface of the skin in the abdominal area, the unhealed post-operative wounds were visible. In the place of subcutaneous dressing placement, the skin bulge of regular colour and look was visible. After the incision, the implants were found to be connected with the subcutaneous tissue structures. The tissues surrounding the implant were soft and had a regular look. The implant was covered with a tissue capsule with red hyperpigmentation through which the dressing was visible. After two weeks of the implantation, the implanted dressings were found to be covered with a tissue capsule connected to fascia and subcutaneous tissue. After four weeks of implantation, it was found that the tissue capsule surrounding the implants was covered slightly with blood vessels. After 8 weeks, it was observed that the implant was covered with a tissue capsule with blood vessels and was more flattened and more connected to the surrounding tissues, compared to the previous research term. Macroscopic images at weeks 12, 16, 20, 24 and 28 after implantation was similar. The skin surface, at the site of Medisorb R implantation, had a regular colour. The place of implant engraftment was either slightly visible or remained invisible. The implanted dressings were surrounded with a tissue capsule with thin blood vessels. They were connected to the subcutaneous tissue tightly and had a regular look. Over the course of time, the implants flattened and became smaller. They remained visible in the macroscopic images at week 28 after implantation.In the microscopic image, in the first week after dressing implantation, the irregular spaces were observed in the implant material. It was the result of the histopathological process of rinsing. Moreover, the fragments with loose connective tissue were observed, in which the presence of granulocytes, macrophages and a small amount of lymphocytes and fibroblasts was stated. In the direct implant neighbourhood, multinucleated macrophages containing elements of material were present .After two weeks, the single irregular fibres and all implants were visible, and were surrounded with the band of connective tissue which separated them from subcutaneous tissue with a regular shape. The connective tissue had a fibrous shape from the side of the lateral muscles of the abdominal wall and connective tissue of the skin and loose structure rich in cells between the dressing fibres . The average width of the implant up to 4 weeks after implantation differed from the data observed from 8 to 28 weeks. The observed differences were statistically significant.In order to evaluate the influence of Medisorb dressing on the wound healing process, comparative macroscopic and histopathological tests were made on 3rd, 6th, 10th, 14th, 18th and 21st day after the operation in relation to wounds treated with gauze dressings . The resOn the 10th day of observation, the wounds under the Medisorb R dressing were covered with a dry scab and was a bit wider and harder compared to the control group. The edges of the wound were harder and shrunk, compared to wounds treated with the gauze dressing . In the All wounds were shrunk and covered with the scab. In the wounds with no scab, a white scar was observed . After 1Along with macroscopic tests, the microscopic  tests of wounds treated with Medisorb R and gauze dressing as control dressing were run. In the microscopic tests, on the third day after operation, the wounds were covered with dressing and thin layer of homogenous mass, which were the image of a scab. Directly beneath them, in the centre of the wound, was the effusion of the fibre–cell with the advantage of eosinophilia. Short fragments of stratified epithelium were observed by the wound edges a. In theOn the sixth day after operation, in the case of wounds supplied with Medisorb R, the histopathological tests showed an in-growth of newly-formed epithelium that covered the fresh connective tissue under a homogenous mass and the remnants of fibre–cell effusion. Multiple fibroblasts and collagen fibres were observed under the newly-formed epithelium. The centre of the wound, which was not covered with epithelium, was filled with rich-in-cells connective tissue with blood vessels of granulation tissue character, in which many fibroblasts and eosinophils and less neutrophilia and lymphocytes were present b. HoweveIn controlled tests after 10 days from the operation, the loss was covered with a homogenous mass (scab). The epidermis reached the wound edge, between the connective tissue rich in cells and the homogenous mass. The epithelial surface was visible on a bigger surface in comparison to the previous research terms. In the wound centre, no fibre–cell effusion and granulation tissue were observed. At the bottom of the wound, the formation of connective tissue with collagen fibres was observed a.On the 14th day, the wounds covered with Medisorb R showed the multiplication of fibrous connective tissue that contained numerous fibroblasts and collagen fibre, which almost completely covered the wide space of keratosis of the epithelium. In the centre of the wound, the epithelium was thinner. Some space of the wound was not covered with epithelium b. In conTwenty-one days after operation, the skin wounds were treated with Medisorb R dressing, and in most animals, the wounds were covered with a layer of epithelium, which was wider at the edges and thinner at the centre. Under the epithelium, connective tissue with collagen fibre was found, and less numerous fibrocytes were growing. In part of the wound, there was a homogenous mass with the remnants of effusion. In the case of the wounds that were treated with the control gauze dressing, on the 21st day after operation, the wound was filled with connective tissue, which was almost completely covered with epithelium with keratosis in which glands and skin elements were built. Newly-grown epithelium was wide at the edges and narrow down to the centre. In part of the preparation, the epithelium was covered with homogenous mass (scab), which was disconnected by migrating layer of epithelium. Under the newly-grown epithelium, the presence of connective tissue rich in cells was observed and contained eosinophils and less numerous lymphoid cells. In the histological examination, changes in the wound healing process using Medisorb R dressing and control one were stated. In the early stage, Medisorb R accelerated the epithelisation process, evoked less intense inflammatory effect with less intense induction of granulocytes and lowered the effusion phase and accelerated the creation phase.5 to 3 × 105; (b) the difference for three controlled samples, direct after inoculation was ≤1; (c) the value of growth F for controlled device was ≥1. Meeting the requirements allowed establishing conditions for marking the A value for Medisorb Ag  inoculum concentration was from 1 × 10isorb Ag .S. aureus, which indicated that the material had significant antibacterial activity. For K. pneumoniae, the value was A = 4.63, which indicated the strong antibacterial activity of the device.The result of antibacterial activity for Medisorb Ag was established at A = 2.83 for Medisorb Ag was also submitted to in vivo tests, alike Medisorb R. It was confirmed that the dressing that contained silver did not show any cytotoxic allergic reaction. The dressing in the subcutaneous tissue underwent degradation and cramped. However, the small residue of dressing was found till the end of the observation phase (up to 28 weeks). The process of wound healing with the use of dressing evoked slight inflammation, which was connected to material degradation and led to filling the implant with connective tissues rich in cells. The dressing adhered to the wound and allowed for its edges to integrate with the bottom of the wound. The effusive phase was shortened and the creation phase was accelerated. The epithelisation phase under the dressing was shorter in resemblance to the control sample. On the basis of the tests it was stated that it was characterised by high biocompatibility.The problem of difficult-to-heal wounds is trying to be solved with the use of target therapies or with the use of innovative dressings which show bioactivity features . UnfortuThe conducted research proves that porous dressing materials Medisorb R and Medisorb Ag are safe materials used in wound healing process. Medisorb R undergoes slow degradation in human plasma, which was confirmed using FTIR tests, although in tests based on material mass determination in physiological fluids, no mass loss was observed. In animal studies, the dressing caused no skin irritation or sensitisation, so the risk of adverse local reactions in humans can be considered clinically insignificant.In all scheduled tests (up to week 28), Medisorb R dressing covered with thin bag was visible in macroscopic image in unchanged subcutaneous tissue.Medisorb R dressing underwent degradation and gradual reduction in subcutaneous tissue.The healing process of Medisorb R evoked little inflammation effect, which was connected to material degradation and led to fulfilling the implant with rich-in-cell tissue of granulation feature.The process of degradation and Medisorb R dressing healing was not finished by week 28 after implantation, and in the material neighbourhood, rich-in-cell connective tissue with multinuclear macrophages was present,Medisorb R dressing was characterised by high biocompatibility.The most important conclusions derived from the research on the healing process with the use of Medisorb R in subcutaneous tissue in the period of up to 6 months are as follows:From the conducted research, due to the influence of Medisorb R on operational skin loss, it is stated that the tested material has a more positive effect on the repair process of skin loss in comparison to control material. In the early stage, it accelerated the epithelisation process, evoked less intense inflammatory effect with less effusion, and enhanced the creation phase.S. aureus, which indicates significant bacterial activity. For K. pneumoniae the value was A = 4.63, which indicates high antibacterial activity of the product. The criteria of PN-EN ISO 20743:2016 were used in the test.The antibacterial activity of material Medisorb Ag with microsilver as an antibacterial additive was determined at A = 2.83 for


