ObjectiveThis study aimed to evaluate the clinical efficacy of Ruyi Zhenbaowan（RYZBW）in the treatment of initial and early knee osteoarthritis （KOA） through a prospective multicenter，randomized，double-blind，and placebo-parallel controlled trial. MethodFrom October 13^th， 2021 to December 25^th， 2021， 240 KOA subjects meeting the acceptance criteria were enrolled in 15 sub-centers including Wangjing Hospital， Chinese Academy of Chinese Medical Sciences， and they were randomly divided into observation group and control group， with 120 cases in each group. The intervention measures for the observation group were RYZBW + health education， and the intervention measures for the control group were RYZBW placebo + health education. The intervention period in both groups was four weeks， and they were followed up for four weeks after the intervention. The primary outcome measure was the total score of Western Ontario and McMaster University Osteoarthritis Index score （WOMAC score）， and the secondary outcome measures were the response rate of visual scale （VAS） pain score， WOMAC sub item scores （joint pain， joint stiffness， and joint function）， quality of life （SF-12） score， and traditional Chinese medicine （TCM） syndrome score. Result（1） Efficacy evaluation. The marginal model results showed that the observation group was better than the control group in improving the WOMAC total score and WOMAC pain score in the treatment of KOA with RYZBW， and the difference was statistically significant （P<0.05）. There was no significant difference between the two groups in improving VAS score response rate， WOMAC function score， WOMAC stiffness score， SF12-PCS （quality of life-physical health） score， SF12-MCS （quality of life-mental health） score， and TCM syndrome score. （2） Subgroup analysis. ① In terms of VAS score response rate， the response rate of the observation group was higher than that of the control group for subjects with baseline VAS score of （4， 5］， and the difference was statistically significant （P<0.05）. ② In terms of TCM syndrome score， for subjects aged ［56， 60］ and ［61， 65］， the decrease in total TCM syndrome score in the observation group was better than that in the control group， and the difference was statistically significant （P<0.05）. ConclusionTibetan medicine RYZBW has good clinical efficacy in improving WOMAC total score， VAS score response rate， WOMAC pain score， WOMAC function score， and TCM syndrome score for patients with initial and early KOA， which can fill the lack of Tibetan medicine RYZBW in the treatment of KOA and make a demonstration study for the inheritance and development of ethnic medicine.

This study explores the effects and possible mechanisms of nuclear factor E2 related factor 2 (NRF2) on ovarian granulosa cells, providing a scientific basis to prevent premature ovarian failure. An ovarian cell injury model was constructed by treating human ovarian granulosa cell (KGN cell) with 4-Vinylcyclohexene dioxide (VCD). Firstly, KGN cells were treated with different concentrations of VCD, and cell counting kit 8 (CCK-8) was used to detect ovarian cell proliferation. After determining IC 50 by CCK8, the levels of estradiol and progesterone in the cell supernatant were detected using enzyme-linked immunosorbent assay (ELISA), reactive oxygen species (ROS) assay kit was used to detect the content of ROS in ovarian cells, real-time fluorescence quantitative polymerase chain reaction (qRT PCR) was used to detect the mRNA expression level of NRF2, and Western blot was used to detect the protein expression level of NRF2. Further, NRF2 silence (siNRF2) and overexpression (NRF2-OE) cell models were constructed through lentivirus transfection, and the effects of regulating NRF2 on VCD treated cell models were investigated by detecting hormone levels, oxidative stress indicators (ROS, SOD, GSH-Px), and autophagy (LC3B level). The results showed that VCD intervention inhibited the proliferation of ovarian granulosa cells in a time-dependent and dose-dependent manner ( F>100, P<0.05), with an IC 50 of 1.2 mmol/L at 24 hours. After VCD treatment, the level of estradiol