•A strategy for in situ metabolically synthesized active drug-based probes was proposed.•The potential purgative targets of SA were successfully hooked and identified.•The work provided a new insight for studying the direct targets of unstable active drugs.

Objective To investigate the effects of kuwanon G(KG)on proliferation,apoptosis,migration and invasion of gastric cancer cells and the molecular mechanisms.Methods The effects of KG on proliferation and growth of gastric cancer cells were assessed with CCK-8 assay and cell clone formation assay,by observing tumor formation on the back of nude mice and using immunohistochemical analysis of Ki-67.The effect of KG on cell apoptosis was analyzed using Annexin V-FITC/PI apoptosis detection kit,Western blotting and TUNEL staining.The effects of KG on cell migration and invasion were detected using Transwell migration and invasion assay and Western blotting for matrix metalloproteinase(MMP).The role of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)pathway in KG-mediated regulation of gastric cancer cell proliferation,migration,and invasion was verified by Western blotting and rescue assay.Results KG significantly inhibited proliferation and reduced clone formation ability of gastric cancer cells in a concentration-dependent manner(P<0.05).KG treatment also increased apoptosis,enhanced the expressions of cleaved caspase-3 and Bax,down-regulated Bcl-2,lowered migration and invasion capacities and inhibited the expression of MMP2 and MMP9 in gastric cancer cells(P<0.05).Mechanistic validation showed that KG inhibited the activation of the PI3K/AKT/mTOR pathway,and IGF-1,an activator of the PI3K/AKT/mTOR pathway,reversed the effects of KG on proliferation,migration and invasion of gastric cancer cells(P<0.05).Conclusion KG inhibits proliferation,migration and invasion and promotes apoptosis of gastric cancer cells at least in part by inhibiting the activation of the PI3K/AKT/mTOR pathway.

Objective To investigate the effects of kuwanon G(KG)on proliferation,apoptosis,migration and invasion of gastric cancer cells and the molecular mechanisms.Methods The effects of KG on proliferation and growth of gastric cancer cells were assessed with CCK-8 assay and cell clone formation assay,by observing tumor formation on the back of nude mice and using immunohistochemical analysis of Ki-67.The effect of KG on cell apoptosis was analyzed using Annexin V-FITC/PI apoptosis detection kit,Western blotting and TUNEL staining.The effects of KG on cell migration and invasion were detected using Transwell migration and invasion assay and Western blotting for matrix metalloproteinase(MMP).The role of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)pathway in KG-mediated regulation of gastric cancer cell proliferation,migration,and invasion was verified by Western blotting and rescue assay.Results KG significantly inhibited proliferation and reduced clone formation ability of gastric cancer cells in a concentration-dependent manner(P<0.05).KG treatment also increased apoptosis,enhanced the expressions of cleaved caspase-3 and Bax,down-regulated Bcl-2,lowered migration and invasion capacities and inhibited the expression of MMP2 and MMP9 in gastric cancer cells(P<0.05).Mechanistic validation showed that KG inhibited the activation of the PI3K/AKT/mTOR pathway,and IGF-1,an activator of the PI3K/AKT/mTOR pathway,reversed the effects of KG on proliferation,migration and invasion of gastric cancer cells(P<0.05).Conclusion KG inhibits proliferation,migration and invasion and promotes apoptosis of gastric cancer cells at least in part by inhibiting the activation of the PI3K/AKT/mTOR pathway.

[摘 要] 目的：探讨肝转移对晚期胃癌患者免疫治疗效果的影响。方法：收集2019年2月至2022年1月在南京医科大学附属常州第二人民医院肿瘤中心接受过免疫治疗的晚期胃癌患者的临床资料，进行回顾性分析，利用卡方检验或Fisher确切概率法进行基线特征比较，利用卡方检验和Kaplan-Meier生存分析方法进行有肝转移与无肝转移胃癌患者的疗效和生存期的比较。结果：共有48例晚期胃癌患者纳入分析，根据有无肝转移将患者分为肝转移队列（n=20）和无肝转移队列（n=28）。有肝转移较无肝转移胃癌患者体力状况更差。肝转移队列与无肝转移队列的ORR分别为15.0%和35.7%（P>0.05），DCR分别为65.0%和82.1%（P>0.05）；中位PFS在两组分别为5.0个月和11.2个月（HR=0.40，P<0.05），中位OS分别为12.0个月和19.0个月（P>0.05）。结论：胃癌肝转移患者免疫治疗的疗效差于无肝转移的患者。