The banana is one of the most important fruits in the world. Bananas undergo a rapid ripening process after harvest, resulting in a short shelf. In this study, the mechanism underlying pulp ripening of harvested bananas was investigated using integrated transcriptomic, proteomic, and metabolomic analysis. Ribonucleic acid sequencing (RNA-Seq) revealed that a great number of genes related to transcriptional regulation, signal transduction, cell wall modification, and secondary metabolism were up-regulated during pulp ripening. At the protein level, 84 proteins were differentially expressed during pulp ripening, most of which were associated with energy metabolism, oxidation-reduction, cell wall metabolism, and starch degradation. According to partial least squares discriminant analysis, 33 proteins were identified as potential markers for separating different ripening stages of the fruit. In addition to ethylene’s central role, auxin signal transduction might be involved in regulating pulp ripening. Moreover, secondary metabolism, energy metabolism, and the protein metabolic process also played an important role in pulp ripening. In all, this study provided a better understanding of pulp ripening of harvested bananas. Musa spp.) is one of the most important fruits in the world  reductase), GSMUA_Achr8T11740_001 (Aldehyde dehydrogenase family 3 member F1), and GSMUA_Achr10T12920_001 (Trans-cinnamate 4-monooxygenase) were up-regulated in the banana maturation process. Here, the up-regulation of aldehyde dehydrogenase gene suggested anaerobic respiration might play a vital role during banana pulp ripening.For oxidation-reduction process, four genes and 16 proteins were differentially expressed in the whole process of banana pulp ripening. Four genes, GSMUA_Achr7T24700_001 (peroxidase 4) and GSMUA_Achr3T16520_001 (peroxidase 43) up-regulated in stage F4, GSMUA_Achr5T25040_001 (peroxidase 55) and GSMUA_Achr6T21530_001 (peroxidase 52) up-regulated in stage F3, and GSMUA_Achr7T11330_001 (peroxidase 15) and GSMUA_Achr9T29540_001 (peroxidase 12) up-regulated in stage F2. Additionally, GSMUA_Achr5T20330_001 (peroxidase5) and GSMUA_Achr6T14620_001 (peroxidase 52) were both up-regulated in F2 and F3. On the other hand, GSMUA_Achr6T33120_001 (L-ascorbate peroxidase) was down-regulated in stages F3 and F4. Four proteins of L-ascorbate peroxidase  were identified with different expression patterns  was observed up-regulated in stage F2. Interestingly, three heat shock proteins (heat shock cognate 70 kDa protein) were up-regulated  and one glutaredoxin protein (spot F70) were up-regulated in the proteomics data. Unfortunately, only one gene of egulated .Despite the previous application of proteomics in banana ripening research , only a A comparison between differentially expressed genes and proteins showed that the genes involved in signaling pathways, especially the auxin signaling pathway, were induced in stage F2, and the expression of most genes and proteins involved in ripening metabolic pathways was triggered in stages F3 and F4. It is suggested that banana pulp ripening is the result of a series of precise and coordinated physiological and biochemical processes, and the regulation of certain metabolism process occurred in certain stage. In addition, it is obvious that the F3 stage was characterized as having the most differentially regulated genes and proteins from transcriptomic and proteomic data. The respiration rate peak appeared before F3 at day 15 , so it iERF1B gene was identified with upregulation as being involved in the ethylene signal transduction pathway. Interestingly, the protein of S-adenosylmethionine synthetase, which was involved in ethylene synthesis [serine/threonine-protein kinases were greatly up-regulated in late stage of banana pulp ripening, including one senescence-induced receptor-like serine/threonine-protein kinase (GSMUA_Achr9T05970_001)  were greatly up-regulated in the early stage of pulp maturation, while two were down-regulated in the late stage. Previous research indicated auxin response factors played important role in regulating auxin-responsive transcription in plants [Ethylene was well proved to play a crucial role in banana fruit ripening and softening ,12. Reasynthesis , was als970_001) a. Choudhng stage b. All thresearch . Recentlresearch . Specifiresearch ,33. Auxiresearch . In thisn plants . A recenn plants . MoreoveTaken together, banana pulp ripening is likely to be regulated by the auxin and ERF-mediated ethylene signal pathways. Sugar signal might also play important role in regulating banana pulp ripening. Therefore, the stimulation of genes and proteins involved in signal transduction and regulation at early ripening stage might lead to the downstream process of banana pulp ripening .Softening is an important determinant of banana ripening , which ipolygalacturonase genes, genes encoding pectinesterase/pectinesterase inhibitor, and two genes of pectate lyase were greatly up-regulated, which induced the degradation of pectin during banana ripening period  is another cell wall–modifying enzyme that can either cut and rejoin xyloglucan by xyloglucan endotransglucosylase action or hydrolyse xyloglucan by xyloglucan hydrolase action . Thereforipening a,e. Accoripening , ML almoexpansin gene family, LeExp2, is regulated by auxin [expansin-A8 and expansin-A2 were up-regulated at all stages  was up-regulated in stage F2 at the transcriptome level. PLS-DA analysis also confirmed the important role of F62 in banana fruit ripening  were up-regulated , but onlripening . Moreoveripening . This miExcessive accumulation of ROS can result in oxidative damage to macromolecules that, in turn, causes a loss of structure and function or even cell death . Wang etIn short, banana pulp ripening was accompanied by ROS stress, which caused protein oxidation, especially for proteins containing thiol. Up-regulation of peroxidases and glutaredoxins help maintain protein function by protecting proteins from oxidative stress to indirectly regulate the pulp ripening. Post-translational modification via HSPs also played an important role in protecting proteins from degradation.The proteomic and transcriptomic analysis accompanied with metabolic and ultrastructure analysis of banana pulp were reported. The analysis of the gene/protein expression changes indicated an important role for proteins and genes involved in cell wall metabolism, sugar metabolism, ROS, and energy metabolism in banana pulp softening. Moreover, besides ethylene signaling, auxin signaling was also involved in banana pulp ripening by regulating cell wall and starch metabolism. Thus, the integrated analysis enabled us to get comprehensive information on the biological events that are relevant to the regulation of banana fruit ripening.


A constitutive activation of the Wnt/β-catenin pathway is an initiating event in colon carcinogenesis. We developed colon cancer cells models that highlight the non-selectivity of previously described inhibitors of the Wnt pathway and we propose our model as a suitable screening system for inhibitors of the pathway. A constitutive activation of the Wnt/β-catenin signaling pathway is admitted as an initiating event of carcinogenesis in at least 90% of colorectal cancers . This coLuciferase-based reporter assays are widely used for studying gene expression at the transcriptional level. Here, we use such a system to set up a high-throughput screening assay for inhibitors of the Wnt/β-catenin signaling pathway by using DLD-1 cells stably transfected with a luciferase TCF reporter plasmid . The choTwo types of available Wnt inhibitors were used in order to validate the model: the tankyrase (TNKS) inhibitors XAV939 , IWR-1  and WIKIFigure 1A), IWR-1 , and WIKI4  specifically inhibited the activity of the Wnt/β-catenin signaling, with and IC50 of 0.13, 0.21 and 0.28 μM, respectively. However, a side activating effect was observed at doses higher than 1 μM as evidenced by the increase of the luciferase activity observed in the control conditions. Besides, both ICRT14  and PNU-74654  behaved as non-selective inhibitors as evidenced by the inhibition of both Wnt dependent and independent luciferase activities. In addition, PNU-74654 was poorly efficient. To further determine whether the apparent inhibitory effect of ICRT14 on the Wnt independent luciferase activity was due to a toxicity, or not, we evaluated the impact of ICRT14 on cells viability by using the MTT system in parallel with measurement of the luciferase activity. As shown in Figure 1F, ICRT14 again decreased both Wnt dependent and independent luciferase activities in a dose dependent manner but had no significant effect on cells viability.XAV939 (Supplementary Material.Luciferase and MTT assays were done as we previously described . More deWith respects to the use of inhibitors previously reported as specific, studies have concluded that biological activities were regulated by the Wnt/β-catenin pathway. In the present study, we demonstrate that the destabilizers of the TCF/β-catenin complex ICRT14 and PNU-74654 are unspecific inhibitors of the Wnt/β-catenin pathway. Therefore, to test the implication of the Wnt pathway in a biological mechanism, it seems more rationable to use at least one of the specific inhibitors confirmed here. Compared with the original reference system dedicated to test the impact of compounds on the activity of the Wnt/β-catenin signaling pathway, our method was set-up with an adequate control that lowers the number of false positives resulting from a non-specific inhibition of the luciferase enzymatic activity. For example, using our method points out ICRT14 as a non-specific inhibitor of the Wnt/β-catenin signaling pathway. Besides, true positives will have to be dose-dependent tested, and their ability to decrease the proliferation of colon cancer cells will have to be evaluated for further potential therapeutic purposes.CP, PhB, and PaB designed the study. CP, PhB, and AT drafted the manuscript. MG, AB, and PhB performed the experimental work.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. The reviewer ZD and handling Editor declared their shared affiliation.


