Objective To investigate the effects of the gene expression of carbohydrate response element binding protein(ChREBP)on human retinal microvascular endothelial cell(hRMEC)injury and angiogenesis under high-glucose conditions.Methods The hRMECs after 4-6 passages were added to a serum-free medium and cultured for 6 hours.Then,these hRMECs were treated by a normal concentration of glucose(5.6 mmol·L-1),a normal concentration of glu-cose+mannitol(24.4 mmol·L-1),a high concentration of glucose(30.0 mmol·L-1),blank siRNA+a high concentra-tion of glucose,and ChREBP siRNA+a high concentration of glucose,which were designed as the control group,hyperos-motic group,high-glucose alone group,blank siRNA group,and ChREBP knockdown group,respectively.Subsequently,these hRMECs were transfected with Lipofectamine RNAiMAX for 24 hours.Western blot was used to analyze the expression of ChREBP in hRMECs under high-glucose conditions.The terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)assay,cell scratch assay,Transwell migration assay,and tube formation assay were conducted to analyze the apoptosis and migration of these cells and capillary angiogenesis.Normal ChREBP-expressing wild-type mice and ChREBP-knockout mutant mice were selected to construct corresponding animal models,and the number of retinal angiogenic sprouts was counted under a microscope.Results The relative protein expression level of ChREBP in hRMECs after 30 minutes of high-glucose stimulation was significantly higher than that after 15 minutes,1 hour,2 hours,and 3 hours of stimulation(all P＜0.05).Meanwhile,the relative protein expression level of TXNIP in hRMECs after 2 hours of high-glu-cose stimulation was significantly higher than that after 15 minutes,30 minutes,1 hour,and 3 hours of stimulation(all P＜0.05).Under high-glucose conditions,the relative protein expression level of TXNIP and NLRP3 in hRMECs of the ChREBP knockdown group was significantly lower than that of the blank siRNA group and the high-glucose alone group(all P＜0.05).The results of TUNEL assay,cell scratch assay,and capillary tube formation assay showed that the relative apopto-sis rate,relative cell coverage area percentage,and relative migrating cell number percentage of the ChREBP knockdown group were significantly lower than those of the blank siRNA group and the high-glucose alone group,whereas the number of ring structures was significantly lower than that of the blank siRNA group,the high-glucose alone group,and the hyper-osmotic group(all P＜0.05).The relative expression level of p-AKT/AKT and p-mTOR/mTOR in the ChREBP knockdown group was significantly lower than that in the blank siRNA group and the high glucose alone group(all P＜0.05).The num-ber of retinal angiogenic sprouts in ChREBP-knockout mutant mice was(22.81±4.03),which was significantly lower than that of normal ChREBP-expressing wild-type mice(64.35±11.69)(P＜0.05).Conclusion ChREBP can affect the oc-currence and development of diabetic retinopathy by regulating the apoptosis,migration,and tubulogenesis of hRMECs and the development of retinal neovascularization under high-glucose conditions,and hence it can be regarded as a potential target for the treatment of this disease.

Objective To investigate the effects of the gene expression of carbohydrate response element binding protein(ChREBP)on human retinal microvascular endothelial cell(hRMEC)injury and angiogenesis under high-glucose conditions.Methods The hRMECs after 4-6 passages were added to a serum-free medium and cultured for 6 hours.Then,these hRMECs were treated by a normal concentration of glucose(5.6 mmol·L-1),a normal concentration of glu-cose+mannitol(24.4 mmol·L-1),a high concentration of glucose(30.0 mmol·L-1),blank siRNA+a high concentra-tion of glucose,and ChREBP siRNA+a high concentration of glucose,which were designed as the control group,hyperos-motic group,high-glucose alone group,blank siRNA group,and ChREBP knockdown group,respectively.Subsequently,these hRMECs were transfected with Lipofectamine RNAiMAX for 24 hours.Western blot was used to analyze the expression of ChREBP in hRMECs under high-glucose conditions.The terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)assay,cell scratch assay,Transwell migration assay,and tube formation assay were conducted to analyze the apoptosis and migration of these cells and capillary angiogenesis.Normal ChREBP-expressing wild-type mice and ChREBP-knockout mutant mice were selected to construct corresponding animal models,and the number of retinal angiogenic sprouts was counted under a microscope.Results The relative protein expression level of ChREBP in hRMECs after 30 minutes of high-glucose stimulation was significantly higher than that after 15 minutes,1 hour,2 hours,and 3 hours of stimulation(all P＜0.05).Meanwhile,the relative protein expression level of TXNIP in hRMECs after 2 hours of high-glu-cose stimulation was significantly higher than that after 15 minutes,30 minutes,1 hour,and 3 hours of stimulation(all P＜0.05).Under high-glucose conditions,the relative protein expression