AIM:To explore the effect and mechanism of hydrogen sulfide(H2S)on autophagy and angiogene-sis in skin wound of diabetic rats.METHODS:Among 36 healthy 8-week-old male Sprague-Dawley rats,12 rats were se-lected as control group,and the remaining rats were intraperitoneally injected with streptozotocin(STZ)to induce diabetic model and were randomly divided into diabetes mellitus(DM)group and NaHS(H2S donor)intervention(DM+NaHS)group,with 12 rats in each group.A skin trauma model was established by excising the skin of the back of rats in each group.The rats in DM+NaHS group were intraperitoneally injected with NaHS(56 μmol/kg),and the rats in control and DM groups were daily received the same volume of normal saline for 21 consecutive days.The healing of skin wound was measured on days 0,7,14 and 21 after operation.On the 21st day after surgery,the content of H2S in skin tissues was de-tected by C-7Az fluorescent probe,and the morphological changes and angiogenesis of wound tissues were observed by HE staining.The expression of CD31 was detected by immunofluorescence staining,and endothelial autophagy was detected by double staining of CD31 and beclin-1.The protein levels of cystathionine γ-lyase(CSE),CD31,microtubule-associated protein 1 light chain 3(LC3),beclin-1,P62,Bcl-2,Bax,phosphatidylinositol 3-kinase(PI3K),protein kinase B(PKB/Akt)and mammalian target of rapamycin(mTOR)in wound tissues were determined by Western blot.Caspase-3 and propidium iodide(PI)staining was used to detect cell apoptosis,and apoptosis of vascular endothelial cells was deter-mined with CD31 and TUNEL double immunofluorescence staining.RESULTS:Compared with DM group,the wound healing rate,H2S content and CSE protein expression were significantly increased in DM+NaHS group(P＜0.01),but still lower than those in control group(P＜0.01).HE staining showed that the wound surface in DM group was thin and wide,with few capillary,while that in DM+NaHS group was thicker with lots of capillary and wound width was reduced.Com-pared with DM group,CD31 expression was markedly increased(P＜0.01),the fluorescence intensity of caspase-3 and PI was significantly decreased(P＜0.01),and CD31+/beclin-1+ as well as CD31+/TUNEL+ cells were decreased(P＜0.01)in DM+NaHS group.Western blot analysis showed that compared with DM group,the levels of beclin-1,Bax and LC3-Ⅱ/LC3-Ⅰ were significantly decreased(P＜0.01),while the levels of P62 and Bcl-2,as well as ratios of p-PI3K/PI3K,p-Akt/Akt and p-mTOR/mTOR were significantly increased(P＜0.01)in DM+NaHS group.CONCLUSION:H2S can promote skin wound healing,which may be related to activation of PI3K/Akt/mTOR signaling pathway,inhibition of endothelial au-tophagy and apoptosis,and promotion of angiogenesis in diabetic rats.

  Objective  To summarize the clinical manifestations, pathological features and gene mutation diversity of Blau syndrome/early-onset sarcoidosis.  Methods  We collected general data, clinical manifestations, and auxiliary examination results from 8 patients who were diagnosed of Blau syndrome/early-onset sarcoidosis and treated in our hospital from January 2011 to December 2022, and then summarized and analyzed their characteristics and diversity.  Results  Among the 8 patients, 4 were males and 4 were females. The onset age was 3 to 8 months old. Rash was the first symptom in 7 patients(87.5%). 6 patients(75.0%) had papules and erythema.3 cases(37.5%) had arthritis. 2 cases(25.0%) had uveitis and other eye inflammation. 4 cases (50.0%) also showed intermittent fever. 3 cases (37.5%) showed symptoms in nerve and respiratory system, and hypertension respectively. The skin histopathology of 8 patients showed non-caseous granuloma formation. In laboratory detection, CRP and TNF-α were significantly increased before treatment, while IL-6, IL-8, TNF-α and IL-2 receptor(IL-2R) were significantly decreased in 5 patients after glucocorticoid therapy. The results of genetic testing showed that 4 of the 7 patients had p.R334W(c.1000C > T) mutation, 1 had p.H313R(c.938A > G) and p.R471C(c.1411C > T)double mutation, and 1 had p.476_477del (c.1427_1429delcct).  Conclusions  Blau syndrome/early-onset sarcoidosis has significant features in clinical manifestations, histopathology and gene mutation, but it also has diversity.