Human scalp HFs were treated with different concentrations of DHT  for 10 days. The effects of DHT on hair shaft elongation, the proliferation of hair matrix cells, and the levels of β-catenin, GSK-3β, and phosphorylated GSK-3β (ser9) were evaluated in the cultured HFs. The effects of DHT were further investigated in C57BL/6 mice. Moreover, the growth of cultured human HFs was observed after interfering with the β-catenin pathway through inhibitors or activators in the presence or absence of DHT. We found that different concentrations of DHT had different effects on human HFs in vitro and C57BL/6 mice. At 10-6 mol/L, DHT inhibited HF growth and β-catenin/p-GSK-3β expression, whereas 10-7 mol/L DHT induced HF growth and β-catenin/p-GSK-3β expression. In addition, a β-catenin inhibitor (21H7) inhibited HF growth in vitro, while a β-catenin activator (IM12) promoted HF growth in vitro and antagonized the inhibition of HFs by high levels of DHT. These results suggest that DHT plays a pivotal role in region-specific hair growth, which may be related to the Wnt/β-catenin pathway.Dihydrotestosterone (DHT) is the most potent androgen that regulates hair cycling. Hair cycling involves cross-talk between the androgen and Wnt/β-catenin pathways. However, how DHT regulates hair follicle (HF) growth through the Wnt/β-catenin pathway has not been well investigated. This study aimed to investigate the roles of DHT in hair growth  Many hormones participate in the regulation of hair follicle (HF) growth and cycling, of which androgens are the most representative. AndrogeAs mentioned before, androgens stimulate hair growth in many areas and have paradoxical effects on human HFs. They cause males to have more hair on the face and promote pubic and axillary hair development in both sexes, while often causing balding in the same individual . Howevervia inhibition of the Wnt/β-catenin pathway in DPCs, which involves inhibiting GSK-3β activity  signed the informed consent form for participation in this study.2/95% air.Anagen VI HFs  were iso-5~10-9 mol/L, MP Biomedicals, 521-18-6, purity≥98% (HPLC), USA) for 10 days. The hair shaft length was measured every 2 days, and the mean growth rate of the hair shaft was calculated. At 10 days, partially isolated HFs cocultured in 10-6 mol/L or 10-7 mol/L DHT were analyzed for the proliferation of hair matrix cells by Ki-67 staining. Ethanol (0.1%) dissolved in serum-free Williams’ E medium served as the vehicle in the negative control group.After 24 h, the live HFs that grew 0.3–0.5 mm were cultured with DHT . All mice were administered 200 μl solution to the test area every day for 17 days, and the back skin of these mice was observed and photographed. After depilation at day 14, the mouse back skin was isolated to examine histological features by hematoxylin and eosin staining and related-proteins expression by Western blot. The individual mouse skin samples were fixed in 4% paraformaldehyde  for 24 h, cut into 3-μm sections with a pathology microtome , and stained with hematoxylin and eosin; the histological morphology of the skin was examined by light microscopy .Male 7-week-old C57BL/6 mice (n = 40) purchased from Nanjing University Model Animal Research Institute  were housed in plastic cages in a room with controlled temperature (22°C ± 1°C) and humidity (55 ± 15%). Mice were maintained on a regular pellet diet with access to fresh water. After acclimatization for 7 days, all mice were depilated with 6% sodium sulfide solution in the dorsal area (approximately 2 cm in width and 4 cm in length) before being anesthetized. Mice were randomly divided into 4 groups administered different treatments: negative control group, 40% DMSO dissolved in saline solution; experimental groups, DHT  was employed to isolate the nucleoproteins of the back skin or cell lysis buffer to lyse the total proteins; after that, the concentrations of these nucleoproteins and total proteins were measured by a micro BCA protein assay kit . Afterwards, a total of 50 μg per lane of nucleoproteins or total proteins was added into the well of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (Invitrogen), and immunoblotting was performed using an anti-β-catenin , anti-GSK3β , and anti-p-GSK3β  antibodies. Lamin B and GAPDH levels were used for the normalization of nucleoproteins or total proteins, respectively. Finally, scanned and quantified the protein bands using a ChemiDoc image analysis system (Bio-Rad Laboratories). The relative protein levels were determined by calculating the ratio of the value of the protein band of interest to that of the corresponding GAPDH or Lamin B band.2O2 for 10 min and preincubated with normal goat serum  for 30 min. Then, the paraffin sections were incubated with primary antibodies against Ki67 , β-catenin , GSK3β , p-GSK3β , or androgen receptor  at 4°C overnight. Afterwards, the sections were incubated with secondary FITC- or CY3-conjugated goat anti-mouse/rabbit antibody for 50 min. Next, the sections were stained with 10 μg/ml DAPI . These sections were observed under an inverted fluorescence microscope , and images were collected . The relative expression levels of Ki67, β-catenin, GSK3β, and p-GSK3β were analyzed by the Image pro-plus 6.0 software: first of all, the immunofluorescence image is converted into a black and white image; then the integrated optical density (IOD) of the positive region is analyzed; afterwards, calculate the area of positive region (AREA); finally, mean density is obtained as follows: Mean density = IOD/AREA.The cultured HFs were fixed in 4% paraformaldehyde (China) for 24 h and then cut into 3-μm sections. After dewaxing, the sections were soaked with 3% H-6 mol/L), IM12 (500 nM) or 10-6 mol/L DHT and 500 nM IM12. After 10 days of culture, the HFs were collected to analyze β-catenin expression using an immunofluorescence assay.21H7 is a selective inhibitor of the Wnt/β-catenin pathway that destabilizes β-catenin, while IM12 is an activator of the Wnt/β-catenin pathway that significantly increases β-catenin levels . After 2t-test. Multiple comparisons among ≥3 groups were performed using one-way ANOVA followed by the Bonferroni post hoc test. The nonparametric Mann-Whitney U test was used if data were not normally distributed. P < 0.05 was considered statistically significant.Data were statistically analyzed and graphed using GraphPad Prism 5 . All results are presented as the mean ± standard deviation. Statistically significant differences between groups were determined by Student’s -5 and 10-6 mol/L DHT inhibited HF growth, while 10-7 mol/L DHT induced HF growth. Additionally, compared with control-treated HFs, HFs had a considerably thinner HM, a more onion-shaped DP, less melanin content and a lower percentage of Ki-67-positive cells by exposing to 10-6 mol/L DHT. While HFs which was treated with 10-7 mol/L DHT showed a HM with a larger DP volume, maximal melanin content and a significantly higher number of Ki-67-positive cells.  and the dermal papilla (DP), and HF proliferative activity. As shown in in vivo. Depilation induces HFs to enter the next growth cycle synchronously in C57BL/6 mice. Depilated dorsal mouse skin is pink in the telogen phase, is gray when anagen is initiated, and then gradually darkens. As shown in -8 mol/L DHT group exhibited light gray skin at day 9 after depilation, the low-dose DHT (10-7 mol/L) group had darker skin than the other groups, and the 10-6 mol/L DHT group still exhibited pink skin at day 9. At day 12, hair regeneration was obvious in the low-dose DHT (10-7 mol/L) group compared with the control group, while the skin of mice in the high-dose DHT (10-6 mol/L) group remained pink at this time point. HE staining (-7 mol/L) group than in the other groups. These results suggest that the effects of DHT on HF growth depend on its concentration: the entry of HFs into the anagen phase in C57BL/6 mice is induced by 10-7 mol/L DHT but inhibited by 10-6 mol/L DHT.Then, we investigated the effects of DHT on HF cycling -5 and 10-6 mol/L) failed to induce the phosphorylation of GSK3β at Ser-9 and thus inhibited β-catenin translocation into the nucleus, while low concentrations of DHT (10-7 mol/L) induced the phosphorylation of GSK3β at Ser-9 and thereby promoted the nuclear translocation of β-catenin (P > 0.05). Furthermore, HFs growth was inhibited by 21H7 and induced by IM12 in a dose-dependent manner, and IM12 promoted the growth of HFs treated with 10-6 mol/L DHT . These results suggest that DHT affects HF growth through the Wnt/β-catenin pathway.The effects of DHT on β-catenin, GSK3β, and p-GSK3β (ser-9) levels in cultured human HFs and mouse HFs were analyzed using