ObjectiveTo investigate the mechanism of action of modified Shaofu Zhuyutang in antagonizing cellular pyroptosis and fibrosis in ectopic endometrial tissues of endometriosis through the Toll-like receptor 4/nuclear factor-κB/NOD-like receptor protein 3 （TRL4/NF-κB/NLPR3） signaling pathway. MethodsSeventy-two SPF-grade female SD rats were randomly divided into a sham-operated group （n = 12） and a modeling group （n = 60）. The rats in the sham-operated group underwent a caesarean section， while the rats in the modeling group were used to establish an endometriosis model through the auto-transplantation method. After successful modeling， the animals were randomly divided into the model group， progesterone group （0.25 mg·kg^-1）， and modified Shaofu Zhuyutang low-， medium-， and high-dose groups （7.5， 15， 30 g·kg^-1）， with 12 animals in each group. After 4 weeks of drug administration， voluntary activity and heat pain latency were observed. The rats were sacrificed for tissue collection， and Masson staining were used to observe histopathological changes in the endometrial tissues. Enzyme-linked immunosorbent assay （ELISA） was used to measure serum levels of interleukin-18 （IL-18）， interleukin-1β （IL-1β）， tumor necrosis factor-α （TNF-α）， and transforming growth factor-β （TGF-β）. Immunohistochemistry （IHC） was used to detect the protein expression area of tumor necrosis factor-related factor 6 （TRAF6） and NLPR3 in the endometrial tissues. Immunofluorescence （IF） was used to detect the relative fluorescence intensity of Caspase-1 and gasdermin D （GSDMD） in the endometrial tissues. Western blot was employed to measure the relative expression of TRL4， myeloid differentiation factor 88 （MyD88）， TRAF6， NF-κB p65， phosphorylated NF-κB p65 （p-NF-κB p65）， and NLPR3 proteins in endometrial tissues. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of TRL4， MyD88， TRAF6， NF-κB， and NLPR3 in the endometrial tissues. ResultsCompared with the sham-operated group， rats in the model group showed reduced voluntary activity and shorter heat pain latency. Serum levels of IL-18， IL-1β， TNF-α， and TGF-β were elevated. The relative expression areas of TRAF6 and NLPR3 proteins were increased， and the relative fluorescence intensity of Caspase-1 and GSDMD was enhanced. The relative expression of TRL4， MyD88， TRAF6， NF-κB p65， p-NF-κB p65， and NLPR3 proteins， along with the expression of TRL4， MyD88， TRAF6， NF-κB， and NLPR3 mRNA， were significantly increased （P<0.01）. Compared with the model group， rats in the progesterone group and the modified Shaofu Zhuyutang medium- and high-dose groups exhibited improved voluntary activity， longer heat pain latency， the fibrosis of endometrial tissue is alleviated. Serum levels of IL-18， IL-1β， TNF-α， and TGF-β were decreased. The relative expression areas of TRAF6 and NLPR3 proteins decreased， and the relative fluorescence intensity of Caspase-1 and GSDMD weakened. The relative expression of TRL4， MyD88， TRAF6， p-NF-κB p65， NLPR3 proteins， and TRL4， MyD88， TRAF6， NF-κB， and NLPR3 mRNA expression were reduced （P<0.05， P<0.01）. ConclusionModified Shaofu Zhuyutang may play a therapeutic role in endometriosis by interfering with key proteins in the TRL4/NF-κB/NLPR3 signaling pathway， reducing NLRP3 inflammasome-induced cellular pyroptosis， antagonizing the fibrosis process in ectopic endometrial tissues， improving the inflammatory microenvironment in the pelvic cavity， and alleviating pain.