in the cell supernatant decreased from (56.32±10.18) ng/ml to (24.59±8.75) ng/ml ( t=5.78, P<0.05). Progesterone decreased from (50.25±7.03) ng/ml to (25.13±6.67) ng/ml ( t=6.54, P<0.05). After VCD treatment, the SOD of cells decreased from (44.47±7.71) ng/ml to (30.92±4.97) ng/ml ( t=3.61, P<0.05). GSH-Px decreased from (68.51±10.17) ng/ml to (35.19±6.59) ng/ml ( t=5.73, P<0.05). Simultaneously accompanied by an increase in autophagy and a decrease in NRF2. This study successfully constructed KGN cell models that silenced NRF2 and overexpressed NRF2. Subsequently, this study treated each group of cells with VCD and found that the cell proliferation activity of the siNRF2 group was significantly reduced ( t=8.37, P<0.05), while NRF2-OE could reverse the cell activity damage caused by VCD ( t=3.37, P<0.05). The siNRF2 group had the lowest level of estradiol ( t=5.78, P<0.05), while NRF2-OE could reverse the decrease in cellular estradiol levels caused by VCD ( t=5.58, P<0.05). The siNRF2 group had the lowest progesterone levels ( t=3.02, P<0.05), while NRF2-OE could reverse the decrease in cellular progesterone levels caused by VCD ( t=2.41, P<0.05). The ROS level in the siNRF2 group was the highest ( t=2.86, P<0.05), NRF2-OE could reverse the increase in ROS caused by VCD ( t=3.14, P<0.05), the SOD enzyme content in the siNRF2 group was the lowest ( t=2.98, P<0.05), and NRF2-OE could reverse the decrease in SOD enzyme content caused by VCD ( t=4.72, P<0.05). The GSH-Px enzyme content in the siNRF2 group was the lowest ( t=3.67, P<0.05), and NRF2-OE could reverse the decrease in antioxidant enzyme content caused by VCD ( t=2.71, P<0.05). The LC3B level was highest in the siNRF2 group ( t=2.45, P<0.05), and NRF2-OE was able to reverse the LC3B elevation caused by VCD ( t=9.64, P<0.05). In conclusion, NRF2 inhibits ROS induced autophagy, thereby playing a role in reducing ovarian granulosa cell damage, which may be a potential target for premature ovarian failure.

This study explores the effects and possible mechanisms of nuclear factor E2 related factor 2 (NRF2) on ovarian granulosa cells, providing a scientific basis to prevent premature ovarian failure. An ovarian cell injury model was constructed by treating human ovarian granulosa cell (KGN cell) with 4-Vinylcyclohexene dioxide (VCD). Firstly, KGN cells were treated with different concentrations of VCD, and cell counting kit 8 (CCK-8) was used to detect ovarian cell proliferation. After determining IC 50 by CCK8, the levels of estradiol and progesterone in the cell supernatant were detected using enzyme-linked immunosorbent assay (ELISA), reactive oxygen species (ROS) assay kit was used to detect the content of ROS in ovarian cells, real-time fluorescence quantitative polymerase chain reaction (qRT PCR) was used to detect the mRNA expression level of NRF2, and Western blot was used to detect the protein expression level of NRF2. Further, NRF2 silence (siNRF2) and overexpression (NRF2-OE) cell models were constructed through lentivirus transfection, and the effects of regulating NRF2 on VCD treated cell models were investigated by detecting hormone levels, oxidative stress indicators (ROS, SOD, GSH-Px), and autophagy (LC3B level). The results showed that VCD intervention inhibited the proliferation of ovarian granulosa cells in a time-dependent and dose-dependent manner ( F>100, P<0.05), with an IC 50 of 1.2 mmol/L at 24 hours. After VCD treatment, the level of estradiol in the cell supernatant decreased from (56.32±10.18) ng/ml to (24.59±8.75) ng/ml ( t=5.78, P<0.05). Progesterone decreased from (50.25±7.03) ng/ml to (25.13±6.67) ng/ml ( t=6.54, P<0.05). After VCD treatment, the SOD of cells decreased from (44.47±7.71) ng/ml to (30.92±4.97) ng/ml ( t=3.61, P<0.05). GSH-Px decreased from (68.51±10.17) ng/ml to (35.19±6.59) ng/ml ( t=5.73, P<0.05). Simultaneously accompanied by an increase in autophagy and a decrease in NRF2. This study successfully constructed KGN cell models that silenced NRF2 and overexpressed NRF2. Subsequently, this study treated each group of cells with VCD and found that the cell proliferation