Objective To investigate the clinical features and prognosis related risk factors in nonalcoholic steatosis liver cirrhosis(NASLC).Methods From January 1st,2006 to December 31st, 2013,in a prospective cohort of 12 489 patients with liver cirrhosis set,174 patients were with NASLC and 306 patients with hepatitis B were the control.The patients were followed up every three months. The clinical data of patients were collected,including gender,age,height,body weight,blood pressure, history of hypertension,history of diabetes,family history of tumor,blood glucose level,high density lipoprotein cholesterol(HDL-C)level,low density lipoprotein cholesterol(LDL-C)level,triglyceride level,white blood cell,platelet,prothrombin time activity,total bilirubin,albumin,cholinesterase,blood urea nitrogen,creatinine,alpha-fetoprotein,abdominal ultrasound,abdominal computer tomography and endoscopy.Body mass index(BMI)and Child-Pugh scores were calculated.The differences between the two groups were analyzed in the incidence of ascites,hepatic encephalopathy,hepatorenal syndrome, esophageal varices bleeding,liver failure,hepatocellular carcinoma and mortality.Chi square test and t test were performed for statistical analysis.logistic regression analysis was used to analyze the risk factors associated with hepatocellular carcinoma in patients with NASLC.Results The proportion of female in NASLC group was higher than that in posthepatic cirrhosis group(56.0%,47/84 vs 28.7%,49/171), and the difference was statistically significant(χ2 =17.653,P<0.01).BMI,systolic pressure,diastolic pressure,level of fasting blood glucose,LDL-C,triglyceride,prothrombin time activity,albumin, cholinesterase,cases number of hypertension,diabetes and metabolic syndrome of NASLC group were all significantly higher than those of posthepatic cirrhosis group(t=6.267,4.091,5.773,2.914,1.877, 2.044,2.326,1.935 and 2.023;χ2=7.241,9.399 and 81.367;all P<0.05),however,serum levels of HDL-C,total bilirubin and creatinine were significantly lower than those of posthepatic cirrhosis group (t=6.127,8.487 and 3.261;all P < 0.05).T he three-year accumulative incidences of hepatic encephalopathy,hepatorenal syndrome and liver failure of NASLC group(8.3%,7/84;1.2%,1/84;0) were all lower than those of posthepatic cirrhosis control group(22.2%,38/171;9.9%,17/171 and 5.8%, 10/171;χ2 = 5.751,3.862 and 3.927,all P< 0.05).The three-year accumulative incidence of hepatocellular carcinoma of NASLC was 8.3%(7/84).The three-year accumulative incidence of mortality was lower than that of posthepatic cirrhosis group(2.4%,2/84 vs 13.5%,23/171;χ2 = 3.884,P=0.049).The results of logistic regression analysis showed that BMI(odds ratio(OR)= 1.469,95%confidence interval(CI)1.093 to 2.176,P=0.016)and diabetes(OR=1.734,95% CI 1.269 to 2.388, P=0.012)were independent risk factors associated with hepatocellular carcinoma in NASLC patients. Conclusions NASLC occurrs mainly in female with good liver function.BMI and diabetes are the risk factors associated with hepatocellular carcinoma in patients with NASLC.

Objective: To analyze the therapeutic effect on HBeAg-negative chronic hepatitis B patients treated with Peg-IFNα-2a combined with NAs to obtain the influencing factors for predicting HBsAg clearance. Methods: A retrospective study was conducted to investigate the effect of pegylated interferon alpha-2a combined with nucleoside analogues (lamivudine/adefovir dipivoxil) on HBeAg-negative chronic hepatitis B. The treatment course was 96 weeks. Patients were followed up 120 weeks after the treatment. HBsAg clearance at 120 weeks was taken as the objective of the study. Logistic regression and receiver operating characteristic curve analysis screened the related factors affecting HBsAg clearance. χ ² test was used to compare count data. Results: 111 patients were treated with pegylated interferon alpha-2a combined with nucleoside analogues, and 107 patients completed the scheduled course of treatment and follow-up. HBsAg clearance rate at120 week was 29.0% (31/107). The influencing factors for analysis were: (1) gender had no effect on HBsAg clearance rate; age and baseline levels of HBV DNA and alanine aminotransferase had no significant effect on HBsAg clearance; low baseline level of HBsAg (< 3.023 lgIU/ml) was beneficial to HBsAg clearance. The area under the working characteristic curve of the subjects was 0.746, the positive predictive value was 44.4%, and the negative predictive value was 86.8%. (2) HBsAg quantification or decline in 24 weeks and 48 weeks of treatment had a good predictive effect on HBsAg clearance, and the 48 weeks predicted value was higher than 24 weeks. When the HBsAg quantification was≤2.070 lgIU/ml at 48 weeks, the area under the receiver operating characteristic curve was 0.931, the positive predictive value was 52.8%, and the negative predictive value was 94.4%. When HBsAg decreased from baseline to≥0.991 lgIU/ml, the area under the receiver operating characteristic curve was 0.888, the positive predictive value was 50.8%, and the negative predictive value was 97.9%. (3) The analysis of HBsAg subgroup levels at 48 weeks suggested that the "interval analysis" can forecast HBsAg clearance more exactly than "nodal analysis" .The final HBsAg clearance rate of 100 IU/ml < HBsAg≤1 000 IU/ml, 10 IU/ml < HBsAg≤100 IU/ml and HBsAg≤10 IU/ml groups reached 6.7%, 31.8% and 67.7%, respectively. (4) The ALT abnormal group in the course of treatment obtained a higher HBsAg clearance rate (48.0%, 12/25). Conclusion 96-weeks long-term treatment with pegylated interferon-alpha -alpha-2a combined with nucleoside analogues for HBeAg-negative chronic hepatitis B has a good predictive value for HBsAg clearance at baseline and during treatment. The "interval level" of HBsAg at 48-weeks is more accurate in predicting HBsAg clearance, suggesting that HBeAg-negative chronic hepatitis B patients with low HBsAg levels at 48-weeks are the advantageous populations with HBsAg clearance. These patients are worthy of prolonged treatment to pursue "clinical cure".