semi-quantitative fluorescence and Western blot. In human HFs, we found that high concentrations of DHT  in cultured HFs. Our results are in agreement with those of Leirós GJ et al., who found that DHT can downregulate the expression of p-GSK3β (ser9) and β-catenin in HFs from AGA patients  levels in cultured HFs, followed by β-catenin translocation into the nucleus and activation of the transcription of downstream target genes (here means activate Wnt/β-catenin pathway), which prompted faster HF growth. As shown in our study, high-dose DHT had a negative effect on HFs that was diminished by cotreatment with a β-catenin activator.in vitro, and further confirmed that the growth of hair follicles was indeed regulated by androgen and Wnt/β-catenin pathway at the organ level.Previous studies have found that androgen has different effects on hair follicles in different regions, and appropriate concentrations of androgens can promote hair growth, while excessive concentrations of androgens can inhibit hair growth . Dermal Unfortunately, our experiments have not found the cause of the regional effects of DHT on HFs. As mentioned above, the concentration of DHT explains these differences, and AR is also involved in the development of AGA, so there may be some unknown mechanisms which regulate the growth of HF. Therefore, in the coming studies, it’s necessary to elucidate the mechanism by which DHT regulates GSK3β activity, and which type of DHT target cells in the hair follicle will be, and what pathway of DHT could activate or inhibit the Wnt pathway. Until now, according to previous studies and our findings, we infer that the kinases that participate in phosphorylating GSK3β at ser-9 may be target genes of DHT.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The Institutional Review Boards of all participating centers approved the protocol used in this study, and all procedures were performed in accordance with the ethical standards established in the Declaration of Helsinki. All volunteers provided written informed consent before starting the study. The experimental studies were approved by the Institutional Animal Care and Use Committee of The Third Affiliated Hospital of Sun Yat-sen University .MW and XC designed the experiments. XC and BL wrote the manuscript. XC, YL, XT, and LH performed experiments. WD and WL analyzed the data. All authors have read and approved the final manuscript.This work was supported by the National Natural Science Foundation of China (No. 81271769 and 81872542).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.


Inflammatory reaction in the dysfunction of retinal endotheliocytes has been considered to play a vital role in diabetic retinopathy (DR). Anti-inflammatory therapy so far gains poor outcome as DR treatment. This study aims to identify a novel therapeutic target of DR from the OMICs studies of a traditional anti-DR botanical products TNTL.Hyperglycemic mice were treated with TNTL. The anti-hyperglycemic effect of TNTL was validated to confirm the biological consistency of the herbal products from batches. Improvement of DR by TNTL was examined by various assays on the retina. Next-generation transcriptome sequencing and cytokine array was used to identify the therapeutic targets. In vitro study was performed to validate the target.We observed that TNTL at its high doses possessed anti-hyperglycemic effect in murine type I diabetic model, while at its doses without reducing blood glucose, it suppressed DR incidence. TNTL restored the blood-retina barrier integrity, suppressed retinal neovascularization, and attenuated the retinal ganglion cell degeneration. Transcriptomic analysis on the retina tissue of hyperglycemic mice with or without TNTL revealed that the inflammatory retina microenvironment was significantly repressed. TNTL treatment suppressed pro-inflammatory macrophages in the retina, which resulted in the inactivation of endothelial cell migration, restoration of endothelial cell monolayer integrity, and prevention of leakage. Cytokine array analysis suggested that TNTL could significantly inhibit the secretion of MIP1γ from pro-inflammatory macrophages. Prevention of endothelial dysfunction by TNTL may be mediated by the inhibition of MIP1γ/CCR1 axis. More specifically, TNTL suppressed MIP1γ release from pro-inflammatory macrophages, which in turn inhibited the activation of CCR1-associated signaling pathways in endothelial cells.Our findings demonstrated that TNTL might be an alternative treatment to DR, and the primary source of potential drug candidates against DR targeting MIP1γ/CCR1 axis in the retinal microenvironment. As one of the characteristic microvascular complications of diabetes mellitus (DM), diabetic retinopathy (DR) is the leading cause of vision loss in diabetic patients . In partp = 0.013), CRP (p = 0.002) and VEGF (p = 0.003) were significantly higher in Type II DM patients with DR than those without DR [p < 0.001), proliferative DR (p < 0.001) and the incidence of diabetic macular edema (DME) (p < 0.001) [Inflammation has been considered as a central role player in the initiation and development of DR in DM patients. Of note, increasing clinical evidence has postulated the association between inflammation and clinical features of DR. Up-regulation of pro-inflammatory cytokines and chemokines, such as TNFα, IL6, IL1β, and MCP-1, were detected in plasma and vitreous samples of patients with DR . Serum lthout DR . A study< 0.001) . Meanwhi< 0.001) . The ear< 0.001) . Increas< 0.001) . High glIn this study, we used multiple OMICs approaches to identify the novel therapeutic target in the treatment of diabetic retinopathy by a herbal product Tang-Ning-Tong-Luo (TNTL). TNTL has been clinically used as traditional Chinese Miao medicine for centuries to treat diabetic-like symptoms of indigenous people in the mountain area. We observed that high doses of TNTL might possess hypoglycemic effects, while at non-hypoglycemic doses, TNTL was able to suppress DR incidence and progression in type I diabetic murine model. Transcriptomic analysis was then performed to identify the global change of retinal gene expression after TNTL treatment. Gene ontology and pathway analysis revealed that TNTL primarily suppressed inflammation in the retina of diabetic mice. Histological analysis showed that TNTL reduced the presence of inflammatory macrophages in the retina, and resulted in inactivation of endothelial cells. Cytokine profile analysis suggested that suppression of MIP1γ release from macrophages by TNTL may contribute to its inhibitory effects on endothelial dysfunction. Our present study suggests that TNTL may be an alternative treatment for DR, and a potential lead for the discovery of novel anti-inflammatory agents that are suitable for DR treatment.TNTL is an ethnomedical formula commonly used in traditional Chinese Miao medicine by the indigenous people in mountainous areas of southwestern China. It was composed of Plantagins Herba (Cheqiancao in Chinese), Lonicerae Flos (Shanyinghua in Chinese), Agrimoniae Herba (Xianhecao in Chinese) and Trichosanthis Radix (Tianhuafen in Chinese). Production of TNTL was performed in GMP manufacturing in Bailing Pharmaceutical Co. .2.Retinal Endothelial Cells (RECs) were purchased from Sciencell. RECs were cultured in Endothelial Cell Medium  with 5% fetal bovine serum, 1% endothelial cell growth supplement and 1% penicillin/streptomycin solution at 37 °C incubator with 5% COProtocols of animal study were reviewed and approved by the Committee on the Use of Live Animals in Teaching and Research of the University of Hong Kong. Streptozotocin (STZ)-induced hyperglycemic mice were utilized as type I diabetic-like model associated with retinopathy. The 10-week C57/BL/J male mice received 5 constitutive intraperitoneal injections of 50 mg/kg STZ in a citric buffer (pH 4.5). Five days after the last injection, 4 h-fasting blood glucose (FBG) was determined, and only FBG within 15.0 to 20.0 nmol/L were included. Mice only injecting citric buffer was used as normal control. Hyperglycemic mice were randomized into 5 groups: model, insulin treatment, and three TNTL treatment groups. For insulin group, mice received a daily injection of insulin . For TNTL treatment groups, mice received different doses of TNTL solution in water  via gavage. Normal and model groups of mice received the same volume of water. Treatment lasted 4 weeks. Body weight and random blood glucose were tested weekly. At the end of this experiment, FBG and glucose tolerance tests were performed. To measure glucose tolerance, mice were fasted for 12 h and then received 2 g/kg b.w. glucose via intraperitoneal injection. The blood glucose level at 0, 30, 60, 90, and 120 min post-injection was determined and plotted. Evans blue assay was used as a direct indicator of retinal vascular leakage. Evans blue was intravenously injected to the mice and dye leakage onto the retina due to blood-retinal barrier breakdown was determined by dissecting the retina and extraction of Evans blue dye according to our previous publication . The areEyeballs were collected from sacrificed mice. The retina was enucleated and placed on 4% paraformaldehyde overnight. The fixed retina was dehydrated and embedded. 