ObjectiveTo observe the effects of modified Shaofu Zhuyutang on key proteins of the epidermal growth factor receptor （EGFR）/phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway in SD rats with endometriosis. MethodsAfter successful establishment of an endometriosis model in 60 female SD rats of SPF grade via the auto-transplantation method， the rats were randomly divided into a model group， modified Shaofu Zhuyutang high-， medium-， and low-dose groups， and a gestrinone group， with another 12 rats serving as a blank group. The blank and model groups were administered 10 mL·kg^-1 normal saline， while the high-， medium-， and low-dose groups received 30， 15， and 7.5 g·kg^-1 modified Shaofu Zhuyutang， respectively. The gestrinone group was administered 0.25 mg·kg^-1 gestrinone suspension. After four weeks of treatment， uterine contractions were induced with 2 U of oxytocin， and the writhing response of rats was observed. After 24 h， the rats were euthanized， and the weight and volume of ectopic endometrial tissue were recorded. Hematoxylin-eosin （HE） staining was used to observe pathological changes in endometrial tissues， while the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling （TUNEL） assay was used to evaluate the apoptosis rate of endometrial tissues. Immunofluorescence was used to detect the relative expression areas of the B-cell lymphoma-2 gene-associated promoter （Bad） and B-cell lymphoma-2 （Bcl-2） proteins in endometrial tissues. Serum levels of interleukin-1β （IL-1β）， interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α）， epidermal growth factor （EGF）， and EGFR were measured by enzyme-linked immunosorbent assay （ELISA）. The relative protein expression levels of EGFR， PI3K， phosphorylated PI3K （p-PI3K）， Akt， and phosphorylated Akt （p-Akt） in endometrial tissues were analyzed by Western blot. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression levels of EGFR， PI3K， and Akt. ResultsCompared with the blank group， the model group showed endometrial thickening， glandular and mesenchymal hyperplasia， a significant decrease in the relative expression area of Bad in ectopic endometrial tissues， a significant increase in the relative expression area of Bcl-2， and a significant reduction in the apoptosis rate as indicated by TUNEL staining. Serum levels of IL-1β， IL-6， TNF-α， EGF， and EGFR were significantly elevated （P<0.01）. The relative protein expression levels of EGFR， PI3K， p-PI3K， Akt， and p-Akt， as well as the mRNA expression levels of EGFR， PI3K， and Akt， were also significantly increased （P<0.01）. Compared with the model group， the high- and medium-dose groups of modified Shaofu Zhuyutang and the gestrinone group exhibited reduced glandular and mesenchymal hyperplasia to varying degrees， with dilated glandular lumens. The number of writhing responses was significantly reduced， the latency to writhing response was significantly prolonged， and the weight and volume of ectopic endometrial tissue were significantly decreased. The relative expression area of Bad in ectopic endometrial tissue was significantly increased， the relative expression area of Bcl-2 was significantly decreased， and the apoptosis rate was significantly elevated as shown by TUNEL staining. Serum levels of IL-1β， IL-6， TNF-α， EGF， and EGFR were significantly reduced， and the relative protein expression levels of EGFR， PI3K， p-PI3K， Akt， and p-Akt， as well as the mRNA expression levels of EGFR， PI3K， and Akt， were significantly decreased （P<0.05，P<0.01）. ConclusionModified Shaofu Zhuyutang may exert therapeutic effects on endometriosis by interfering with key proteins of the EGFR/PI3K/Akt signaling pathway and inducing apoptosis in ectopic endometrial tissue.

