HBV特异性抗原对HBV携带者T淋巴细胞免疫功能的影响

目的 通过分析不同类型HBV携带者外周血单个核细胞(PBMCs)的细胞免疫功能,分析HBV抗原对其的影响,探索HBV慢性感染的机制并寻求可能的免疫治疗方法.方法 用不同的抗原和(或)细胞因子刺激培养的无症状HBV携带者PBMCs,酶联免疫吸附法检测细胞培养上清液中不同细胞因子的水平;流式细胞术检测PBMCs的细胞表型.对数据进行t检验分析和相关性分析.结果 HBsAg刺激无症状HBV携带者PBMCs后产生的干扰素(IFN)γ为(48.3±19.8)Pg/ml,较健康对照人群低[(196.2±104.3)Pg/ml(t=3.023,P＜0.05)].HBsAg和HBcAg刺激HBeAg阳性患者PBMCs分泌的IFN y水平分别为(50.4±51.6)Pg/ml和(63.2±36.9)pg/ml,明显低于HBeAg阴性组[(86.2±42.3)Pg/ml和(101.4±32.5)pg/ml],t值分别为2.468和3.184,P值均＜0.05;HBeAg阳性患者组分泌白细胞介素(IL)-12 P70明显低于HBeAg阴性组(P＜0.05);补偿外源性IL-12可明显促进HBV携带者PBMCs分泌IFN γ(P＜0.01),IL-12协同HBV抗原可激活CD8+CD45RA+CCR7及CD8+CD45RA CD62L+细胞.结论 HBeAg阳性患者PBMCs分泌IL-12减少,这可能是HBV携带者持续感染的重要原因;外源性IL-12可促进HBV携带者PBMCs中的中枢记忆性T淋巴细胞的免疫功能.

Abstract：

Objective To investigate the effect of HBV antigens and pathological mechanism of chronic HBV infection by analyzing the cellular immune function of peripheral blood mononuclear cells (PBMCs) from HBsAg carriers. Methods PBMCs were prepared from individuals with chronic asymptomatic HBV infection and cultured in the presence of different antigens and/or cytokines. The levels of cytokines in culture supernatants were detected by ELISA method. The phenotype of the cells was detected by FACS.Results The levels of IFN γ secreted by PBMCs from HBsAg carriers were (48.3 ± 19.8) pg/ml, significantly lower than that from healthy controls (t = 3.023, P ＜ 0.05＝; The IFN γ produced by PBMCs from HBeAg positive patients due to HBsAg and HBcAg stimulation were (50.4±51.6) pg/ml and (63.2 ± 36.9)pg/ml, significantly lower than that of HBeAg negative patients (t = 2.468 and 3.184, P ＜ 0.05, respectively＝.The IL-12p70 secreted by PBMCs from HBeAg positive patients was also significantly lower than that of HBeAg negative patients (P ＜ 0.05＝; Exogenous IL-12 promoted significantly PBMCs to secrete IFN γ (P ＜0.01＝ and IL-12 combined with HBV antigens activated CD8+CD45RA+CCR7+ and CD8+CD45RA-CD62L+cells. Conclusion IL-12 secreted by PBMCs decreased in HBeAg positive patients, which may be the crucial reason of viral persistence in chronic HBV carriers. Exogenous IL-12 combined with specific HBV antigen could promote the central memory CD8+ T cells to produce IFN γ.     

生长抑素和生长激素联用对兔重症急性胰腺炎肠黏膜屏障损伤的保护作用

目的 观察生长抑素(SS)和生长激素(GH)联合应用对重症急性胰腺炎(SAP)兔肠黏膜屏障损伤的保护作用,探讨其对治疗SAP的意义.方法 72只新西兰大白兔平均分为3组,SAP模型组(SAP组)、SS治疗组(SS组)以及SS和GH联合治疗组(SS+GH组).经胰管开口逆行注入5%牛磺胆酸钠溶液诱导兔SAP模型,造模后3组均每日予5%葡萄糖氯化钠(GNS)治疗,SS组造模后按3.5μg·kg-1·h-1持续48 h泵入SS治疗,SS+GH组造模后第1、24 h在持续泵入SS的基础上,按0.15 IU/kg皮下注射GH治疗.观察各组动物造模后第6、12、24、48小时血清淀粉酶、肿瘤坏死因子(TNF)-α,血浆二胺氧化酶水平的变化,观察兔胰腺和肠黏膜的病理学变化及存活率.采用SPSS 16.0统计软件进行分析,组间比较采用单因素方差分析.结果 SS+GH组兔血清TNF-α和血浆二胺氧化酶水平较SAP组和SS组均明显降低,造模后24 h[分别为(2.43±0.14)pg/ml和(4.61±0.45)U/L]和48 h[分别为(2.08±0.23)pg/rl和(3.75±0.47)U/L]较SS组[24 h分别为(2.80±0.30)pg/ml和(8.74±1.77)U/L,48 h分别为(2.45±0.12)pg/m1和(5.02±0.95)U/L]显著降低,差异均有统计学意义(P＜0.05).SS+GH组较SAP组和SS组兔肠黏膜炎性反应减轻,肠黏膜的完整性增加,胰腺组织炎性反应减轻,存活率提高,但血清淀粉酶在各时间点与SS组相比差异均无统计学意义.结论 SS和GH联合应用可增强兔肠黏膜屏障功能,改善SAP预后.