4 μm paraffin section of the retina was stained with hematoxylin and eosin dye for histological measurement. For retinal vascular preparation, the fixed retina was digested with 3% of trypsin dissolved in 0.1 M Tris buffer (pH = 8.2), and the preserved vascular architecture was stained with hematoxylin-eosin dye and imaged under 400x magnification by a digital camera (CoolSNAP) linked to the microscope (LEICA). The number of endothelial cells/pericytes and acellular capillaries were quantified in each sample.+ /Tuj1+ RGC cells from eight random fields taken from four angles of the retina , four at both 1 mm and 2 mm from the optic nerve head respectively. Each selected area was 0.076 mm2 (250 × 250 μm). All images were captured by a confocal laser microscope .Survival RGCs were labeled by specific markers RBPMS and Tuj1 following a previous procedure with modifications , 16. Brihttps://www.networkanalyst.ca) [p-value< 0.05, log2(fold change) > 1) were shortlisted for the generation of heatmap and volcano plot, as well as the analysis of protein-protein interaction network and gene set enrichment analysis (GSEA). Gene ontology (GO) and KEGG pathway analysis were performed on DAVID platform (https://david.ncifcrf.gov/home.jsp) with common genes of the three retrieved lists.Total RNA was isolated from the retina tissue of hyperglycemic mice with or without TNTL treatment using RNeasy mini kit . The total RNA was then subject to library construction and transcriptomic analysis . The original data of mRNA sequencing was then uploaded to NetworkAnalyst platform for further analysis (lyst.ca) –20. DiffBMDMs were prepared according to our previous publication . In brieREC cells were seeded onto the apical side of transwell inserts  with 150 μl serum-free medium. 750 μl serum-free medium containing indicated cytokines or collected cell supernatants were added to the receiving chamber and incubated for 6 h. Cells retained on the apical side of the insert membrane were removed, whereas cells at the basolateral side were fixed in 4% paraformaldehyde and stained with crystal violet. Images of migrated cells were captured under a light microscope.The function of endothelial cells was tested by Transepithelial/Transendothelial Electrical Resistance (TEER) and leakage of FITC-Dextran through the endothelial monolayer. RECs were seeded on the apical side of transwell inserts  and grown until full confluence. The endothelial monolayer was incubated with culture supernatant of treated BMDMs for 48 h. TEER of the endothelial monolayer was tested by Ohm’s Law Method as described previously . To testTotal RNA was extracted by Trizol  and cDNA was synthesized from total RNA with first strand synthesis kit . The mRNA expression of target genes was quantitatively measured with SYBR Green reagents  on the LC480 platform . Targeted genes expression data were normalized by the expression level of β-actin. Primer sequence would be available upon request.Protein was extracted with RIPA buffer. 30 μg total protein was loaded onto SDS-PAGE and separated by electrophoresis. Separated proteins were then transferred to PVDF membrane followed by blocking with 5% BSA in TBST buffer  for 2 h at room temperature. Membranes were then incubated with primary antibody of interests overnight at 4 °C and appropriate secondary antibody for 2 h at room temperature. The membrane was probed with ECL select substrate  on the Chemidoc chemiluminescent platform .p < 0.05 were considered as statistically significant.For the animal study, the sample size was 6 in each group; for in vitro experiment, studies were performed in triplicate. Data was present in mean ± SD. For multiple group comparison, differences were measured with an ordinary two-way ANOVA with LSD multiple comparisons, while for comparison between two groups, differences were measured with student t-test. Several previous studies have proven that TNTL improved insulin sensitivity and reduced blood glucose in type II diabetic models , 24. To DR is the major microvascular complication in diabetic patients. Proper glycemic control in patients with diabetes mellitus correlates to the amelioration of DR incidence . RetinalThe initiation of DR, as well as its progression and end-stage vision loss, involves several physiopathological mechanisms such as biochemical, endocrine and hemodynamic anatomical alterations, which they interact with each other in a time sequence . Among tInflammation in the retinal microenvironment was majorly contributed by the infiltration of macrophages and release of pro-inflammatory cytokines . To examTo further explore the underlying anti-inflammatory mechanism of TNTL in preventing endothelial dysfunction in the retina, we conducted the antibody array on serum from mice treated with TNTL and SN collected from pro-inflammatory BMDMs treated with TNTL. The cytokines down-regulated by TNTL were highlighted in red while those up-regulated by TNTL were highlighted in blue Fig. a & b. ItMIP1γ is a cytokine constitutively secreted by macrophages and myeloid cells . MIP1γ aclinicaltrial.gov registration No. NCT02161276 & NCT02174042). Findings from our studying combining the clinical observation on efficacy and safety of TNTL may further justify its potential as an adjuvant therapy to intravitreal injection of anti-VEGF in DR treatment.Insulin therapy remains to be an effective mean for glycemic control in diabetic patients. However, insulin therapy is deemed to be unsuccessful in well controlling the incidence of DR, conversely, acts as one of the contributors of DR . InsulinAlthough in our study, we found that TNTL exhibited a protective effect against hyperglycemia-induced retinopathy at the dose which it did not reduce blood glucose, it cannot fully conclude that the two actions could be separated. This could occur due to the low sensitivity of the assays but may also be due to the complicated crosstalk of hyperglycemia and inflammation in the mechanism initiating retinopathy during diabetes. It was previously shown that hyperglycemia could significantly provoke inflammation via an oxidative mechanism . HoweverAn opportunity for prevention and treatment of DR by blocking inflammation has been partially evident by clinical observations on the use of corticosteroids in the treatment of proliferative DR. It was observed that intravitreal injection of triamcinolone before laser panretinal photocoagulation could significantly reduce the neovascularization  and was We noticed that in addition to pathways-related to inflammation and immune system, TNTL might also target on PI3K/Akt pathway. It was previously found that regulation on the PI3K/Akt pathway may play an essential role in the treatment of diabetic retinopathy. Sheikpranbabu et al. suggested that inhibition of PI3K/Akt could significantly suppress vascular permeability by improving junction protein expression ; controvWe found that macrophage-secreting MIP1γ may mediate the inhibitory effect of TNTL on endothelial dysfunction. The role of MIP1γ in the initiation and progression of DR has yet been understood, however, as a well-known small cytokine secreted by macrophages and myeloid cells, MIP1γ induced movement and infiltration of macrophages during inflammation . Apart fIn conclusion, in this study, we explore the novel therapeutic target of diabetic retinopathy in the intervention of a botanical formula TNTL via transcriptomic analysis in combination with protein array. Consistent quality of TNTL extract was validated by its similar biological activity on hyperglycemia with that reported in previous studies. At its doses without significantly reducing blood glucose in hyperglycemic mice, TNTL was able to ameliorate the initiation and progression of DR, as evidenced by reduced BRB leakage, retinal neovascularization and restored retinal ganglion population. We performed transcriptomic analysis on the gene expression in the retina of hyperglycemic mice with or without TNTL treatment and found that TNTL may inhibit inflammation in the retinal microenvironment. The anti-inflammatory effect of TNTL was validated by experimental observations of reduced infiltration of pro-inflammatory macrophages in the retina. Suppression of inflammation in macrophages reduced the secretion of factors that promoted migration and loss of endothelial cells integrity. Also, antibody array analysis suggested that MIP1γ, whose secretion by macrophage was inhibited by TNTL, may be responsible for the therapeutic effects. Supplementation of MIP1γ neutralized the effect of TNTL on endothelial dysfunction. CCR1-associated pathway in endothelial cells might be responsible for the inhibitory effect of TNTL on MIP1γ-induced disorders in endotheliocytes Fig. . Our stu