Objective To evaluate the feasibility and safety profile of pegylated-interferonα-2a (Peg IFNα-2a) combined with adefovir dipivoxil (ADV) in inactive hepatitis B surface antigen (HBsAg) carriers (IHC).Methods This was a single center, prospective and open-label study.IHC were divided into therapeutic group (T, 112 subjects) and control group (C, 72 subjects) according to personal willingness.Patients with hepatitis B virus (HBV) DNA<20 IU/mL were treated with Peg IFNα-2a monotherapy, and those with HBV DNA ≥20-<2 000 IU/mL were treated with Peg IFNα-2a combined with ADV.Total therapy duration was 96 weeks.For patients who achieved HBsAg seroconversion and continued consolidation treatment for 24 weeks, the treatment duration could be less than 96 weeks.t test was used for continuous variable comparison between the two groups, while chi-square test or Fisher′s exact probability method was used for counting data analysis.The related factors affecting HBsAg clearance was analyzed by univariate or multivariate logistic regression analysis.Results A total of 194 patients were enrolled with 112 in therapeutic group and 72 in control group.The HBsAg clearance rate and seroconversion rate at week 48 in therapeutic group were 30.8% (32/104) and 26.0% (27/104), respectively.The rates at week 96 increased to 45.2% (47/104) and 38.5% (40/104), respectively.The HBsAg clearance rates at weeks 48 and 96 in control group were both 1.5% (1/68).HBsAg seroconversion was not achieved in control group.The HBsAg clearance rate in treatment group was significantly higher than that in control group (χ2=39.066, P<0.01).The quantitative HBsAg levels at baseline (OR=2.313, 95%CI: 1.258-4.251, P=0.007), week 12 (OR=3.159, 95%CI: 1.826-5.466, P<0.01) and week 24 (OR=3.347, 95%CI: 2.050-5.465, P<0.01), the decline of HBsAg at week 12 (OR=5.343, 95%CI: 2.085-13.689, P<0.01), and week 24 (OR=4.855, 95%CI: 2.380-9.902, P<0.01), and alanine transaminase (ALT) elevation at week 12 (OR=3.520, 95%CI: 1.369-9.052, P=0.009) were independent predictors for HBsAg clearance.Conclusions Peg IFNα-2a-based treatment for IHC could achieve higher HBsAg clearance rate and seroconversion rate, and has a safety profile.Decline of HBsAg at week 12 and week 24 with ALT elevation at week 12 could predict a higher HBsAg clearance rate.

Objective To study the expressions of B7-H1 and PD-1 which are costimulatory molecules in the pathogenesis of chronic hepatitis B (CHB). Methods The heparin anticoagulant blood samples from 11 CHB patients and 16 healthy controls were collected. The expressions of B7-H1 and PD-1 on myloid dendritic cells (mDC) and T lymphocytes in the peripheral blood were analyzed by flow cytometry. The data comparison was done by t test. Results The expressions of B7-H1 on CD3~+ ,CD4~+ , CD8~+ T lymphocytes and mDC in CHB patients were (40. 69± 14. 49)%, (42. 84 ±11. 19)%,(33. 48±14.07)% and (16. 60±4. 04) % , respectively, and those were (14. 66±10. 11) % , (4. 62±3.84)%, (1.89±2.31)% and (0. 49±0. 37)% , respectively in healthy controls. The expressions of B7-H1 on T-lymphocytes and mDC in CHB patients were significantly higher than those in healthy controls, and the differences were significant (t=-2.884,t=-10.894, t=-7.378, t=-13. 182,respectively; all P<0. 05). The expressions of PD-1 on CD3~+ , CD4~+ and CD8~+ T lymphocytes in CHB patients were (12. 45±6. 36)%, (11. 42 ± 6. 20)% and (13. 03± 6. 71) %, respectively, and those were (7. 80±3. 53)%, (7. 12±2. 60)% and (7. 88±3. 74)% , respectively in healthy controls.The expressions of PD-1 on T lymphocytes in CHB patients were significantly higher than those of the healthy controls, the differences were significant (t = -2.323, t =- 2. 355, t =- 2. 439,respectively) all P<0. 05). Conclusion The expressions of B7-H1 and PD-1 on T lymphocytes and mDC in CHB patients are higher than the healthy people.