Abstract：

Objective To investigate the protective effect of somatostatin (SS)combined with growth hormone (GH) in treatment of intestinal mucosal barrier injury in rabbits with severe acute pancreatitis (SAP), as well as its clinical significance. Methods Seventy-two rabbits were equally assigned into model group (SAP group), SS treated group (SS group) and SS combined with GH treated group (SS + GH group). SAP models were induced by retro-injection of 5% sodium taurocholate into the pancreatic duct. After modeling, all rabbits were given 5 % glucose saline daily.The rabbits in SS group and SS+GH group were continuously Given SS (3.5μg·kg-1·h-1)for 48 hours. Besides, the rabbits in SS+GH group were subcutaneously injected with 0.15 IU/kg of GH at the 1st and the 24th hours after modeling. The levels of serum amylase, serum tumor necrosis factor-α (TNF-α) and plasma diamine oxidase were measured at the 6th, 12th, 24th and 48th hours after modeling. The pathological changes of pancreatic tissue and ileal mucosa were observed. Survival rate was calculated. Data were analyzed using SPSS 16.0 software. The univariate analysis was used to compare the difference among groups. Results In SS+GH group, the levels of serum TNF-α and plasma diamine oxidase were (2. 43 ± 0. 14) pg/ml and (4. 61 ± 0. 45) U/L at the 24th hour respectively, and were (2.08±0.23) pg/ml and (3.75±0.47) U/L at the 48th hour, respectively,which were lower than those in SAP group and SS group [(2.80 0.30) pg/ml and (8.74 ± 1.77)U/L, respectively, at the 24th hour; (2. 45±0.12) pg/ml and (5. 02±0.95) U/L, respectively, at the 48th hour)]with significant difference (P＜0.05). The inflammation in pancreas and ileal mucosa was alleviated, and the integrity of bowel mucosa was improved. Survival rate of SS+GH group was significantly higher than SAP group and SS group. There was no significant difference in level of serum amylase between SS+GH group and SS group. Conclusion The combination of SS with GH may enhance the function of intestinal mucosa barrier and improve the prognosis of SAP in rabbits.     

羟氯喹抑制紫外线诱导的系统性红斑狼疮CD4+T细胞白细胞介素-10和干扰素-γ的表达

目的 研究紫外线对系统性红斑狼疮(SLE)CD4+T细胞因子的影响和羟氯喹的抑制作用.方法 选择SLE 30例,健康对照10名.磁珠分选SLE患者和健康人的CD4+T细胞,紫外线311 nm窄谱中波紫外线暴露,加入羟氯喹共培养,酶联免疫吸附试验(ELISA)检测培养上清白细胞介素(IL)-10和干扰素-γ的表达水平.采用t检验进行统计学分析.结果 SLE患者CD4+T细胞IL-10表达高于健康对照[(27±4)和(18±3) pg/ml,P=0.011];经45、100 mJ/cm2紫外线暴露后,SLE活动患者CD4+T细胞IL-10表达升高[(27±4)和(77±42) pg/ml,(40±18)和(77±42) pg/ml,P=0.022,P=0.048],经100 mJ/cm2紫外线暴露后,活动患者CD4+T细胞IL-10表达高于稳定患者[(77±42)和(24±4)pg/ml,P=0.029];羟氯喹降低SLE活动患者CD4+T细胞IL-10和干扰素-γ表达[(2.6±4.0)和(17.9±2.3)pg/ml,P=0.018,P=-0.017)];羟氯喹降低经45,100 mJ/cm2紫外线暴露后SLE活动患者T细胞IL-10表达[(40±18)和(22±6)pg/ml,(77±42)和(21±5) pg/ml,P=0.037,P=0.04];羟氯喹降低经100 mJ/cm2紫外线暴露的SLE活动和稳定患者T细胞干扰素-γ表达[(18±3)和(13±14) pg/ml,(19±7)和(12±5) pg/ml,P=0.013,P=0.049].结论 紫外线加重SLE患者体内Th1/Th2细胞因子的比例失衡;羟氯喹抑制了紫外线诱发SLE患者干扰素-γ和IL-10的表达.

Abstract：

Objective To explore the role of hydroxychloroquine (HCQ) in ultraviolet B (UVB)- induced expression of interleukin (IL)-10 and interferon (IFN)-γ from CD4+T cells in patients with systemic lupus erythematosus (SLE). Methods Thirty patients with SLE and 10 healthy controls were enrolled in the study. CD4+ T cells were isolated using magnetic beads from SLE patients and healthy controls. HCQ was added in culture media before and after irradiation with UVB 311 nm narrow band ultraviolet B (NB-UVB). The levels of IL-10 and IFN-γ in the supernatant were detected with enzyme-linked immunosorbent (ELISA). Comparisons between groups were performed by t-test. Results The level of IL-10 was higher in SLE patients [(27±4) pg/ml] than that in healthy controls [(18±3) pg/ml, P=0.011]. After exposure of CD4+T cells to UVB in 45 or 100 mJ/cm2 dosages, the level of IL-10 was increased significantly in patients with active disease (P=0.022, P=0.048). After exposure of CD4+T cells to UVB in 100 mJ/cm2 dosages, the levels of IL-10 was higher in patients with active disease [(77±42) pg/ml] than patients with stable disease [(24± 4) pg/ml, P=0.029]. When CD4+ T cell were cultured with HCQ, IL-10 and IFN-γ levels in patients with active disease [(2.6±4.0), (17.5±2.3) pg/ml] were decreased significantly (P=0.018, P=0.017). HCQ reversed UVB-induced IL-10 expression in active SLE patients after exposure of CD4+T cells to UVB in 45 or 100 mJ/cm2 dosages (P=0.037, P=0.04). HCQ also reversed UVB-induced IFN-7 expression in active SLE patients and stable SLE patients after exposure to CD4+T cells with UVB in 100 mJ/cm2 dosages (P=0.013, P= 0.049). Conclusion UVB can aggravate the imbalance of Th1 and Th2 cytokines. HCQ inhibits UVB-induced IL-10 and IFN-7 expression of CD4+T cells in patients with SLE, especially in patients with active disease.     