Inhibition of PRMT5, the major Type II arginine methyltransferase, suppresses pathogenic T cell responses and EAE. PRMT5 is transiently induced in proliferating memory inflammatory Th1 cells and during EAE. However, the mechanisms driving PRMT5 protein induction and repression as T cells expand and return to resting is currently unknown. Here, we used naive mouse and memory mouse and human Th1/Th2 cells as models to identify mechanisms controlling PRMT5 protein expression in initial and recall T cell activation. Initial activation of naive mouse T cells resulted in NF-κB-dependent transient Prmt5 transcription and NF-κB, mTOR and MYC-dependent PRMT5 protein induction. In murine memory Th cells, transcription and miRNA loss supported PRMT5 induction to a lesser extent than in naive T cells. In contrast, NF-κB/MYC/mTOR-dependent non-transcriptional PRMT5 induction played a major role. These results highlight the importance of the NF-κB/mTOR/MYC axis in PRMT5-driven pathogenic T cell expansion and may guide targeted therapeutic strategies for MS.Multiple sclerosis is an autoimmune disease of the central nervous system (CNS) mediated by CD4 Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS), thought to be driven by myelin-reactive inflammatory T cells. T cell responses against myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and proteolipid protein (PLP) antigens are thought to underlie MS. In support of this notion, immunization against these antigens induces the MS-like disease experimental autoimmune encephalomyelitis (EAE) in mice and other rodents –3. MS anWe recently reported that protein arginine methyltransferase 5 (PRMT5) plays a crucial role in inflammatory T cell expansion and EAE disease . PRMT5 iPRMT5-targeting miR-96 and miR-92b in Epstein Barr Virus-transformed cell lines was driven by nuclear factor (NF)-κB signaling and resulted in PRMT5 protein induction , phosphoinositide 3-kinase (PI3K), and mammalian target of rapamycin (mTOR) pathways. The Erk pathway drives transcription and translocation of transcription factor Fos into the nucleus, which together with Jun, forms the functional Activator Protein 1 (AP-1) complex. Transcription factors NF-κB and AP-1 converge to rapidly upregulate IL-2 expression, a growth, and survival cytokine that drives T cell expansion , 25. PI3ys in MS –44. In ays in MS . Given tys in MS , 22 as wPrmt5 transcription and PRMT5 protein induction, the latter mediated by MYC induction and mTOR-induced miR-322 loss. In contrast, in memory Th cells, the NF-κB/MYC/mTOR axis was dispensable for Prmt5 transcription and loss of Prmt5-targeting miRNAs. Rather, NF-κB/MYC/mTOR pathways contributed to PRMT5 protein induction via non-transcriptional/non-miRNA mechanisms. Overall, these data are consistent with a model in which initial murine naive T cell activation induces PRMT5 via both transcriptional and non-transcriptional mechanisms downstream of NF-κB, mTOR and/or MYC. During recall activation, memory T cells would be poised to rapidly induce PRMT5 expression, mainly through NF-κB/MYC/mTOR-dependent non-transcriptional mechanisms, possibly allowing the more robust proliferative response characteristic of memory T cell responses. Overall, this study provides insight into the importance of the NF-κB/mTOR/MYC axis in PRMT5-driven pathogenic Th cell responses and may guide targeted therapeutic strategies for MS.In this study, we explore the signaling pathways and mechanisms driving PRMT5 expression after T cell activation. Using murine naive and memory as well as human memory Th cells as models of initial and recall T cell activation, we show that PRMT5 protein expression is regulated via a combination of transcriptional and non-transcriptional mechanisms. NF-κB, mTOR and MYC pathways promoted PRMT5 protein induction in murine naive and memory T cells. However, some differences in the mechanisms of PRMT5 regulation were observed between naive and memory T cells. In naive T cells, NF-κB induced both Ac1−11-specific TCR-transgenic (Tg) mice [described previously (B10.PL (Jackson Laboratory) and myelin basic protein (MBP)eviously ] were brin vitro studies.Stock Bay11-7082 or Bay11-7085 (SelleckChem), 10058-F4 (Sigma or SelleckChem), LY294002 (SelleckChem), SCH772984 (SelleckChem), and Rapamycin (SelleckChem) were solubilized in DMSO vehicle and diluted ≥1:1,000 for Ac1−11 and irradiated splenocytes in the presence of recombinant human (rh) IL-2 (Miltenyi) every 7–10 days. T cells collected 7–10 days after activation with MBPAc1−11 and irradiated splenocytes provided the resting Th cell condition. To avoid the presence of non-T cells in in vitro experiments, resting Th1 or Th2 cell lines were activated with anti-CD3/CD28 for the indicated lengths of time. Mouse naive CD4+ T cells were isolated from spleens and lymph nodes using the mouse naive CD4+ T cell isolation kit (Miltenyi or STEMCELL Technologies) and activated using 5 μg/ml coated anti-CD3 and 2 μg/ml soluble CD28. Human Th1 and Th2 cells were generated by isolating CD4+ T cells with a CD4+ T Cell Isolation Kit (STEMCELL Technologies) from human whole blood leukocytes from normal donors and activating on anti-CD3/CD28 Dynabeads (Thermo Fisher) under Th1 (rhIL-12 + anti-hIL-4) or Th2 (rhIL-4 + anti-hIL-12 + anti-hIFNγ) conditions for 1 week and reactivated for further experiments, as previously described , according to the manufacturer's instructions, and stored at −80°C until analysis. Optical density (OD) 230, 260, and 280 were obtained with a NanoDrop 2000 to evaluate RNA concentration and quality.Prmt5 (Mm00550472_m1), mouse Hprt (Mm0044968_m1), mouse Myc (Mm00487804_m1) or human PRMT5 (Hs01047356_m1) and human 18S (Hs99999901_s1) primer sets (Life Technologies), as previously described  or random primers and Superscript III (Applied Biosystems) according to manufacturer's instructions; TaqMan quantitative real-time PCR was performed using mouse 5 Mm005502_m1, mou5 Mm005502_m1, mouPrimary naive or memory T cells were transfected with Mirus TransIT-TKO (MIR2150) or TransIT-X2 (MIR6003) transfection reagent according to manufacturer's instructions. Briefly, cells were activated for 4–8 h and subsequently incubated with 25–50 nM of negative control miRNA mimic (4464061 or AM17111), or a combinations of miR-15b (MC10904), miR-140-3p (MC12503), and/or miR-322/424 (MC11080) mimics. Average transfection efficiencies, measured by transfection with Cy3 conjugated negative control miRNA (AM17120), were 30% for naive Th cells, 15–20% for memory Th1 cells and 50% for memory Th2 cells.Prmt5 or human PRMT5 3′-UTR. In some cases, mouse Prmt5 3′UTRs with mutated miRNA-binding sequences were transfected. Mutated 3′UTRs were ordered from IDT. Mouse Prmt5 3′UTR was mutated as follows: The miR-140-3p binding site CUGUGGA was mutated to GCCGCCG, the miR-15/16/195/322 binding site UGCUGCU was mutated to CCGGCGC, and the miR-96 sites UGUAGAACAUCUGCUGGUUCAGU and UGCUCAGCCGCCAGA were deleted. Human PRMT5 3'UTR was mutated at the putative miR-140-3p binding site CCCUGGA to GCCGCCG. The wild-type or mutant plasmids were co-transfected with miR-15a (MC10235), miR-15b (MC10904), miR-16 (MC10339), miR-96 (MC10422), miR-140-3p (MC12503), miR-195 (MC10827), murine miR-322/424 (MC11080), or human miR-424 (MC10306), or control nonsense (NS) pre-miR (4464061 or AM17111) (Thermofisher). Cells media was changed to complete media  + Penicillin/Streptomycin) after 24 h. Cells were collected and lysed in 1X Cell Lysis Buffer (Promega) 4 days post transfection. Luminol was detected using a GloMax 96 Microplate Luminometer (Promega). Firefly luciferase signal was normalized to Renilla luciferase as a measure of transfection efficiency.Cos-7 cells were transfected using Lipofectamine (Life Technologies) in serum-free conditions with pmiRGlo Dual Glow Luciferase plasmid (Promega) containing the wild-type mouse Cells were collected at different time points and cell pellets were frozen at −80°C. Cell pellets were lysed in RIPA buffer  and protein concentration