activity of the siNRF2 group was significantly reduced ( t=8.37, P<0.05), while NRF2-OE could reverse the cell activity damage caused by VCD ( t=3.37, P<0.05). The siNRF2 group had the lowest level of estradiol ( t=5.78, P<0.05), while NRF2-OE could reverse the decrease in cellular estradiol levels caused by VCD ( t=5.58, P<0.05). The siNRF2 group had the lowest progesterone levels ( t=3.02, P<0.05), while NRF2-OE could reverse the decrease in cellular progesterone levels caused by VCD ( t=2.41, P<0.05). The ROS level in the siNRF2 group was the highest ( t=2.86, P<0.05), NRF2-OE could reverse the increase in ROS caused by VCD ( t=3.14, P<0.05), the SOD enzyme content in the siNRF2 group was the lowest ( t=2.98, P<0.05), and NRF2-OE could reverse the decrease in SOD enzyme content caused by VCD ( t=4.72, P<0.05). The GSH-Px enzyme content in the siNRF2 group was the lowest ( t=3.67, P<0.05), and NRF2-OE could reverse the decrease in antioxidant enzyme content caused by VCD ( t=2.71, P<0.05). The LC3B level was highest in the siNRF2 group ( t=2.45, P<0.05), and NRF2-OE was able to reverse the LC3B elevation caused by VCD ( t=9.64, P<0.05). In conclusion, NRF2 inhibits ROS induced autophagy, thereby playing a role in reducing ovarian granulosa cell damage, which may be a potential target for premature ovarian failure.

Objective A rat model of pulmonary fibrosis was constructed using a single or two intratracheal drops of bleomycin(BLM)and the modeling rate and stability of the two modeling modalities were compared.Methods A total of 150 specific pathogen-free SD rats were divided randomly into blank control(control),single intratracheal drop of bleomycin(BLM-S),and two intratracheal drops of bleomycin(BLM-M)groups.Rats in the BLM-S group received a single dose of 3 mg/kg BLM by noninvasive intratracheal instillation,and rats in BLM-M group received 3 mg/kg BLM on day 1 and BLM 2 mg/kg on day 14.Rats in the control group were given intratracheal instillation of 0.9％sodium chloride(1 mL/kg).The rats were euthanized on days 28,42,56,and 84 after modelling,respectively.Deep inspiratory capacity(IC),vital capacity(VC),static lung compliance(Cchord),and dynamic lung complication(Cdyn)were measured in all rats.Pathological changes in lung tissue were observed,and the extent of alveolitis and fibrosis was graded.Collagen-Ⅲ(COL-Ⅲ)expression in rat lung tissue was detected by immunohistochemistry.Results(1)The survival rates in the control,BLM-S,and BLM-M groups were 100％,80％,and 66％,respectively.Rats in the BLM-S and BLM-M groups had significantly lower body weights on days 14～42 compared with the control group(P＜0.05,P＜0.01),and rats in the BLM-M group had significantly lower body weight on days 28～42 than rats in the control and BLM-S groups(P＜0.05,P＜0.01).(2)Regarding lung function,IC,VC,Cchord,and Cdyn were all markedly decreased in the BLM-S group compared with the control group(P＜0.05,P＜0.01)and IC,VC,and Cchord were significantly decreased in the BLM-M group(P＜0.05,P＜0.01)on day 28.IC,VC,and Cchord were significantly decreased in rats in the BLM-S group on day 42(P＜0.05,P＜0.01),and were also significantly decreased in rats in the BLM-M group on days 42～84(P＜0.05,P＜0.01).(3)In terms of lung pathology,inflammatory infiltration and fibrous cords appeared in the BLM-S group from days 28～84 and then gradually decreased(P＜0.05,P＜0.01),while fibrosis and alveolitis were relatively stable in the BLM-M group(P＜0.05,P＜0.01).(4)COL-Ⅲ expression levels in lung tissue were significantly higher in rats in the BLM-S and BLM-M groups compared with the control group(P＜0.05,P＜0.01),and the COL-Ⅲ content in the BLM-S group was significantly lower at 42～84 days than at 28 days(P＜0.05).Conclusions Both method are capable of effectively creating pulmonary fibrosis models.The single-dose approach is straightforward,has a lower death rate,and the degree of fibrosis is clearly visible by day 28,but progressively recovers after 42 days.In contrast,the two-dose instillation model has a greater success rate and better stability,with over half the rats still exhibiting visible fibrosis on day 84.