Effects of splenectomy in patients with cirrhosis undergoing hepatic resection for hepatocellular carcinoma

AIM: To study the effects of splenectomy in patients with cirrhosis undergoing hepatic resection for hepatocellular circinoma.METHODS: Twenty-six patients with HCC associated with cirrhosis were divided into hepatectomy with splenectomy group (splenectomy group, n=11) and hepatectomy without splenectomy group (non-splenectomy group, n=15). T lymphocyte subsets such as CD4, CD8, CD4/CD8, helper T (Th) lymphocyte cytokines such as interferon γ(IFN-γ),interleukin 2(IL-2), interleukin 10(IL-10) and white blood cell (WBC), platelet (PLT), total bilirubin (T-Bil) were measured and used as parameters to evaluate the effects of splenectomy.RESULTS: There was no significant difference in CD4, CD8,CD4/CD8, IL2, IFN-γ, IL10, WBC, PLT, T-Bil levels between two groups before surgery. Two months after operation,the levels of CD4 (41.2 %±4.2 % vs 34.7 %±3.8 %), CD4/CD8 (1.7±0.2 vs 1.0±0.2), IFN-γ (102.3±15.9 pg/ml vs 86.5±14.8 pg/ml), IL-2(97.2±15.6 pg/ml vS77.6±14.5 pg/ml) were increased and those of CD8 (25.6±3.9 vs32.8 %±4.1%),IL-10 (56.9±10.4 pg/ml vs 72.6±15.3 pg/ml) were decreased in splenectomy groups as compared with those in nonsplenectomy group (P<0.05). WBC and PLT counts in the splenectomy group were 8.9±1.6×109 and 310±32×109,respectively, which were significantly higher than those in non-splenectomy group (3.7±1.4×109 and 104±41×109) respectively on the 14th post-operative day. T-Bil concentration in the splenectomy group (24±7 μmol/L) was significantly lower than that in the non-splenectomy group (37±13 μmol/L) on the 7th post-operative day (P<0.05).CONCLUSION: Splenectomy combined with hepatectomy for HCC associated with cirrhosis is helpful for the recovery of T-lymphocyte subsets and the maintenance of Th:L/Th2 cytokine balance.     

生长抑素和生长激素联用对兔重症急性胰腺炎肠黏膜屏障损伤的保护作用

Objective To investigate the protective effect of somatostatin (SS)combined with growth hormone (GH) in treatment of intestinal mucosal barrier injury in rabbits with severe acute pancreatitis (SAP), as well as its clinical significance. Methods Seventy-two rabbits were equally assigned into model group (SAP group), SS treated group (SS group) and SS combined with GH treated group (SS + GH group). SAP models were induced by retro-injection of 5% sodium taurocholate into the pancreatic duct. After modeling, all rabbits were given 5 % glucose saline daily.The rabbits in SS group and SS+GH group were continuously Given SS (3.5μg·kg-1·h-1)for 48 hours. Besides, the rabbits in SS+GH group were subcutaneously injected with 0.15 IU/kg of GH at the 1st and the 24th hours after modeling. The levels of serum amylase, serum tumor necrosis factor-α (TNF-α) and plasma diamine oxidase were measured at the 6th, 12th, 24th and 48th hours after modeling. The pathological changes of pancreatic tissue and ileal mucosa were observed. Survival rate was calculated. Data were analyzed using SPSS 16.0 software. The univariate analysis was used to compare the difference among groups. Results In SS+GH group, the levels of serum TNF-α and plasma diamine oxidase were (2. 43 ± 0. 14) pg/ml and (4. 61 ± 0. 45) U/L at the 24th hour respectively, and were (2.08±0.23) pg/ml and (3.75±0.47) U/L at the 48th hour, respectively,which were lower than those in SAP group and SS group [(2.80 0.30) pg/ml and (8.74 ± 1.77)U/L, respectively, at the 24th hour; (2. 45±0.12) pg/ml and (5. 02±0.95) U/L, respectively, at the 48th hour)]with significant difference (P＜0.05). The inflammation in pancreas and ileal mucosa was alleviated, and the integrity of bowel mucosa was improved. Survival rate of SS+GH group was significantly higher than SAP group and SS group. There was no significant difference in level of serum amylase between SS+GH group and SS group. Conclusion The combination of SS with GH may enhance the function of intestinal mucosa barrier and improve the prognosis of SAP in rabbits.     

生长抑素和生长激素联用对兔重症急性胰腺炎肠黏膜屏障损伤的保护作用

Objective To investigate the protective effect of somatostatin (SS)combined with growth hormone (GH) in treatment of intestinal mucosal barrier injury in rabbits with severe acute pancreatitis (SAP), as well as its clinical significance. Methods Seventy-two rabbits were equally assigned into model group (SAP group), SS treated group (SS group) and SS combined with GH treated group (SS + GH group). SAP models were induced by retro-injection of 5% sodium taurocholate into the pancreatic duct. After modeling, all rabbits were given 5 % glucose saline daily.The rabbits in SS group and SS+GH group were continuously Given SS (3.5μg·kg-1·h-1)for 48 hours. Besides, the rabbits in SS+GH group were subcutaneously injected with 0.15 IU/kg of GH at the 1st and the 24th hours after modeling. The levels of serum amylase, serum tumor necrosis factor-α (TNF-α) and plasma diamine oxidase were measured at the 6th, 12th, 24th and 48th hours after modeling. The pathological changes of pancreatic tissue and ileal mucosa were observed. Survival rate was calculated. Data were analyzed using SPSS 16.0 software. The univariate analysis was used to compare the difference among groups. Results In SS+GH group, the levels of serum TNF-α and plasma diamine oxidase were (2. 43 ± 0. 14) pg/ml and (4. 61 ± 0. 45) U/L at the 24th hour respectively, and were (2.08±0.23) pg/ml and (3.75±0.47) U/L at the 48th hour, respectively,which were lower than those in SAP group and SS group [(2.80 0.30) pg/ml and (8.74 ± 1.77)U/L, respectively, at the 24th hour; (2. 45±0.12) pg/ml and (5. 02±0.95) U/L, respectively, at the 48th hour)]with significant difference (P＜0.05). The inflammation in pancreas and ileal mucosa was alleviated, and the integrity of bowel mucosa was improved. Survival rate of SS+GH group was significantly higher than SAP group and SS group. There was no significant difference in level of serum amylase between SS+GH group and SS group. Conclusion The combination of SS with GH may enhance the function of intestinal mucosa barrier and improve the prognosis of SAP in rabbits.     