ObjectiveTo investigate the mechanism of modified Shaofu Zhuyutang in inducing ferroptosis in ectopic endometrial tissues of rats with endometriosis through the murine double minute 4 （MDM4）/tumor suppressor p53/glutathione peroxidase 4 （GPX4） signaling pathway. MethodsSeventy SPF-grade female SD rats were randomly divided into a blank group （n = 10）， a sham-operated group （n = 10）， and a modeling group （n = 50）. The sham-operated group underwent laparotomy， while the modeling group was subjected to the autotransplantation method to establish an endometriosis model. After successful modeling， the animals were randomly assigned to the model group， progesterone group （0.25 mg·kg^-1）， and modified Shaofu Zhuyutang high-， medium-， and low-dose groups （30， 15， and 7.5 g·kg^-1， respectively）， with 10 rats per group. After four weeks of drug administration， the rats were euthanized for sample collection. The weight and volume of ectopic endometrial tissues were recorded for each group. Transmission electron microscopy （TEM） was employed to observe ultrastructural changes in endometrial tissues， while Prussian blue staining was used to assess iron ion deposition. Serum levels of interleukin-6 （IL-6）， interleukin-1β （IL-1β）， and tumor necrosis factor-α （TNF-α） were measured by enzyme-linked immunosorbent assay （ELISA）. The relative levels of Fe²⁺， malondialdehyde （MDA）， and glutathione （GSH） in endometrial tissues were determined by colorimetric assay. Immunofluorescence （IF） was used to detect the relative fluorescence intensities of GSH and GPX4 in endometrial tissues. The relative expression levels of phosphatidylinositol 3-kinase （PI3K）， phosphorylated PI3K （p-PI3K）， protein kinase B （Akt）， phosphorylated Akt （p-Akt）， MDM4， p53， and GPX4 proteins were detected by Western blot. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to assess the mRNA expression of PI3K， Akt， MDM4， p53， and GPX4. ResultsCompared with the blank and sham-operated groups， the model group exhibited reduced ferroptotic damage in ultrastructural observations， decreased ferroptotic aggregates and positive iron ion expression area on Prussian blue staining， elevated serum IL-6， IL-1β， and TNF-α levels， reduced Fe²⁺ and MDA content， increased GSH content in endometrial tissues， and enhanced GSH and GPX4 fluorescence intensities （P<0.01）. The protein and mRNA expression levels of PI3K， p-PI3K， Akt， p-Akt， MDM4， and GPX4 were elevated， while those of p53 were decreased （P<0.01）. Compared with the model group， in the progesterone group and the modified Shaofu Zhuyutang high- and medium-dose groups， ferroptotic damage in ultrastructural observations was exacerbated to varying degrees by TEM， and ferroptotic aggregates and positive iron ion expression areas were increased on Prussian blue staining. Serum IL-6， IL-1β， and TNF-α levels decreased， Fe²⁺ and MDA content increased， and GSH content decreased in endometrial tissues. GSH and GPX4 fluorescence intensities weakened， while the protein and mRNA expression levels of PI3K， p-PI3K， Akt， p-Akt， MDM4， and GPX4 decreased， and those of p53 increased （P<0.05， P<0.01）. Conclusionmodified Shaofu Zhuyutang may exert therapeutic effects in endometriosis by inducing ferroptosis in ectopic endometrial tissues， alleviating inflammatory responses， and modulating key proteins in the MDM4/p53/GPX4 signaling pathway.