was quantified by BCA assay. Protein (5–10 μg) was run on 14% SDS-PAGE gels and transferred onto PVDF membrane. Blots were blocked with Odyssey Blocking Buffer (LICOR) and probed with antibodies against PRMT5 (Abcam ab31751), SYM10 pan-symmetric dimethylarginine (Millipore 07–412), or MYC . β-actin (Sigma) was used as a housekeeping control. Secondary antibodies used were goat anti-rabbit 800CW and goat anti-mouse 680RD (LICOR). Blots were imaged on an Odyssey CLx machine (LICOR) and quantifications were performed using ImageStudio.We previously reported that PRMT5-selective inhibitors suppressed not only memory Th cell proliferation, but also the proliferation of newly activated naive Th cells, albeit higher concentrations were required . This suTo explore which TcR-induced pathways contribute to PRMT5 induction, we targeted major pathways activated via the TcR, namely NF-κB, PI3K, mTOR, and Erk. Out of these, NF-κB has been shown to promote PRMT5 in cancer cells . We founMyc transcript  reduction in PRMT5 protein expression in activated memory Th cells  4-mediated translation initiation , 57, 58 ependent  and IRESependent . While tPrmt5/PRMT5-targeting miRNAs in mouse and human T cells. In the early phases after naive T cell activation, only miR-322/424 decreased significantly, suggesting that most of PRMT5 induction occurs via the transcriptional and non-transcriptional mechanisms described in the paragraph above. However, miR-322/424, miR-15b, and miR-140-3p substantially increase as naive T cells down-regulate PRMT5 and return to the resting non-proliferative state of resting memory cells, suggesting these miRNAs contribute to PRMT5 suppression. Once these resting memory T cells were reactivated, Prmt5-targeting miRNAs decreased, which may contribute to PRMT5 protein induction and fast proliferative responses in recall memory T cell responses. Overexpression of Prmt5-targeting miRNAs in our system resulted in modest decreases in PRMT5 protein. miRNAs suppressing PRMT5 protein expression would be expected to suppress T cell proliferation as well. One limitation is that, since efficient transfection of primary Th cells is difficult to achieve, a majority of untransfected cells continue to proliferate, overwhelming the culture and making it difficult to observe an effect. Thus, it is conceivable that miRNAs may have a larger effect on PRMT5 protein expression than our data may initially suggest.Post-transcriptional miRNA-mediated regulation of PRMT5 expression has long been known to operate in transformed cells , 21 and MYC. Although the implications of this SNP on MYC activity and PRMT5 expression remain to be fully elucidated, we show here that MYC activity is required for full PRMT5 induction in T cells. Finally, miRNAs have also been shown to be dysregulated in MS patients  and the Policy on Humane Care and Use of Laboratory Animals (PHS). Animal use protocol was approved by the Institutional Animal Care and Use Committee (IACUC). Studies using human T cells were all completely de-identified and therefore, exempt from Institutional Review Board (IRB) approval.LW, JN, SA, and MG-d-A formulated research hypothesis and designed experiments. LW, JN, SA, SS, and GN performed experiments and interpreted data. LW drafted the manuscript. MG-d-A revised manuscript. All authors extensively reviewed the manuscript.MG-d-A has a PRMT5 inhibitor patent pending and is a PRMT5 inhibitor inventor on a licensing deal with Prelude Therapeutics. 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.


Further investigation revealed that simvastatin did not alter the expression or phosphorylation of LRP6, but reduced intracellular cholesterol deposition and inhibited endoplasmic reticulum (ER) stress. Inducing ER stress with tunicamycin activated the Wnt/β-catenin pathway, whereas reducing ER stress with 4-phenylbutyric acid inhibited it. We hypothesize that simvastatin does not affect transmembrane signal transduction in the Wnt/β-catenin pathway, but inhibits ER stress by reducing intracellular cholesterol accumulation, which blocks intracellular signal transduction in the Wnt/β-catenin pathway and ameliorates endothelial dysfunction.Atherosclerosis is the pathological basis of cardiovascular disease, whilst endothelial dysfunction (ED) plays a primary role in the occurrence and development of atherosclerosis. Simvastatin has been shown to possess significant anti-atherosclerosis activity. In this study, we evaluated the protective effect of simvastatin on endothelial cells under oxidative stress and elucidated its underlying mechanisms. Simvastatin was found to attenuate H Globally, the incidence and mortality rates of cardiovascular diseases remain very high . AtherosSimvastatin is a common hypolipidemic drug that inhibits hydroxymethylglutaryl coenzyme A (HMG-COA) reductase activity and thus endogenous cholesterol synthesis . Numerou2O2-induced endothelial dysfunction [The classical Wnt/β-catenin pathway is highly conserved and is involved in multiple physiological processes, including embryonic development and organ formation . For exafunction .The endoplasmic reticulum (ER) is an organelle found in eukaryotic cells. Hyperlipidemia, oxidative stress, and calcium imbalance can all disrupt ER homeostasis and lead to ER stress ,20. To r2O2-induced EC dysfunction via the Wnt/β-catenin pathway and ER stress. To investigate this, we treated human umbilical vein endothelial cells (HUVECs) with simvastatin and measured the subsequent levels of EC dysfunction, Wnt/β-catenin signaling, and ER stress.Simvastatin, the Wnt/β-catenin pathway, and ER stress clearly affect AS development; however, the links between these factors and EC dysfunction are unclear. We hypothesized that simvastatin reduces Hp < 0.001, n = 3) and this concentration was used in all future experiments.To investigate the effects of simvastatin, HUVECs were exposed to different doses of the drug to determine its optimal concentration. As shown in p < 0.05, n = 3 or 6) but higher than in the Li group , whilst the opposite result was observed for Bax expression .To evaluate the protective effect of simvastatin on HUVECs under oxidative stress, cell viability and the expression of Bax and Bcl-2 were measured. In the Li group, cell viability and Bcl-2 expression were 35% and 37% lower than that in the C group, respectively a, whilstp < 0.05, n = 3) but higher than those in the Sim group . SOD activity was 43% lower in the Li group and 53% higher in the Sim group than in the C group , and was higher in the Li+Sim group than in the Li group, but lower in the Li+Sim group than in the Sim group .LDH levels in the medium and intracellular MDA concentration and SOD activity were also determined. LDH levels and MDA concentration were 23% and 35% higher in the Li group, and 20% and 38% lower in the Sim group than in the C group, respectively. LDH levels and MDA concentration in the Li+Sim group were lower than those in the Li group . When the Wnt/β-catenin pathway was activated by LiCl, the adhesion ability of HUVECs increased by 48% , and was higher in the Li+Sim group than in the Sim group yet lower than in the Li group . The levels of adhesion molecule expression can affect the adhesion ability of HUVECs. As shown in p < 0.05, n = 3) and 25%, 44%, and 38% higher in the Li group  than in the C group, respectively. In the Li+Sim group, the expression of all of the adhesion molecules was lower and higher than that in the Li and Sim groups, respectively . Taken together, these results indicate that simvastatin reduced the adhesion ability of HUVECs by inhibiting the Wnt/β-catenin pathway.The ability of the HUVECs to adhere to THP-1 cells was assessed by counting the number of THP-1 cells adhered to the HUVECs. As shown in p < 0.05 or 0.001, n = 3). β-catenin protein expression was 34% higher and 20% lower in the Li and Sim groups, respectively, compared to that in the C group. In addition, phospho-β-catenin levels 30% lower and 16% higher in the Li and Sim groups, respectively, compared to that in the C group. The levels of phosphorylated β-catenin in the Li+Sim group were lower than those in the Sim group but higher than those in the Li group.Under oxidative stress, simvastatin can reduce the injury and adhesion of HUVECs. To investigate the possible underlying mechanisms, the levels of β-catenin and phosphorylated β-catenin were determined by western blotting analysis . Likewise, total and phosphorylated LRP6 levels in the Li+Sim group were not significantly different from those in either the Li or Sim groups .LDL receptor-related protein 6 (LRP6) is a coreceptor of the Wnt/β-catenin pathway, and its phosphorylation