乙型肝炎病毒感染肾小管上皮细胞Toll样受体4的表达及其作用

目的 探讨HBV感染人近端肾小管上皮细胞系HK-2后对其表达Toll样受体4(TLR4)的影响,并观察TLR4抗HBV感染的生物学作用.方法 收集HBV DNA拷贝在107-102/ml的患者血清,通过显微镜及免疫荧光法观察HBV阳性血清感染HK-2前、后细胞形态及α抗平滑肌抗体(α-SMA)的变化,应用MTT法检测不同浓度TLR4刺激因子(LPS)及TLR4抑制因子(CLI-095)对HK-2细胞增殖的影响.选取10 μL/ml脂多糖(LPS)及5μg/ml CLI-095作用于HBV感染的HK-2细胞,通过细胞免疫荧光技术及免疫印迹法检测HK-2细胞内TLR4蛋白的变化,ELISA法和荧光定量PCR法观测各组细胞上清液中HBsAg、HBeAg和HBV DNA含量的变化.结果 HBV分别感染HK-2细胞12 h和24 h后,随感染时间延长,细胞形态变得不规则,数量也减少.α-SMA的表达水平与感染24 h后相比,在HBV感染12 h后表达最多.LPS浓度在小于10μg/ml范围内,HBV感染HK-2细胞24 h后其增殖程度与剂量呈正相关,与CLI-095浓度呈负相关(P＜0.05).LPS组HK-2细胞TLR4蛋白的表达高于CLI-095组,其上清液中HBV DNA水平及HBsAg、HBeAg表达水平较CLI-095组降低.结论 TLR4可能通过免疫炎症反应参与抑制HK-2细胞中的HBV复制,当HBV感染肾组织细胞时可发挥抗病毒作用.

Abstract：

Objective To explore the expression and role of Toll receptor 4(TLR4)in human proximal tubular epithelial cell line HK-2,infected by HBV. Methods The serum of HBV DNA copies between 107-108/ml was collected. Before and after infected by HBV DNA positive serum. the HK-2 cells' morphology and the expression of α-smooth muscle actin(α-SMA)were observed by microscopy and immunofluorescence, and the effects of different concentrations of lipopolysaccharides(U)S.TLR4-stimulating factor)and CLI-095(TLR4 Inhibitor)on the proliferation rate of HK-2 cells were observed by MTT assays. After HBV serum and 10μg/ml LPS and 5μl/ml CLI-095 acted on HK-2 cells,TLR4 protein expression was measured by immunofluorescence and Western-blotting assay, and HBsAg and HBeAg in cell culture medium were detected by ELISA. and HBV DNA copies by fluorescence quantitative PCR. Results The longer HBV infected HK-2 cells, the more irregular of the cells' shape, the fewer number of the cells were left. But compared with HBV infected after 24 hours, α-SMA was more expressed after HBV infected 12 hours. After infected by HBV serum in 24 hours.HK-2 cells' proliferation rate was positively correlation in a dose range of LPS, but was negatively correlated with the CLI-095(P＜0.05＝.The levels of HBsAg and HBeAg in cell culture medium were largest when the LPS concentration was at 10μg/ml and CLI-095 at 5μg/ml.The expression of TLR4 significantly increased in HK-2 cells treated with LPS compared with those with CLI-095.but HBV DNA levels and HBsAg and HBeAg expression levels were lower. Conclusions HBV infection may promote cell transdifferentiation and cell injury. The stimulation of HK-2 infected with HBV by LPS may upregulate the expression of TLR4 and reduce the copies of HBV DNA.     

Hydrogen sulfide system in the pathogenesis of renovascular hypertension in rats

Objective To investigate the role of hydrogen sulfide (H2S) synthases/H2S pathway in the pathogenesis of renovascular hypertension.Methods Wistar rats were subdivided into 4 groups:(1) 2-kidney,1-clip (2K-1C group,n=7),(2) control (n=7),(3)sham (n=7),and (4) 2K-1C plus sodium hydrosulfide (NariS) (NariS-treated group,n=7).The systolic blood pressure (SBP) was measured by a tail-cuff method using a pulse transducer once a week.Four weeks later,all rats were killed and the concentration of plasma hydrogen sulfide (H2S),the activity of the H2S syntha.ses in the kidneys on both sides,the plasma angiotensin Ⅱ concentration,and the left-to-whole heart weight ratio were measured.Results The SBP was significantly increased in the 2K-IC group (185.4± 14.0mmHg) comparing with those in the sham group (112.9±6.5mmHg,,or the NariS-treated group(134.8±9.5mmHg) (both P＜0.01).At 4 weeks,the angiotensin Ⅱ concentration in the plasma was increased in the 2K-1C and NariS-treated group,comparing with the control and the sham group (306.92±7.03 pg/ml and 240.73±13.22 pg/ml vs 122.6±25.49 pg/ml and 125.95±10.55 pg/ml,respectively,both P＜0.05).The plasma H2S concentration and the activity of H2S synthases in the left kidney were decreased in the 2K-1C group comparing with those in the sham and the control groups.There was no difference of the activity of the H2S synthases in the right kidneys among the 4 groups.The left-to-whole heart weight ratio was increased in the 2K-1C and the NariS-treated group camparing with that in the sham and natural control groups.Conclusions Dysfunction of the H2S synthases/H2S pathway was involved in the 2K-1C-induced renovascular hypertension in rat.Exogenous administration of H2S donor can attenuate the development of hypertension.These findings suggest that the H2S synthases/H2S pathway participates in the pathogenesis of renovascular hypertension.     