OBJECTIVE To mine and analyze the adverse drug events （ADE） signals of two camptothecin topoisomerase 1 inhibitors， i.e. irinotecan and topotecan， and to provide reference for clinical medication safety. METHODS Based on the U.S. Food and Drug Administration Adverse Event Reporting System （FAERS） database， ADE report data for the aforementioned two drugs were extracted from January 1， 2004 to March 31， 2023. After processing the data， signal mining was conducted by using the reporting odds ratio in conjunction with the Bayesian confidence propagation neural network， followed by analysis. RESULTS A total of 14 738 relevant ADE reports were screened， among which 11 483 were associated with irinotecan and 3 255 with topotecan. The ADE reports for irinotecan were predominantly male， whereas for topotecan， they were predominantly female； the age of patients using the two drugs mainly concentrated in 45-＜75 years old. A total of 847 signals were detected， involving 24 system organ classes （SOCs）. Among them， 565 signals of irinotecan were detected， involving 24 SOCs， primarily concentrating on gastrointestinal disorders， general disorders and administration site conditions， blood and lymphatic system disorders； the most frequently reported ADE was diarrhea， and the ADE with the strongest signal intensity was cholinergic syndrome. A total of 282 signals of topotecan were detected， involving 22 SOCs， primarily concentrating on general disorders and administration site conditions， investigations， blood and lymphatic system disorders， and gastrointestinal disorders； the most frequently reported ADEs were death and anemia， and the ADE with the strongest signal intensity was febrile bone marrow aplasia. ADE signals for irinotecan such as metastatic colorectal cancer， peripheral sensory neuropathy， steatohepatitis， and those for topotecan such as iris atrophy， retinal degeneration， vitreous hemorrhage， were not documented in their respective drug instruction. CONCLUSIONS ADEs of irinotecan and topotecan primarily involve the digestive and hematologic systems， warranting close clinical monitoring. Cholinergic syndrome caused by irinotecan should be concerned. In addition， patients receiving irinotecan should also be monitored for ADE such as metastatic colorectal cancer， peripheral sensory neuropathy， steatohepatitis， and proteinuria； for patients using topotecan， enhanced surveillance of ocular diseases is recommended to ensure medication safety.

Objective To investigate the genetic risk factors of deep vein thrombosis(DVT)after trauma.Methods In a nested case-control study,50 patients with DVT after traumatic lower extremity fractures and 50 patients without DVT were recruited.The two groups were matched with gender,age and fracture sites.Preoperative venography was performed to diagnose DVT in trauma patients.Genome wide association study(GWAS)was used to investigate the genetic risk factors for preoperative DVT after traumatic lower ex-tremity fractures.Genomic DNA in leukocytes from blood sample was extracted and used for GWAS.Results GWAS was conducted based on 2 662 single nucleotide variants(SNV)which were dispersed in 144 interested genes.Ten genes were found to have signifi-cant association with trauma-related DVT,including cofactors of hemostasis mechanism,i.e.,THBD,F5,SERPIND1 and ITGA2,the factors related to vitamin K-dependent(VKD)carboxylation,i.e.,GGCX and CALU,and the members of cytochrome P450 family,i.e.,CYP1A1,CYP3A4,CYP2C19 and CYP2B6.Conclusion DVT after trauma might be regulated by the cofactors of hemostasis mechanism,the factors related to VKD carboxylation and the members of cytochrome P450 family.The results of our study may provide reference and inspiration for genetic susceptibility of preoperative DVT after trauma.

Objective To analyze the characteristics of platelet changes and their influencing factors during postoperative hospitalization in patients who underwent transcatheter aortic valve implantation (TAVI). Methods The patients who underwent TAVI at Beijing Anzhen Hospital Valve Surgery Center between March 2017 and October 2021 were retrospectively selected. The patients were divided into a self-limiting group and a non-self-limiting group according to the characteristics of postoperative platelet decline. In addition, the general preoperative data, preoperative and postoperative ultrasound data, intraoperative data, and the use of anticoagulant drugs during the postoperative stay in the hospital were compared between the two groups. Results A total of 249 patients were enrolled in this study. There were 175 (70.3%) patients in the self-limiting group, including 100 males and 75 females, and there were 74 (29.7%) patients in the non-self-limiting group, including 43 males and 31 females, with no statistical difference between the two groups (P=0.863). The mean age of patients was 73.11±8.88 years in the self-limiting group and 71.54±10.39 years in the non-self-limiting group (P=0.231). The decline of platelets in the self-limiting group generally occurred on the postoperative day 2 and reached the lowest count on the postoperative day 4, and returned to the baseline level on the postoperative day 5-7, while the platelets in the non-self-limiting group changed by simple rise, fall or irregular fluctuation. Patients in the self-limiting group had severer preoperative aortic stenosis (P<0.001) and used more extracorporeal circulation assistance during surgery (P<0.001). Postoperatively, patients in the self-limiting group were more likely to have periaortic valve leakage than those in the non-self-limiting group (P=0.013). Conclusion Platelet changes in most patients after TAVI show a self-limiting decline, which may be related to the severity of patients’ preoperative aortic stenosis, intraoperative extracorporeal circulation device use, and postoperative perivalvular leakage.