level of TXNIP and NLRP3 in hRMECs of the ChREBP knockdown group was significantly lower than that of the blank siRNA group and the high-glucose alone group(all P＜0.05).The results of TUNEL assay,cell scratch assay,and capillary tube formation assay showed that the relative apopto-sis rate,relative cell coverage area percentage,and relative migrating cell number percentage of the ChREBP knockdown group were significantly lower than those of the blank siRNA group and the high-glucose alone group,whereas the number of ring structures was significantly lower than that of the blank siRNA group,the high-glucose alone group,and the hyper-osmotic group(all P＜0.05).The relative expression level of p-AKT/AKT and p-mTOR/mTOR in the ChREBP knockdown group was significantly lower than that in the blank siRNA group and the high glucose alone group(all P＜0.05).The num-ber of retinal angiogenic sprouts in ChREBP-knockout mutant mice was(22.81±4.03),which was significantly lower than that of normal ChREBP-expressing wild-type mice(64.35±11.69)(P＜0.05).Conclusion ChREBP can affect the oc-currence and development of diabetic retinopathy by regulating the apoptosis,migration,and tubulogenesis of hRMECs and the development of retinal neovascularization under high-glucose conditions,and hence it can be regarded as a potential target for the treatment of this disease.

Objective:To study the mechanism of electroacupuncture (EA) on improving insulin resistance and in vitro fertilization and embryo transfer (IVF-ET) pregnancy outcome in polycystic ovary syndrome (PCOS) patients with phlegm-damp syndrome based on DNA methylation and insulin metabolism signaling pathway. Methods:In this prospective randomized controlled study, 100 PCOS patients with phlegm-damp syndrome who underwent IVF-ET in Department of Reproduction and Genetics in Affiliated Hospital of Shandong University of Traditional Chinese Medicine from January 2020 to December 2022 were enrolled and divided into a treatment group (EA therapy) and control group (placebo needling) by random number table, 50 patients in each group. Fixed antagonist regimen was used to promote ovulation in the two groups. Patients received EA therapy and placebo needling respectively twice a week from the menstrual cycle before oocyte retrieval till human chorionic gonadotrophin injection day. The granulosa cells were collected. The improvement of phlegm and dampness syndrome, homeostasis model assessment insulin resistance (HOMA-IR), dosage and number of days of gonadotropins (Gn) used, number of oocytes retrieved and high-quality embryos, fertilization rate, clinical pregnancy rate, early miscarriage rate and live birth rate of patients in the two groups were compared. Bisulfite amplicon sequencing was used to evaluate the methylation levels of the INSR gene promoter region in ovarian granulosa cells of patients in the two groups. Real-time polymerase chain reaction and Western blotting technology were used to detect the expression of INSR, PI3K and GLUT4. Results:The phlegm-dampness score (15.23±1.57) and HOMA-IR (2.82±0.39) of the experimental group after treatment were significantly lower than those before treatment (21.65±3.61 and 3.34±0.56), and the differences were statistically significant ( P<0.001, P=0.014). The differences of the phlegm-dampness score (-5.76±2.86) and HOMA-IR (-2.67±0.06) before and after treatment in the experimental group were significantly greater than those in control group (-1.64±0.84, -0.11±0.04), and the differences were all statistically significant ( P<0.001, P=0.021). In the experimental group, the dosage of Gn used by patients [(2 119.53±338.28) U] and the duration of Gn used [(10.16±1.25) d] were significantly reduced compared with control group [(2 405.65±434.20) U, P=0.005; (10.94±1.46) d, P=0.026], and the number of high-quality embryos (3.54±1.04) was significantly increased compared with control group (2.66±1.87, P=0.014). Fertilization rate [66.91% (552/825)], clinical pregnancy rate [63.27% (31/49)] and live birth rate [51.02% (25/49)] were significantly higher in the experimental group than in control group [60.20% (475/789), 41.67% (20/48), 31.25% (15/48)], and the differences were all statistically significant ( P=0.005, P=0.033, P=0.048). There were no significant differences in the number of oocytes retrieved and early abortion rate between the two groups (all P>0.05). The methylation levels of INSR gene promoter sites 38, 47, 56, 59, 94 and 143 in the experimental group were significantly lower than those in control group (all P<0.05), but there were no significant differences in methylation levels of sites 71, 74, 154, 156 and 162 between the two groups (all P>0.05). Compared with control group, the mRNA and protein expression of INSR, PI3K and GLUT4 was up-regulated in the experimental group (all P<0.05). Conclusion:EA may improve IR by down-regulating INSR methylation levels and up-regulating INSR, PI3K and GLUT4 expression, thereby enhancing the quality of embryos and improving the pregnancy outcome in PCOS patients.