Antiviral effect of Chinese medicine jiaweisinisan in hepatitis B virus transgenic mice

AIM: To study the antiviral effect of Chinese medicine jiaweisinisan (JWSNS) on hepatitis B virus (HBV) infection in transgenic mice (TGM). METHODS: Twenty two 6-8 wk old HBV TGM in the third generation were divided into TGM control group and TGM treated group randomly. The normal control group included ten normal BC 57L/6 mice at the same age. The mice in treated group were administrated with JWSNS at the concentration of 4 g/mL and the dosage of 50 g/kg per d for 30 d, while the mice in TGM control group and normal control group were administrated with normal saline at the same dosage and the same time. Polymerase chain reaction (PCR) was used to assess the contents of HBV DNA in serum of HBV TGM before and after treatments, whereas blot hybridization was utilized to measure the contents of HBV DNA in the liver of both HBV TGM and normal BC 57L/6 mice. RESULTS: The levels of serum HBV DNA in TGM treated group were remarkably decreased after the treatment of JWSNS (7.662±0.78 vs 5.22±3.14, P<0.05), while there was no obvious change after administration of normal saline in TGM control group (7.125±4.26 vs 8.932±5.12, P>0.05). The OD values of HBV DNA in the livers of the mice in TGM treated group were significantly lower than those of TGM control group (0.274±0.096 vs 0.432±0.119,P<0.01). CONCLUSION: JWSNS exerts suppressive effects on HBV DNA in the serum and liver of TGM.     

β-淀粉样肽40/42与2型糖尿病及动脉粥样硬化的关系

检测2型糖尿病、动脉粥样硬化(AS)患者血清β-淀粉样肽(Aβ)40和Aβ42水平.结果 显示,Aβ40在糖尿病组高于对照组[(274.70±159.51对162.63±87.58)pg/ml,P<0.05],糖尿病合并AS组升高更显著[(616.95±195.13)pg/ml,P<0.01];单纯AS组Aβ 40水平也明显高于对照组[(318.52±188.65)pg/ml,P<0.05],提示Aβ40是2型糖尿病合并AS的危险因素.而各组间Aβ42水平差异无统计学意义.

Abstract：

Serum p-amyloid peptide(Aβ)40 and Ap42 levels in patients with type 2 diabetes mellitus (T2DM)and atherosclerosis(AS)were detected by ELISA.The results showed that serum Ap40 level in T2DM group was significantly higher than that in the control group[(274.70±159.51 vs 162.63±87.58)pg/ml,P<0.05],especially in the diabetic patients accompanied with AS[(616.95±195.13)pg/m],P<0.01].Serum Ap40 level in simple AS group was also higher than that in control group[(318.52± 188.65)pg/ml,P<0.05].These results suggest that Ap40 is a risk factor of T2DM complicated with AS.However,there was no difference in serum Ap42 levels among various groups.     

Exogenous carbon monoxide attenuates inflammatory responses in the small intestine of septic mice

AIM:To determine whether the carbon monoxide(CO)-releasing molecules(CORM)-liberated CO suppress inflammatory responses in the small intestine of septic mice.METHODS:The C57BL/6 mice(male,n = 36;weight 20 ± 2 g) were assigned to four groups in three respective experiments.Sepsis in mice was induced by cecal ligation and puncture(CLP)(24 h).Tricarbonyldichlororuthenium(Ⅱ) dimer(CORM-2)(8 mg/kg,i.v.) was administrated immediately after induction of CLP.The levels of inflammatory cytokines [interleukin-1(IL-1) and tumor necrosis factor-(TNF-)] in tissue homogenates were measured with enzyme-linked immunosorbent assay.The levels of malondialdehyde(MDA) in the tissues were determined.The levels of nitric oxide(NO) in tissue homogenate were measured and the expression levels of intercellular adhesion molecule 1(ICAM-1) and inducible nitric oxide synthase(iNOS) in the small intestine were also assessed.NO and IL-8 levels in the supernatants were determined after the human adenocarcinoma cell line Caco-2 was stimulated by lipopolysaccharide(LPS)(10 g/mL) for 4 h in vitro.RESULTS:At 24 h after CLP,histological analysis showed that the ileum and jejunum from CLP mice induced severe edema and sloughing of the villous tips,as well as infiltration of inflammatory cells into the mucosa.Semi-quantitative analysis of histological samples of ileum and jejunum showed that granulocyte infiltration in the septic mice was significantly increased compared to that in the sham group.Administration of CORM-2 significantly decreased granulocyte infiltration.At 24 h after CLP,the tissue MDA levels in the midileum and mid-jejunum significantly increased compared to the sham animals(103.68 ± 23.88 nmol/mL vs 39.66 ± 8.23 nmol/mL,89.66 ± 9.98 nmol/mL vs 32.32 ± 7.43 nmol/mL,P 0.01).In vitro administration of CORM-2,tissue MDA levels were significantly decreased(50.65 ± 11.46 nmol/mL,59.32 ± 6.62 nmol/mL,P 0.05).Meanwhile,the tissue IL-1 and TNF-levels in the mid-ileum significantly increased compared to the sham animals(6.66 ± 1.09 pg/mL vs 1.67 ± 0.45 pg/mL,19.34 ± 3.99 pg/mL vs 3.98 ± 0.87 pg/mL,P 0.01).In vitro administration of CORM-2,tissue IL-1 and TNF-levels were significantly decreased(3.87 ± 1.08 pg/mL,10.45 ± 2.48 pg/mL,P 0.05).The levels of NO in mid-ileum and mid-jejunum tissue homogenate were also decreased(14.69 ± 2.45 nmol/mL vs 24.36 ± 2.97 nmol/mL,18.47 ± 2.47 nmol/mL vs 27.33 ± 3.87 nmol/mL,P 0.05).The expression of iNOS and ICAM-1 in the mid-ileum of septic mice at 24 h after CLP induction significantly increased compared to the sham animals.In vitro administration of CORM-2,expression of iNOS and ICAM-1 were significantly decreased.In parallel,the levels of NO and IL-8 in the supernatants of Caco-2 stimulated by LPS was markedly decreased in CORM-2-treated Caco-2 cells(2.22 ± 0.12 nmol/mL vs 6.25 ± 1.69 nmol/mL,24.97 ± 3.01 pg/mL vs 49.45 ± 5.11 pg/mL,P 0.05).CONCLUSION:CORM-released CO attenuates the inflammatory cytokine production(IL-1 and TNF-),and suppress the oxidative stress in the small intestine during sepsis by interfering with protein expression of ICAM-1 and iNOS.     