Objective: To evaluate the risk of fine particulate matter (PM2.5) on excess mortality among residents. Methods: The data of residential mortality in Yangzhou City, Jiangsu Province from 2015 to 2021 were collected from the Chinese Disease Prevention and Control Information System. The average daily mass concentration of PM2.5 and meteorology data were collected from the Yangzhou Environmental Monitoring Station and Yangzhou Meteorological Bureau, respectively. The effects of PM2.5 on non-accidental mortality, morality of respiratory diseases and mortality of circulatory diseases were evaluated using a generalized additive model. The risk of excess mortality was evaluated using excess risk (ER) and the number of excess mortality. Results: The median average annual mass concentration of PM2.5 was 38.00 (interquartile range, 31.95) µg/m3 in Yangzhou City from 2015 to 2021, decreasing from 51.75 (interquartile range, 32.82) µg/m3 in 2015 to 28.00 (interquartile range, 23.42) µg/m3 in 2021. The median average annual number of non-accidental mortality, mortality of respiratory diseases and mortality of circulatory diseases were 96 (interquartile range, 22), 9 (interquartile range, 5) and 38 (interquartile range, 13) cases, respectively. The greatest effects of per 10 μg/m3 increase in PM2.5 mass concentration on non-accidental mortality, mortality of respiratory diseases, and mortality of circulatory diseases were seen at a cumulative lag of 1 day (ER=0.528%, 95%CI: 0.293%-0.763%), a cumulative lag of 2 days (ER=0.917%, 95%CI: 0.125%-1.714%) and a cumulative lag of 1 day (ER=0.595%, 95%CI: 0.232%-0.961%), respectively. The number of excess mortality caused by PM2.5 on non-accidental mortality, mortality of respiratory diseases, and mortality of circulatory diseases in Yangzhou City from 2015 to 2021 were 2 125, 412 and 977 cases, respectively; compared with 2015, the number of excess mortality in 2021 decreased by 66.95%, 75.53% and 64.42%, respectively. Conclusions An increase in the mass concentration of atmospheric PM2.5 may elevate the risk of excess mortality among residents. Compared to 2015, the number of excess deaths attributed to exposure to atmospheric PM2.5 declined in 2021.

BACKGROUND:High-methionine diet can cause liver injury in Cbs+/-mice,and hyperhomocystinemia is related to the occurrence and progression of various liver-related diseases,such as hepatic steatosis,autoimmune hepatitis,and alcoholic fatty liver disease.MicroRNAs(miRNAs)are involved in various cellular processes including cell survival,differentiation and autophagy,which are of great significance. OBJECTIVE:To investigate the critical role of miR-144-3p on Cbs+/-mouse hepatocyte autophagy induced by high methionine die. METHODS:(1)Ten male cystathione-β-synthase normal(Cbs+/+)mice and another 10 male mice with single gene knockout(Cbs+/-)of similar body mass,4 weeks of age,were fed a high-methionine diet and executed after 12 weeks to take liver tissue.(2)Human hepatocytes(HL-7702)were cultured in vitro and divided into control[0 μmol/L homocysteine(Hcy)],Hcy(100 μmol/L Hcy),mimic-NC(transfected with mimic-NC),mimic-NC + Hcy(mimic-NC transfecton+100 μmol/L Hcy),miR-144-3p mimic(transfected with miR-144-3p mimic),and miR-144-3p mimic + Hcy(miR-144-3p mimic transfection+100 μ mol/L Hcy),inhibitor-NC(transfected with inhibitor-NC),inhibitor-NC + Hcy(inhibitor-NC transfection + 100 μmol/L Hcy),miR-144-3p inhibitor(transfected with miR-144-3p inhibitor),and miR-144-3p inhibitor + Hcy(miR-144-3p inhibitor transfection + 100 μmol/L Hcy).Quantitative real-time PCR was used to detect the expression of miR-144-3p in liver tissue and hepatocytes.After transfection of miR-144-3p mimic or inhibitor,quantitative real-time PCR and western blot were used to detect the transfection efficiency of