ObjectiveTo explore the amelioration of cognitive dysfunction in diabetes mellitus （DM） by Jianpi Qinghua prescription （JPQH） based on type 2 diabetes （T2DM） model rats. MethodFifty healthy male Wistar rats of SPF grade were randomly divided into control group （n=10） and experimental group （n=40）. The rats in the control group were fed conventionally， while those in the experimental group were fed on a high-sugar， high-fat diet for six weeks and administered with streptozotocin （STZ） for the induction of the DM model. The model rats were randomly divided into model group， sitagliptin group （1.2 g·L^-1）， pioglitazone group （0.8 g·L^-1）， and JPQH group （1.3 g·mL^-1）， with 10 rats in each group. After six weeks of drug intervention， the changes in body weight， blood glucose， and other related indexes of each group were recorded. Enzyme-linked immunosorbent assay （ELISA） was performed to detect the levels of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， and interleukin-6 （IL-6） in the peripheral blood and brain. The Morris water maze test was used to evaluate the cognitive function in rats. Hematoxylin-eosin （HE） staining was used to observe the pathological morphology of the hippocampal CA region. The amyloid β-protein 40 （Aβ₄₀） level was detected by immunohistochemistry. The protein expression of t-tau and p-tau in hippocampal neurons of rats was detected by Western blot. ResultCompared with blank group， the body weight of model group was significantly decreased （P<0.05）， blood glucose level was significantly increased （P<0.01）， inflammatory cytokines TNF-α and IL-1β were increased （P<0.05）， learning and spatial ability were significantly decreased （P<0.01）， the arrangement of hippocampal cells was loose and disordered， and the intercellular space was significantly increased. The number of cells decreased significantly， and the expression of Aβ₄₀ increased significantly. and increased t-tau and p-tau protein content in the hippocampus （P<0.01）. Compared with model group， the JPQH group showed reduced blood glucose （P<0.01）， decreased TNF-α and IL-1β levels in the peripheral blood and cerebrospinal fluid （P<0.05）， a downward trend of IL-6 without a statistical difference， improved learning and spatial memory ability （P<0.01）， densely arranged cells in the hippocampal CA1 area， increased cell number， reduced Aβ₄₀ expression， and decreased p-tau protein expression （P<0.05）. ConclusionJPQH can prevent cognitive dysfunction in DM by reducing inflammatory factor levels， decreasing neurotoxicity caused by Aβ₄₀ deposition， and inhibiting hyperphosphorylation of tau protein in DM rats.

Objective: To investigate the correlation between CD47 and cervical lesions. Methods: The expression and overall survival of CD47 in cervical cancer was analyzed using TCGA and TCGA Target GTEx databases. Based on GEO databases , the expression of CD47 was analyzed between cervical sample infected by HPV 16/18 and nonHPV. The differences of CD47 expression in the degrees of cervical lesions infected by HPV 16/18 were compared.The patholgical sections of colposcopy biopsy came from 320 patients who were admitted cervical cancer screening ,including HPV typing and E6/E7 mRNA detection. Immunohistochemistry were used to determine CD47 expression , and histochemistry score(H⁃score) was used to quantify the levels of CD47. The correlation among HPV 16/18 , pathological grades and CD47 was analyzed. Results: Bioinformatics tool analysis and immunohistochemistry showed that CD47 was up⁃regulated in HSIL and CC , associated with the prognosis of CC. The expression levels of CD47 in cervical tissues increased significantly after HPV 16/18 infection ( t = 2. 494 , P < 0. 05 ) , and in HPV 16/18 infected tissues , with the increase of pathological grades. The expression levels of CD47 significantly increased(F = 4. 351 , P < 0. 05 ; rs = 0. 278 , P < 0. 001) . The expression of CD47 in the E6/E7 mRNA( + ) was higher than that in the E6/E7 mRNA( - ) ( t = 5. 710 , P < 0. 000 1) , and the copies of E6/E7 were related to the degree of cervical lesions , and were positively correlated with the expression level of CD47 ( F = 15. 557 , P <0. 000 1 ; rs = 0. 649 , P < 0. 000 1) ( rs = 0. 73 , P < 0. 000 1) . Conclusion CD47 is overexpressed in HSIL and CC after HPV 16/18 infection. The expression levels of CD47 increases with the degree of cervical lesions , and is correlated with E6/E7 mRNA. CD47 may be a potential target for cervical disease progression and therapy.