Objective:To study the mechanism of electroacupuncture (EA) on improving insulin resistance and in vitro fertilization and embryo transfer (IVF-ET) pregnancy outcome in polycystic ovary syndrome (PCOS) patients with phlegm-damp syndrome based on DNA methylation and insulin metabolism signaling pathway. Methods:In this prospective randomized controlled study, 100 PCOS patients with phlegm-damp syndrome who underwent IVF-ET in Department of Reproduction and Genetics in Affiliated Hospital of Shandong University of Traditional Chinese Medicine from January 2020 to December 2022 were enrolled and divided into a treatment group (EA therapy) and control group (placebo needling) by random number table, 50 patients in each group. Fixed antagonist regimen was used to promote ovulation in the two groups. Patients received EA therapy and placebo needling respectively twice a week from the menstrual cycle before oocyte retrieval till human chorionic gonadotrophin injection day. The granulosa cells were collected. The improvement of phlegm and dampness syndrome, homeostasis model assessment insulin resistance (HOMA-IR), dosage and number of days of gonadotropins (Gn) used, number of oocytes retrieved and high-quality embryos, fertilization rate, clinical pregnancy rate, early miscarriage rate and live birth rate of patients in the two groups were compared. Bisulfite amplicon sequencing was used to evaluate the methylation levels of the INSR gene promoter region in ovarian granulosa cells of patients in the two groups. Real-time polymerase chain reaction and Western blotting technology were used to detect the expression of INSR, PI3K and GLUT4. Results:The phlegm-dampness score (15.23±1.57) and HOMA-IR (2.82±0.39) of the experimental group after treatment were significantly lower than those before treatment (21.65±3.61 and 3.34±0.56), and the differences were statistically significant ( P<0.001, P=0.014). The differences of the phlegm-dampness score (-5.76±2.86) and HOMA-IR (-2.67±0.06) before and after treatment in the experimental group were significantly greater than those in control group (-1.64±0.84, -0.11±0.04), and the differences were all statistically significant ( P<0.001, P=0.021). In the experimental group, the dosage of Gn used by patients [(2 119.53±338.28) U] and the duration of Gn used [(10.16±1.25) d] were significantly reduced compared with control group [(2 405.65±434.20) U, P=0.005; (10.94±1.46) d, P=0.026], and the number of high-quality embryos (3.54±1.04) was significantly increased compared with control group (2.66±1.87, P=0.014). Fertilization rate [66.91% (552/825)], clinical pregnancy rate [63.27% (31/49)] and live birth rate [51.02% (25/49)] were significantly higher in the experimental group than in control group [60.20% (475/789), 41.67% (20/48), 31.25% (15/48)], and the differences were all statistically significant ( P=0.005, P=0.033, P=0.048). There were no significant differences in the number of oocytes retrieved and early abortion rate between the two groups (all P>0.05). The methylation levels of INSR gene promoter sites 38, 47, 56, 59, 94 and 143 in the experimental group were significantly lower than those in control group (all P<0.05), but there were no significant differences in methylation levels of sites 71, 74, 154, 156 and 162 between the two groups (all P>0.05). Compared with control group, the mRNA and protein expression of INSR, PI3K and GLUT4 was up-regulated in the experimental group (all P<0.05). Conclusion:EA may improve IR by down-regulating INSR methylation levels and up-regulating INSR, PI3K and GLUT4 expression, thereby enhancing the quality of embryos and improving the pregnancy outcome in PCOS patients.