miR-144-3p and its effect on the expression of autophagy-related proteins LC3B and p62.The levels of alanine transferase and aspartate aminotransferase in hepatocyte supernatants were determined by enzyme linked immunosorbent assay.The correlation between the expression of miR-144-3 in hepatocyte and the levels of alanine transferase and aspartate aminotransferase in hepatocyte supernatants were analyzed by Pearson correlation analysis. RESULTS AND CONCLUSION:Compared with the Cbs+/+ group and control group,the expression of miR-144-3p in the liver tissue of the Cbs+/-group and in hepatocytes of the Hcy group was decreased(P<0.01).The expression of LC3B-Ⅱ/Ⅰ was decreased in hepatocyte after transfection of miR-144-3p mimic,while the protein expression of p62 was increased(P<0.01).The opposite results were obtained after transfection of miR-144-3p inhibitor(P<0.01).Compared with the mimic-NC group,the levels of alanine transferase and aspartate aminotransferase were decreased in the miR-144-3p mimic group(P<0.01),while the opposite results were obtained in the inhibitor-NC group(P<0.01).The expression of miR-144-3p in hepatocytes was negatively correlated with the levels of alanine transferase(P<0.01,r=-0.887 6)and aspartate aminotransferase(P<0.01,r=-0.829 9)in the supernatant of hepatocytes.To conclude,Hcy promotes hepatocyte autophagy by inhibiting the expression of miR-144-3p,which subsequently aggravates liver injury.

Objective To investigate the potential molecular mechanism of modified Shaofu Zhuyu Decoction in treating endometriosis from the perspective of autophagy in ectopic endometrial tissue cells mediated by the epidermal growth factor receptor (EGFR)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. Methods Seventy-two female SD rats were divided into the blank group,the model group,the gestrinone group and the modified Shaofu Zhuyu Decoction high-,medium-,and low-dose groups according to the random number table method,12 rats in each group. Construction of an endometriosis model used auto-transplantation method. The rats in the gestrinone group were gavaged with 0.25mg/(kg·d) of gestrinone suspension,the rats in the modified Shaofu Zhuyu Decoction high-,medium-,and low-dose groups were gavaged with 30,15,and 7.5g/(kg·d) of modified Shaofu Zhuyu Decoction,respectively,and the rats in the blank and model groups were gavaged with an equal amount of physiological saline,respectively. 4 weeks of continuous treatment. At the end of drug administration,2 U of oxytocin was given to each rat to induce contractions,and the pain of the rats was observed;the weight and volume of ectopic endometrial tissue of the rats were recorded. Hematoxylin-eosin (HE) staining method was used to observe the pathological changes of rat endometrial tissue in each group;transmission electron microscope was used to observe the ultrastructure of rat endometrial tissue. Enzyme-linked immunosorbent assay was used to detect the serum levels of oestradiol (E2),progesterone (P),interleukin-1β (IL-β),interleukin-6 (IL-6),tumour necrosis factor-α (TNF-α),transforming growth factor-β (TGF-β),epidermal growth factor (EGF),and EGFR in the rat serum of each group. Mean fluorescence intensity of autophagy-associated protein yeast Atg6 homologue (Beclin-1) and microtubule-associated protein 1 light chain 3 (LC3B) in endometrial tissues of rat endometrium in each group by immunofluorescence assay. Protein expression levels of EGFR,PI3K,phosphorylated phosphatidylinositol 3-kinase (p-PI3K),AKT,and phosphorylated protein kinase B (p-AKT) in endometrial tissues of rats in each group were examined by protein blotting. Real-time fluorescence PCR was used to detect EGFR,PI3K,and AKT mRNA expression levels.Results Compared with the blank group,the rats in the model group showed typical torsion