Hepatitis B surface antigen clearance in inactive hepatitis B surface antigen carriers treated with peginterferon alfa-2a

AIM: To examine the association between interferon(IFN) therapy and loss of hepatitis B surface antigen(HBs Ag) in inactive HBs Ag carriers. METHODS: This was a retrospective cohort study in inactive HBs Ag carriers, who were treatment-naive, with a serum HBs Ag level 100 IU/m L and an undetectable hepatitis B virus(HBV) DNA level( 100 IU/m L). All the 20 treated patients received subcutaneous PEG-IFN alfa-2a 180 μg/wk for 72 wk and were then followed for 24 wk. There were 40 untreated controls matched with 96 wk of observation. Serum HBs Ag, HBV DNA, and alanine aminotransferases were monitored every 3 mo in the treatment group and every 3-6 mo in the control group. RESULTS: Thirteen(65.0%) of 20 treated patients achieved HBs Ag loss, 12 of whom achieved HBs Ag seroconversion. Mean HBs Ag level in treated patients decreased to 6.69 ± 13.04 IU/m L after 24 wk of treatment from a baseline level of 26.22 ± 33.00 IU/m L. Serum HBV DNA level remained undetectable( 100 IU/m L) in all treated patients during the study. HBs Ag level of the control group decreased from 25.72 ± 25.58 IU/m L at baseline to 17.11 ± 21.62 IU/m L at week 96(P = 0.108). In the control group, no patient experienced HBs Ag loss/seroconversion, and two(5.0%) developed HBV reactivation.CONCLUSION: IFN treatment results in HBs Ag loss and seroconversion in a considerable proportion of inactive HBs Ag carriers with low HBs Ag concentrations.     

Chronic hepatitis B serum promotes apoptotic damage in human renal tubular cells

AIM: To investigate the effect of the serum of patients with chronic hepatitis B (CHB) on apoptosis of renal tubular epithelial cells in vitro and to study the role of hepatitis B virus (HBV) and transforming growth factor-β1 (TGF-β1) in the pathogenesis of hepatitis B virus associated glomerulonephritis (HBV-GN). METHODS: The levels of serum TGF-β1 were measured by specific enzyme linked immunosorbent assay (ELISA) and HBV DNA was tested by polymerase chain reaction (PCR) in 44 patients with CHB ,and 20 healthy persons as the control. The normal human kidney proximal tubular cell (HK-2) was cultured together with the sera of healthy persons, CHB patients with HBV-DNA nega-tive(20 cases) and HBV-DNA positive (24 cases) for up to 72 h. Apoptosis and Fas expression of the HK-2 were detected by flow cytometer. RESULTS: The apoptosis rate and Fas expression of HK-2 cells were significantly higher in HBV DNA positive serum group 19.01±5.85% and 17.58±8.35%, HBV DNA negative serum group 8.12±2.80% and 6.96±2.76% than those in control group 4.25±0.65% and 2.33±1.09%, respectively (P < 0.01). The apoptosis rate and Fas expression of HK-2 in HBV DNA positive serum group was significantly higher than those in HBV DNA negative serum (P < 0.01). Apoptosis rate of HK-2 cells in HBV DNA positive serum group was positively correlated with the level of HBV-DNA (r = 0.657). The level of serum TGF-β1 in CHB group was 163.05±91.35μg/L, significantly higher as compared with 81.40±40.75μg/L in the control group (P < 0.01). CONCLUSION: The serum of patients with chronic hepatitis B promotes apoptotic damage in human renal tubular cells by triggering a pathway of Fas up-regula-tion. HBV and TGF-β1 may play important roles in the mechanism of hepatitis B virus associated glomerulone-phritis.     

凝血酶引起人支气管上皮细胞内活性氧及血小板源生长因子-AB分泌的改变

目的 探讨凝血酶对永生化人支气管上皮细胞(BEP2D细胞)内活性氧产生及血小板源生长因子-AB(PDGF-AB)分泌的影响.方法 不同浓度凝血酶刺激指数生长期的BEP2D细胞,通过检测氧化型氢化乙啶及二氯荧光素荧光强度测定活性氧簇含量变化.采用双抗体夹心ELISA法检测BEP2D细胞分泌PDGF-AB的变化.结果 随着凝血酶浓度增加,反应体系中活性氧明显升高,且有明显的剂量反应关系.凝血酶处理组BEP2D细胞上清液中PDGF-AB含量较对照组上清液显著升高[(770.33+24.29)ng/L vs(117.42±10.85)ng/L,P＜0.01].凝血酶在一定范围内有剂量反应关系.10 U/ml凝血酶刺激48 h后BEP2D细胞分泌PDGF-AB量最大[(817.63+22.53)ng/L].结论 凝血酶既可以引起细胞内氧化应激导致基因毒性,又可以通过刺激BEP2D细胞分泌PDGF-AB发挥自分泌及旁分泌作用.