Women undergoing in vitro fertilization-embryo transfer (IVF-ET) may experience repeated implantation failure (RIF). Under the guidance of "Yu Pei Qi Sun" theory, Traditional Chinese Medicine (TCM) compounds can improve the quality of sperm and eggs of both husband and wife by anti-oxidative stress, improving mitochondrial function and reducing excessive apoptosis before IVF-ET to obtain high-quality embryos; TCM compounds can also promote angiogenesis, anti-oxidative stress, regulation of estrogen and progesterone receptor content, regulate immunity to improve female endometrial receptivity to facilitate embryo implantation; it can also invigorate the spleen and kidney after transplantation and caring for the fetus to help implantation, as well as alleviate the anxiety of patients during transplantation.

ObjectiveTo investigate the clinical efficacy and possible mechanism of Erzhi Tiangui prescription on repeated implantation failure （RIF） of kidney deficiency syndrome. MethodSeventy patients with RIF of kidney deficiency syndrome who underwent natural cycle frozen-thawed embryo transfer （FET） in the Reproductive and Genetic Center of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine were enrolled and randomly divided into a treatment group （35 cases） and a control group （35 cases）. Patients in the treatment group took oral Erzhi Tiangui prescription from the third day of each menstrual cycle two months before the FET cycle and continued to take it until the day of transplantation from the third day of the menstrual cycle in the month of transplantation. Those in the control group did not accept traditional Chinese medicine （TCM）. In addition，10 patients who successfully achieved clinical pregnancy after the first natural cycle FET were screened from the reproductive medical record bank of this hospital and assigned to the normal group. Peripheral blood samples of patients in the three groups on the day of embryo transfer were collected from the specimen bank of the Reproductive and Genetic Center. Serum soluble programmed death molecule-1 （sPD-1），soluble programmed death molecule-ligand 1 （sPD-L1），transforming growth factor-β （TGF-β），interleukin-17 （IL-17）， and interleukin-10 （IL-10） levels were measured by enzyme-linked immunosorbent assay （ELISA）. The changes in kidney deficiency syndrome scores， the final biochemical pregnancy rates， clinical pregnancy rates， and embryo implantation rates of the treatment group and the control group before and after treatment were observed. ResultCompared with the normal group，the model group showed increased serum levels of sPD-1 and IL-17（r=0.347，P<0.05），decreased levels of IL-10 and TGF-β （P<0.01），and non-significant change in sPD-L1 level. Serum sPD-1 was positively correlated with IL-17 （P<0.05） and negatively correlated with IL-10（r=-0.521，P<0.01） and TGF-β（r=-0.457，P<0.01） in RIF patients with kidney deficiency syndrome. After TCM treatment，compared with the control group， the treatment group showed improved TCM syndrome score （P<0.05） and increased clinical pregnancy rate and embryo transfer rate（P<0.05），but there was no statistically significant difference in the biochemical pregnancy rate between the two groups. ConclusionAbnormal expression of sPD-1 in patients with RIF of kidney deficiency syndrome breaks the balance of T helper 17 （Th17）/regulatory T cell （Treg），which is not conducive to embryo implantation and pregnancy maintenance. Erzhi Tiangui prescription，a TCM for tonifying the kidney，can significantly improve the symptoms of kidney deficiency in patients with RIF of kidney deficiency syndrome，reduce the concentrations of sPD-1 and IL-17 in the peripheral serum，increase the levels of TGF-β and IL-10，regulate the peripheral Th17/Treg immune balance，and increase the implantation rate and clinical pregnancy rate，which has a high clinical value.

Endometriosis (EMS) is a common and frequently-occurring disease in gynecology, which has the characteristics of strong invasiveness, wide range of lesions and easy recurrence. However, its pathogenesis is not clear. Exocrine bodies are microvesicles that widely exist in a variety of body fluids and cells, and have the functions of secretion, uptake and transport. This paper summarized the related studies of exosomes and their non-coding RNA in EMS, and their roles in cell proliferation and apoptosis, intimal migration, angiogenesis and fibrosis in the occurrence and development of EMS. Exosomes may become a non-invasive diagnostic markers of EMS with high specificity, sensitivity and accuracy, which provide ideas for clinical targeted therapy of EMS in the future.

Endometriosis (EMS) is a common and frequently-occurring disease in gynecology, which has the characteristics of strong invasiveness, wide range of lesions and easy recurrence. However, its pathogenesis is not clear. Exocrine bodies are microvesicles that widely exist in a variety of body fluids and cells, and have the functions of secretion, uptake and transport. This paper summarized the related studies of exosomes and their non-coding RNA in EMS, and their roles in cell proliferation and apoptosis, intimal migration, angiogenesis and fibrosis in the occurrence and development of EMS. Exosomes may become a non-invasive diagnostic markers of EMS with high specificity, sensitivity and accuracy, which provide ideas for clinical targeted therapy of EMS in the future.