Objective:To investigate the influence of the depth of invasion on the prognosis of pT1 stage mid-thoracic esophageal cancer patients undergoing left thoracotomy.Methods:Retrospectively analyze the clinicopathological data of 139 patients with pT1N0M0 stage of mid-thoracic esophageal cancer who meet the enrollment criteria. Firstly, the prognosis and influencing factors of the whole group were analyzed. The differences in prognosis, local recurrence and distant metastasis between PT1A and PT1B patients were compared, and the influence of different infiltration depth on prognosis and treatment failure of patients was analyzed. SPSS 19.0 statistical software was used for statistical analysis.Results:The 1-year, 3-year and 5-year overall survival(OS) and disease-free survival(DFS) were 95.0%, 87.8%, 82.0% and 91.4%, 84.2%, 77.0%, respectively. There were significant differences in OS( χ2=7.500, P=0.006) and DFS( χ2=7.354, P=0.007) at 1, 3 and 5 years between pT1a and pT1b patients. Cox multivariate analysis showed that pT stage and pathological type were independent prognostic factors for OS and DFS( P<0.05). There were no significant differences in OS( χ2=0.734, P=0.693) and DFS( χ2=0.7690, P=0.681) of pT1a tumors with different invasion depths. There were significant differences in OS( χ2=15.368, P<0.001) and DFS( χ2=27.470, P<0.001) at 1, 3 and 5 years of pT1b tumors with different invasion depths. The recurrence rate of pT1b(23.8%) was significantly higher than that of pT1a(5.3%)( χ2=5.274, P=0.022). The distant metastasis rate of the former(10.9%) was also significantly higher than that of the latter(0)( χ2=4.494, P=0.034). There were significant differences in local recurrence rate( χ2=17.051, P<0.001) and distant metastasis rate( χ2=15.460, P<0.001) among pT1b patients with different infiltration depths. Logistic multivariate analysis showed that the depth of infiltration was an independent factor affecting the occurrence of local recurrence in stage pT1b patients after treatment( P<0.001). Pathological type( P=0.003) and infiltration depth( P=0.027) were independent factors affecting the occurrence of distant metastasis. Conclusion:pT1a period and pT1b period after the prognosis and treatment of patients with different failure modes, and pT1b period in patients with different infiltration depth and the prognosis of patients and its failure mode after treatment significantly related, infiltration depth of pT1b period after treatment in patients with the independence of the influencing factors of failure, suggest that clinical doctors should pay attention to pT1b period in patients with postoperative adjuvant therapy. This conclusion needs to be confirmed by large prospective studies of cases.

OBJECTIVE: To assess the efficacy and safety of mulberry twig alkaloids (Sangzhi alkaloids, SZ-A) for treatment of type 2 diabetes in a randomized, double-blind, placebo-controlled multicenter clinical trial. METHODS: A total of 200 patients were randomized to receive SZ-A (n=100) or placebo (n=100) for 16 weeks. The data analysis system for electronic data capture clinical trial central randomization system was used for randomization and dispensing of drugs. The primary outcome was the change in glycosylated hemoglobin (HbA1c) level. The secondary outcome included the proportions of cases with HbA1c <7.0% and HbA1c <6.5%, fasting blood glucose (FBG), postprandial blood glucose (PBG), area under curve for the PBG (AUC_0-2h), body weight, and body mass index (BMI). Adverse events (AEs), severe adverse events (SAEs), treatment-related adverse events (TAEs), gastrointestinal disorders (GDs), blood pressure, routine blood tests, and liver and kidney function were monitored. RESULTS: Compared with baseline, the change of HbA1c at week 16 was -0.80% (95% CI: -0.98% to -0.62%) and -0.09% (95% CI: -0.27% to 0.09%) in SZ-A group and placebo group, respectively. The proportion of patients with HbA1c <7% and <6.5% was higher in the SZ-A group than in the placebo group (46.8% vs. 21.6% and 29.9% vs. 10.8%). The observed values and changes in FBG, 1 h-PBG, 2 h-PBG, and AUC_0-2h differed significantly between groups (P<0.001), but differences were not significant in body weight and BMI (P>0.05). The incidence rates of AEs, TAEs, and GDs differed significantly between groups (P=0.010, P=0.005, and P=0.006, respectively), whereas the incidence rates of SAEs showed no significant differences between groups (P=1.000). CONCLUSION SZ-A are effective and safe for treatment of type 2 diabetes. The protocol was registered in http://www.chictr.org.cn/showproj.aspx?proj=60117 (ChiCTR2000038550).