Abstract：

Objective To investigate the effects of thrombin on reactive oxygen species and plateletderived growth factor-AB (PDGF-AB) in immortalized human bronchial epithelial cells (BEP2D cells).Methods BEP2D cells at exponential growth phase were stimulated by different concentrations of thrombin. The content of active oxygen species was determined by detecting the fluorescence intensity of hydroxyethidium and dichlorofluorescein. The changes of PDGF-AB were measured by double antibody sandwich ELISA assay. Results With the increase of the thrombin concentration, reactive oxygen species significantly increased, and there was a dose-response relationship. The concentration of PDGF-AB in culture supematants in thrombin-treated group was higher than that in control group [(770. 33 +24. 29 )ng/L vs (117. 42+ 10.85) ng/L, P ＜ 0. 01], and there was a dose-response relationship. The concentration of PDGF-AB was maximal [(817.63 +22.53) ng/L] when BEP2D cells were stimulated with 10 U/ml thrombin for 48 hours. Conclusions Thrombin can induce cellular oxidative stress to lead to genotoxicity,and play autocrine and paracrine role by stimulating the secretion of PDGF-AB in BEP2D cells.     

Hydrogen-rich water protects against acetaminopheninduced hepatotoxicity in mice

AIM: To investigate the hepatoprotective effects and mechanisms of hydrogen-rich water(HRW) in acetaminophen(APAP)-induced liver injury in mice.METHODS: Male mice were randomly divided into the following four groups: normal saline(NS) control group, mice received equivalent volumes of NS intraperitoneally(ip); HRW control group, mice were given HRW(same volume as the NS group); APAP + NS group, mice received NS ip for 3 d(5 mL /kg body weight, twice a day at 8 am and 5 pm) after APAP injection; APAP + HRW group, mice received HRW for 3 d(same as NS treatment) after APAP challenge.In the first experiment, mice were injected ip with a lethal dose of 750 mg/kg APAP to determine the 5-d survival rates.In the second experiment, mice were injected ip with a sub-lethal dose of 500 mg/kg.Blood and liver samples were collected at 24, 48, and 72 h after APAP injection to determine the degree of liver injury.RESULTS :Treatment with HRW resulted ina significant increase in the 5-d survival rate compared with the APAP + NS treatment group(60% vs 26.67%, P 0.05).HRW could significantly decrease the serum alanine aminotransferase level(24 h: 4442 ± 714.3 U/L vs 6909 ± 304.8 U/L, P 0.01; 48 h: 3782 ± 557.5 U/L vs 5111 ± 404 U/L, P 0.01; and3255 ± 337.4 U/L vs 3814 ± 250.2 U/L, P 0.05, respectively) and aspartate aminotransferase level(24 h: 4683 ± 443.4 U/L vs 5307 ± 408.4 U/L, P 0.05; 48 h: 3392 ± 377.6 U/L vs 4458 ± 423.6 U/L, P 0.01; and 3354 ± 399.4 U/L vs 3778 ± 358 U/L, respectively) compared with the APAP treatment group.The alkaline phosphatase, total bilirubin and lactate dehydrogenase levels had the same result.Seventy-two hours after APAP administration, liver samples were collected for pathological examination and serum was collected to detect the cytokine levels.The liver index(5.16% ± 0.26% vs 5.88% ± 0.073%, P 0.05) and percentage of liver necrosis area(27.73% ± 0.58% vs 36.87% ± 0.49%, P 0.01) were significantly lower in the HRW-treated animals.The malonyldialdehyde(MDA) contents were significantly reduced in the HRW pretreatment group, but they were increased in the APAP-treated group(10.44 ± 1.339 nmol/mg protein vs 16.70 ± 1.646 nmol/mg protein, P 0.05).A decrease in superoxide dismutase(SOD) activity in the APAP treatment group and an increase of SOD in the HRW treatment group were also detected(9.74 ± 0.46 U/mg protein vs 12.1 ± 0.67 U/mg protein, P 0.05).Furthermore, HRW could significantly increase the glutathione(GSH) contents(878.7 ± 76.73 mg/g protein vs 499.2 ± 48.87 mg/g protein) compared with the APAP treatment group.Meanwhile, HRW could reduce the inflammation level(serum TNF-α: 399.3 ± 45.50 pg/L vs 542.8 ± 22.38 pg/L, P 0.05; and serum IL-6: 1056 ± 77.01 pg/L vs 1565 ± 42.11 pg/L, P 0.01, respectively).In addition, HRW could inhibit 4-HNE, nitrotyrosine formation, JNK phosphorylation, connexin 32 and cytochrome P4502 E expression.Simultaneously, HRW could facilitate hepatocyte mitosis to promote liver regeneration.CONCLUSION: HRW has significant therapeutic potential in APAP-induced hepatotoxicity by inhibiting oxidative stress and inflammation and promoting liver regeneration.     