activates the downstream Wnt/β-catenin pathway . Lipid rp < 0.05 or 0.001, n = 3), indicating that ER stress may be activated by the Wnt/β-catenin pathway. GRP78, ATF6, and CHOP are three key molecules in ER stress. In the Li group, GRP78, ATF6, and CHOP expression was increased by 29%, 61%, and 47%, respectively, compared to that in the C group , whilst in the Li+Sim group, their expression was significantly lower than those in the Li group, but higher than those in the Sim group . These results indicate that simvastatin inhibits ER stress and that the Wnt/β-catenin pathway affects ER stress. In addition, GRP78, ATF6, and CHOP expression levels in the Sim group were 27%, 19%, and 24% lower, respectively, than those in the C group ; however, phosphorylated β-catenin levels decreased by 24% and increased by 30% after tunicamycin and 4-PBA treatment, respectively . GRP78, ATF6, and CHOP expression increased by 58%, 76%, and 42%, respectively, after tunicamycin treatment, but decreased by 42%, 53%, and 26%, respectively, after 4-PBA treatment . Thus, endoplasmic reticulum stress affects the Wnt/β-catenin pathway.Tunicamycin is an ER stress activator, whereas 4-PBA is an ER stress inhibitor. As shown in p < 0.001, n = 3) and 35% lower in the Sim group  than in the C group, whilst it was significantly lower in the Li+Sim group than that in the Li group and higher than that in the Sim group .To explore the possible mechanism underlying the inhibition of ER stress by simvastatin, intracellular cholesterol was measured by Filipin staining, as fluorescence intensity is proportional to intracellular cholesterol content. As shown in Simvastatin treatment reduces the risk of cardiovascular events and cardiovascular disease-related deaths, predominantly by decreasing intracellular and circulating cholesterol levels ,8,9,10; To determine whether simvastatin could reduce Wnt/β-catenin pathway-induced EC dysfunction, LiCl was used to activate this pathway in HUVECs. LiCl inhibited the phosphorylation of cytoplasmic β-catenin, confirming Wnt/β-catenin pathway activation a. ReduceCholesterol is one of the main components of lipid rafts, which are involved in numerous cellular and signal transduction processes since many receptors and signal transduction-related proteins are localized in or associated with them . For exaER stress occurs when the morphology and function of the ER are altered as a result of endogenous or exogenous stimuli, and may lead to the accumulation of misfolded proteins in the ER. During ER stress, the expression of GRP78, CHOP, and ATF6 increases . Recent To further investigate the mechanism underlying the inhibition of the Wnt/β-catenin pathway by simvastatin, HUVECs were treated with tunicamycin (an ER stress activator) or 4-PBA (an ER stress inhibitor). When ER stress was induced by tunicamycin, total β-catenin levels increased and phosphorylated β-catenin levels decreased. When ER stress was inhibited by 4-PBA, total β-catenin levels decreased and phosphorylated β-catenin levels increased . TherefoAltogether, our results show that simvastatin did not affect transmembrane signal transduction in the Wnt/β-catenin pathway, but inhibited ER stress by reducing intracellular cholesterol accumulation, which blocked intracellular signal transduction in the Wnt/β-catenin pathway and reduced endothelial dysfunction .2. THP-1 cells (Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences) were cultured in RPMI-1640 and incubated under the same conditions as the HUVECs.HUVECs were purchased from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences  and were cultured in DMEM containing penicillin (100 U/mL), streptomycin (0.1 mg/mL), and 10% fetal calf serum at 37 °C in an incubator containing 5% CO2O2; Li, treated with LiCl + H2O2; Sim, treated with simvastatin + H2O2; and Li+Sim, treated with simvastatin + LiCl + H2O2. After cells reached 70% confluence, simvastatin (0.2 μmol/L) was added to the Sim and Li+Sim groups. Two hours later, LiCl (20 μmol/L) was added to the Li and Li+Sim groups. After 24 h, 500 μmol/L H2O2 was added to the C, Li, Sim, and Li+Sim groups. Then after another 24 h, the cells were collected for further analysis. To investigate whether the Wnt/β-catenin pathway affected ER stress, HUVECs were treated with tunicamycin  or 4-phenylbutyric acid . The protein expression of β-catenin, phospho-β-catenin, and the ER stress-related molecules, GRP78, ATF6, and CHOP, was measured.To determine the optimal simvastatin concentration, HUVECs were cultured in 6-well plates. When the cells reached 70% confluence they were treated with different doses of simvastatin  for 24 h. The lactate dehydrogenase (LDH) level in the medium and β-catenin, Bax, and Bcl-2 protein levels were then quantified. To determine whether simvastatin had a protective effect on endothelial cells under oxidative stress and to identify the underlying mechanism, HUVECs were treated with either lithium chloride (LiCl), simvastatin, or both. HUVECs were divided into the following four groups: C, treated with HThe LDH level in the culture medium and the intracellular SOD activity and MDA concentration can reflect EC dysfunction. After the HUVECs were treated as described above, the medium was collected from the six-well plate and its LDH level was measured using a commercially available kit. The cells from the six-well plate were digested with trypsin, collected, disrupted using ultrasound, and centrifuged at 10,000 rpm for 10 min at 4 °C. The SOD activity and MDA concentration of the resulting supernatant were measured using a commercially available kit according to the manufacturer’s instructions.2O2 for 24 h, THP-1 cells (1 × 105 cells/well) were added. After 2 h, the medium in each well was removed and unattached THP-1 cells were washed away with PBS. The number of THP-1 cells adhered to the HUVECs was counted using a microscope .HUVECs were cultured in 24-well plates as described above. After incubation with HTo measure protein expression levels, cells were lysed and total protein was collected. Samples were then boiled in protein loading buffer at 100 °C for 5 min. Total proteins were extracted from the cells, subjected to 8–12% SDS-PAGE, and transferred to polyvinyl fluoride microporous membranes. Membranes were blocked with 5% skim milk or 5% bovine serum albumin, dissolved in Tris-buffered saline/Tween 20 (TBST) for 1 h, and incubated at 4 °C overnight with primary antibodies  at a 1:1000 dilution. Membranes were then washed three times with TBST and incubated with a secondary antibody for 1 h at room temperature. After washing three times, an enhanced chemiluminescent reagent was added to the membrane and the blot was visualized using an autoradiography system . Each set of experiments was performed three times. 5 cells/well and cultured as described above. Next, 20 µL of MTT (5 mg/mL) was added to each well, the cells were incubated at 37 °C for 4 h, the medium was removed, and 200 µL of dimethyl sulfoxide was added to each well to dissolve the blue precipitate. The absorbance was measured at 490 nm using a microplate reader . A total of 6 wells were measured per group and the experiment was performed three times.To determine the effect of simvastatin on the viability of HUVECs under oxidative stress, they were seeded onto 96-well plates at a density of 1 × 104 cells were cultured in the 24-well plate after the glass lips were added. When HUVECs reached 70% confluence in the 24-well plate, the four cell groups were treated with different drugs, as described above. The medium in each well was then removed and the cells were washed three times with PBS. After being fixed with 4% paraformaldehyde for 30 min, the cells were washed with PBS three more times, 1.5 mg/mL of glycine was added to each well, and the cells were incubated in the glycine solution for 10 min. Finally, 500 μL of Filipin (0.05 mg/mL Filipin in PBS) was added to each well, incubated for 2 h, then the fluorescence intensity of each group was observed using a fluorescence microscope . The experiment was performed three times [2.5 × 10ee times .p values < 0.05 were considered statistically significant.The data were analyzed using the statistical software, SPSS 22.0 . Significant differences between the groups were determined using one-way analysis of variance. All results represent at least 3 independent experiments. Differences with Simvastatin did not affect transmembrane signal transduction in the Wnt/β-catenin pathway, but inhibited ER stress by reducing intracellular cholesterol accumulation, which blocked intracellular signal transduction in the Wnt/β-catenin pathway and reduced endothelial dysfunction.