reaction,ectopic endometrial tissue was visible in the abdominal wall,and the HE result showed thickening of the ectopic endometrial tissue,mesenchymal hyperplasia,and glandular dilatation;only autophagic vesicles were seen in the ultrastructure,and no obvious autophagic structures were seen in some of the fields of view;Serum E2,IL-6,IL-1β,TNF-α,TGF-β,EGF,and EGFR levels were elevated and P levels were decreased in rats;autophagy-related proteins Beclin-1 and LC3B decreased in average fluorescence intensity;protein expressions of EGFR,PI3K,AKT,p-PI3K,p-AKT was elevated;and mRNA expressions of EGFR,PI3K,AKT was elevated (P<0.05). Compared with the model group,the gestrinone group and modified Shaofu Zhuyu Decoction high-,medium-dose groups showed a decrease in the number of torsion and a prolongation of the latency time of the torsion response,a decrease in the volume and mass of ectopic endometrial tissues,a decrease in the degree of pathological damage as shown by HE,and the appearance of lysosomes,autophagolysosomes and autophagic vesicles in different degrees in the ultrastructure;Serum E2,IL-6,IL-1β,TNF-α,TGF-β,EGF,EGFR levels were decreased,and P levels were increased;the mean fluorescence intensity of autophagy-related proteins Beclin-1 and LC3B was increased;the protein expression of EGFR,PI3K,AKT,p-PI3K,and p-AKT was decreased;and the mRNA expression of EGFR,PI3K,and AKT was decreased (P<0.05). Conclusion The mechanism of modified modified Shaofu Zhuyu Decoction for the treatment of endometriosis may be related to the induction of autophagy of ectopic endometrial tissues mediated by the EGFR/PI3K/AKT signaling pathway and the attenuation of inflammatory responses.

Objective:To prepare advanced platelet-rich fibrin (A-PRF)/chitosan thermosensitive hydrogel (hereinafter referred to as composite hydrogel) and explore the effects of composite hydrogel on full-thickness skin defect wound healing in diabetic rats.Methods:This study was an experimental study. The composite hydrogel with porous mesh structure and thermosensitive characteristics was successfully prepared, containing A-PRF with mass concentrations of 10, 15, 20, 50, and 100 g/L. Diabetic model was successfully established in male Sprague-Dawley rats aged 6-8 weeks by intraperitoneal injection of streptozotocin, and 4 full-thickness skin defect wounds were established on the back of each rat (finally the model was successfully established in 36 rats). Three wounds of each rat were divided into blank group (no drug intervention), positive control group (dropping recombinant human granulocyte-macrophage stimulating factor gel), and chitosan hydrogel group (dropping chitosan hydrogel solution). Thirty rats were collected, and the remaining one wound of each rat (totally 30 wounds) was divided into 10, 15, 20, 50, and 100 g/L composite hydrogel groups, with 6 wounds in each group, which were dropped with composite hydrogel solution containing 10, 15, 20, 50, and 100 g/L A-PRF, respectively. Taking the remaining six rats, the remaining one wound from each rat was dropped with composite hydrogel solution containing 100 g/L A-PRF. On 14 d after injury, 6 rats with one wound dropped with composite hydrogel containing 100 g/L A-PRF were selected for hematoxylin-eosin (HE) staining to observe the inflammation, hemorrhage, or necrosis of the heart, liver, spleen, lung, and kidney. On 10 d after injury, 6 rats with one wound dropped with composite hydrogel containing 15 g/L A-PRF were selected to observe the blood perfusion of wounds in the four groups (with sample size of 6). On 7 and 14 d after injury, the wound healing rates in the eight groups were calculated. On 14 d after injury, the wound tissue in the eight groups was taken for HE and Masson staining to observe the formation of new epithelium