BACKGROUND: Neuroleptic agents are effective in treating Tourette syndrome (TS). Side effects such as acute myodystonia and oculogyric crisis as well as the risk of delayed dyskinesia have led to search for alternative therapy.OBJECTIVE: To explore the effects of metoclopramide in controlling the symptoms of TS and improving the intelligence and memory function without causing extrapyramidal side effects.DESIGN: Randomized controlled trial and blind method.SETTING: Neurological Department of Xiangya Hospital Affiliated to Central South University; Institute of Traditional Chinese Medicine of Hunan Province; Neurological Department of the First People's Hospital of Nanning City; Public Health College of Central South University.PARTICIPANTS: Fifty patients with TS came from Tic Specialty Clinic,Neurological Department of Xiangya Hospital, between January 2000 and June 2001. Patients and their family members agreed toaccept our medical treatment and reexamination on fixed dates. There were 38 males and 12 females aged 5-21 years with the mean age of (10.4±4.2) years. All of them were divided into metoclopramide group and haloperidol group with 25 in each group.METHODS: Metoclopramide and haloperidol were white tablets bottled without tag (100 tablets of white color medicine in each bottle). The two types of medicine in each bottle were marked Ⅰ or Ⅱ, respectively. The blind method (patients, their parents and evaluation blinded) was used to test the real biochemical nature of the medicine. Medicine was given by a specific doctor on regular days (2, 4, 8, 16 weeks) to patients on their revisit. The daily dose of metoclopramide for body weight was 0.5-2 mg/(kg· d) and haloperidol was 0.05 mg/(kg· d) and was assigned 3-4 times (principle of dose individuality). In addition, TS patients with severe tics in metoclopramide group would receive metoclopramide 10 mg by intramuscular injection twice a day, at the initial treatment for 2-4 days. ①Before and after treatment, TS severity of patients at weeks 2, 4, 8 and 16 was evaluated by YGTSS (the total score was 100 points; the higher the score, the more severe the condition and overall impairment). ② The clinical curative effect of the medicines was evaluated with YGTSS deduction rate before and after treatment. ③ Asberg side-effect rating score: The lower the score, the fewer and milder the side effects. ④ The cognition function was evaluated with WISC score before treatment and after 3 months of treatment. WISC consisted of language and performance components with 11 subtests, while memory scale category had 10 subtests (memory score lower than the standard indicated problem in memory function).MAIN OUTCOME MEASURES: ① Comparison of YGTSS in patients before and after treatment; ② comparison of efficacy in patients of the two groups; ③ comparison of intelligence quotient and memory quotient of patients before and after treatment; ④ adverse events and side effects.RESULTS: Totally 50 patients entered the final analysis. ① Comparison of Tourette' s syndrome in patients before and after treatment: YGTSS score in the two groups decreased significantly after treatment. The comparison of the differences suggested that metoclopramide had better curative effect than haloperidol. In metoclopramide group, YGTSS score was reduced steadily at weeks 2, 4, 8 and 16. In haloperidol group, YGTSS score was also reduced steadily at weeks 2, 8 and 16, but it was similar at week 2 and week 4. ② Comparison of efficacy in patients of the two groups: The total clinical curative effect rate was 92% (23/25) in both groups withoutany difference (P ＞ 0.05). ③ Comparison of intelligence quotient beforeand after treatment: In both metoclopramide group and haloperidol group itwas higher after treatment than before treatment (93.0±15.1, 87.0±14.6; 93.2±17.0, 87.3±13.6, t=3.43, 2.3, P ＜ 0.01). ④ Comparison of memory quo tient of patients before and after treatment: It was higher only in metoclo pramide group after treatment than before treatment (87.8±12.8, 75.8±15.5, t=3.30, P ＜ 0.01). ⑤ Score of Asberg side effects: It was lower in metoclo pramide group than in haloperidol group [(1.00±0.76), (3.24±1.40) points, t=7.05, P ＜ 0.01]. CONCLUSION: The results suggest that metoclopramide is effective in con trolling the symptoms of TS and improving intelligence and memory function. Metoclopramide causes mild side effects, but no extrapyramidal side effects.