Alkaloids ; Blood Glucose ; Diabetes Mellitus, Type 2/drug therapy* ; Double-Blind Method ; Glycated Hemoglobin A ; Humans ; Hypoglycemic Agents/therapeutic use* ; Morus ; Tablets/therapeutic use* ; Treatment Outcome

The constitutive androstane receptor (CAR, NR3I1) belongs to nuclear receptor superfamily. It was reported that CAR agonist TCPOBOP induces hepatomegaly but the underlying mechanism remains largely unknown. Yes-associated protein (YAP) is a potent regulator of organ size. The aim of this study is to explore the role of YAP in CAR activation-induced hepatomegaly and liver regeneration. TCPOBOP-induced CAR activation on hepatomegaly and liver regeneration was evaluated in wild-type (WT) mice, liver-specific YAP-deficient mice, and partial hepatectomy (PHx) mice. The results demonstrate that TCPOBOP can increase the liver-to-body weight ratio in wild-type mice and PHx mice. Hepatocytes enlargement around central vein (CV) area was observed, meanwhile hepatocytes proliferation was promoted as evidenced by the increased number of KI67

Objective:To investigate the effect of Elian granule on autophagy and the phosphatidylinositol -3 kinase （PI3K）/protein kinase B （PKB/Akt）/mammalian target of rapamycin （mTOR） signaling pathway in gastric tissue of rats with gastric cancer. Method:SPF SD rats were randomly divided into the normal， model， Elian granule， and Weifuchun groups. In addition to the routine feeding in the normal group， the model， Elian granule， and Weifuchun groups received N-methyl-N'-nitro-N-nitrosoguanidine （MNNG） to induce gastric cancer in rats， and they were respectively given normal saline， Elian granule aqueous solution （3.240 g·kg^-1） and Weifuchun aqueous solution （0.390 g·kg^-1） by gavage （ig） for 48 weeks. The gross changes of the stomach taken by laparotomy were observed by naked eyes. Hematoxylin-eosin （HE） staining was performed to observe the histopathological changes of the gastric tissue in rats. Real-time quantitative polymerase chain reaction （Real-time PCR） and Western blot （WB） were used to detect the mRNA and protein expression of microtubule-associated protein 1 light chain 3 beta （LC3B）， Beclin1， p62， PI3K， Akt， mTOR in rat gastric tissue. Result:Compared with the normal group， the model group showed gastric distension， thinner gastric wall， pale gastric mucosa， atrophied and flat folds， disordered course， and visible nodules and vegetations. Compared with the model group， the Elian granule group demonstrated alleviated gastric distension， dark gastric mucosa， reduced folds， and regular course， with the thinned gastric wall improved and granular nodules observed occasionally. According to HE staining， compared with the normal group， the model group showed crowded and disordered rat gastric glands， diverse in shape， varied cell morphology， basophilic cytoplasm， large irregular hyperchromatic nuclei， visible mitosis， and infiltrated and destroyed muscularis mucosae. While compared with the model group， the arrangement of gastric glands was regular， and a few mildly atypical cells could be observed in rats of the Elian granule group. Compared with the normal group， the model group exhibited decreased expression of LC3B and Beclin1 mRNA and protein in gastric tissue （P<0.05）， and increased expression of PI3K， p62， Akt， and mTOR mRNA and protein （P<0.05）. Compared with the model group， the Elian granule group showed increased expression of LC3B and Beclin1 mRNA and protein in gastric tissue （P<0.05）， and decreased expression of PI3K mRNA and p62， Akt， and mTOR mRNA and protein （P<0.05）. Conclusion:Elian granule can improve the cell atypia of gastric tissue in rats with gastric cancer， and the mechanism may be related to the inhibition of the PI3K/Akt/mTOR signaling pathway to promote autophagy.