and collagen formation, respectively; the positive expressions of CD31 and vascular endothelial growth factor A (VEGFA) were detected by immunohistochemistry, and the percentages of positive areas were calculated; the protein expressions of CD31 and VEGFA were detected by Western blotting; the mRNA expressions of CD31 and VEGFA were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction method (with all sample sizes of 4).Results:On 14 d after injury, no obvious inflammation, hemorrhage, or necrosis was observed in the heart, liver, spleen, lung, and kidney in the 6 rats. On 10 d after injury, the blood perfusion volume of wound in 15 g/L composite hydrogel group was significantly more than that in blank group, positive control group, and chitosan hydrogel group, respectively (with P values all <0.05). On 7 and 14 d after injury, the wound healing rates of blank group were (26.0±8.9)% and (75.0±1.8)%, which were significantly lower than those of positive control group, chitosan hydrogel group, and 10, 15, 20, 50, and 100 g/L composite hydrogel groups, respectively ((45.8±3.2)%, (49.8±3.7)%, (51.2±2.9)%, (68.5±2.4)%, (68.8±1.5)%, (72.7±2.1)%, (75.0±3.7)% and (79.1±1.9)%, (77.2±1.7)%, (82.3±1.3)%, (89.6±1.9)%, (89.8±1.3)%, (87.3±1.1)%, (87.9±1.3)%), P<0.05; the wound healing rates of positive control group, chitosan hydrogel group, and 10 g/L composite hydrogel group were significantly lower than those of 15, 20, 50, and 100 g/L composite hydrogel groups ( P<0.05). On 14 d after injury, the wound epithelialization degrees of 15, 20, 50, and 100 g/L composite hydrogel groups were higher than those of the other 4 groups, the new microvascular situation was better, and the collagen was more abundant and arranged more neatly. On 14 d after injury, the percentages of CD31 and VEGFA positive areas in wounds in positive control group and the percentage of VEGFA positive area in wounds in chitosan hydrogel group were significantly higher than those in blank group ( P<0.05), the percentage of VEGFA positive area in wounds in 10 g/L composite hydrogel group was significantly higher than that in blank group, chitosan hydrogel group, and positive control group (with P values all <0.05), and the percentages of CD31 and VEGFA positive areas in wounds in 15, 20, 50, and 100 g/L composite hydrogel groups were significantly higher than those in blank group, positive control group, chitosan hydrogel group, and 10 g/L composite hydrogel group ( P<0.05). On 14 d after injury, the protein and mRNA expressions of CD31 and VEGFA in wound tissue in chitosan hydrogel group, positive control group, and 10 g/L composite hydrogel group were significantly higher than those in blank group ( P<0.05); the protein expression of VEGFA in wound tissue in 10 g/L composite hydrogel group was significantly higher than that in positive control group ( P<0.05), and the mRNA expressions of CD31 and VEGFA in wound tissue in 10 g/L composite hydrogel group were significantly higher than those in positive control group and chitosan hydrogel group ( P<0.05); the protein and mRNA expressions of CD31 and VEGFA in wound tissue in 15, 20, 50, and 100 g/L composite hydrogel groups were significantly higher than those in blank group, positive control group, chitosan hydrogel group, and 10 g/L composite hydrogel group ( P<0.05); the mRNA expressions of CD31 and VEGFA in wound tissue in chitosan hydrogel group were significantly lower than those in positive control group ( P<0.05). Conclusions:The composite hydrogel has high biological safety, can improve wound blood perfusion, effectively promote the formation of blood vessels and collagen in wound tissue, thus promoting the wound healing of full-thickness skin defects in diabetic rats. 15 g/L is the optimal mass concentration of A-PRF in composite hydrogel.