蛋白激酶C-胞外信号调节激酶1/2信号通路在尼古丁诱导的血管内皮细胞表达纤维溶解酶原激活物抑制物-1中的作用

目的 探讨蛋白激酶C(PKC)-胞外信号调节激酶(ERK)1/2信号通路在尼古丁诱导人脐静脉内皮细胞(HUVECs)表达纤维溶解酶原激活物抑制物-1(PAI-1)中的作用.方法 体外培养HUVECs,采用不同实验条件尼古丁进行干预,ELISA法测定细胞上清液中PAI-1的浓度,观察尼古丁作用的最佳浓度和时间.进一步分别用PKC的抑制剂星型胞菌素staurosporine (STS)和ERK的抑制剂PD98059干预HUVECs,观察PKC或ERK被阻断后对尼古丁诱导的HUVECs 表达PAI-1的影响,ELISA测定各组细胞上清液中PAI-1蛋白的表达水平,RT-PCR检测各组细胞PAI-1 mRNA的表达.结果 100 μmol/L尼古丁组PAI-1蛋白水平[(22.6 ± 1.1)μg/L]明显高于对照组[(14.2± 2.8)μg/L,q=5.64,P＜0.05];以100 μmol/L 尼古丁分别与HUVECs孵育0、4、6、8、12及24 h,各组PAI-1蛋白表达呈时间依赖性升高,并在12 h达到高峰(F=32.063,P＜0.05);尼古丁组PAI-1 mRNA及蛋白含量[(1.32±0.20),(21.08±0.83)μg/L]明显高于对照组[(0.73±0.10),(13.39± 0.93)μg/L,q=8.43、11.97,均P＜0.05];尼古丁+STS组PAI-1 mRNA及蛋白含量[(1.07±0.10),(16.19±2.15)μg/L]较尼古丁组降低(q=5.61、7.61,均P＜0.05),但仍高于对照组(q=7.84、4.36,均P＜0.05);尼古丁+ PD98059组PAI-1 mRNA及蛋白表达[(1.12±0.11),(17.52±1.72)μg/L]低于尼古丁组(q=4.68、5.54,均P＜0.05),仍高于对照组(q=8.77、6.43,均P＜0.05).结论 PKC-ERK1/2信号通路在尼古丁诱导的血管内皮细胞PAI-1表达上调中发挥一定作用.

Abstract：

Objective To explore the role of protein kinase C (PKC)- extracellular signal-regulated kinase (ERK)1/2 signal pathway in the process of plasminogen activator inhibitor-1(PAI-1) protein and mRNA expression in cultured human umbilical vein endothelial cells(HUVECs) induced by nicotine. Methods HUVECs were cultured to examine the effect of nicotine on the expression of secreting PAI-1 in HUVECs on different experimental conditions. The expression of PAI-1 protein was measured by ELISA. PKC inhibitor staurosporine (STS)and ERK1/2 inhibitor PD98059 were used to detect PKC or ERK1/2 function on the expression of PAI-1 in HUVECs induced by nicotine. The PAI-1 mRNA expression was determined by RT-PCR. Results The expression level of PAI-1 protein in 100 μmol/L nicotine treated group [(22.6±1.1) μg/L] increased significantly compared to the control group [(14.2±2.8) μg/L; q=5.64,P<0.05]. After stimulation with 100 μmol/L nicotine for 0,4,6,8,12 and 24 h, the levels of PAI-1 protein increased over time and reached the peak at 12 h (F=32.063,P<0.05).The PAI-1 mRNA and protein expression in nicotine treated group [(1.32±0.20), (21.08 ± 0.83) μg/L] increased significantly compared to the control group [(0.73±0.10), (13.39±0.93) μg/L; q=8.43,11.97,all P<0.05].Compared with nicotine treated group , the PAI-1 mRNA and protein expression in nicotine and STS treated group [(1.07±0.10),(16.19±2.15) μg/L] decreased significantly(q=5.61,7.61, all P<0.05), but still higher than the control group (q=7.84,4.36, all P<0.05). In nicotine and PD98059 treated group, the PAI-1 mRNA and protein expression [(1.12±0.11),(17.52±1.72) μg/L] decreased significantly compared to the nicotine treated group(q= 4.68,5.54, all P<0.05), still higher than the control group (q=8.77,6.43, all P<0.05). Conclusion PKC-ERK1/2 signal pathway may play a partial role in the up-regulation of PAI-1 induced by nicotine in HUVECs.     

Therapeutic effects of caspase-1 inhibitors on acute lung injury in experimental severe acute pancreatitis

AIM： To assess the therapeutic effect of Caspase-1 inhibitors （ICE-I） on acute lung injury （ALI） in experimental severe acute pancreatitis （SAP）.
METHODS： Forty-two SD rats were randomly divided into 3 groups： healthy controls （HC, n = 6）; SAP-S group （n = 18）; SAP-ICE-i group （n = 18）. SAP was induced by retrograde infusion of 5% sodium taurocholate into the bile-pancreatic duct. HC rats underwent the same surgical procedures and duct cannulation without sodium taurocholate infusion, in SAP-S group, rats received the first intraperitoneal injection of isotonic saline 2 h after induction of acute pancreatitis and a repeated injection after 12 h. In SAP-ICE-I group, the rats were firstly given ICE inhibitors intraperitoneally 2 h after induction of pancreatitis. As in SAP-S group, the injection was repeated at 12 h. Serum 1L-1β was measured by EUSA. Intrapulmonary expression of Caspase-1, IL-1β and IL-18 mRNA were detected by semi-quantitative RT-PCR. The wet/dry weight ratios and histopathological changes of the lungs were also evaluated.
RESULTS： Serum IL-1β levels in SAP-S group were 276.77 ± 44.92 pg/mL at 6 h, 308.99 ± 34.95 pg/mL at 12 h, and 311.60 ± 46.51 pg/mL at 18 h, which were increased significantly （P 〈 0.01, vs HC）. in SAP- ICE-I group, those values were decreased significantly （P 〈 0.01, vs SAP-S）. intrapulmonary expression of Caspase-1, IL-1β and IL-18 mRNA were observed in the HC group, while they were increased significantly in the SAP-S group （P 〈 0.01, vs HC）. The expression of IL-lβ and IL-18 mRNA were decreased significantly in the SAP- ICE-I group （P 〈 0.01, vs SAP-S）, whereas Caspase-1 mRNA expression had no significant difference （P 〉 0.05）. The wet/dry weight ratios of the lungs in the SAP-S group were increased significantly （P 〈 0.05 at 6 h, P 〈 0.01 at 12 h and 18 h, vs HC） and they were decreased significantly in the SAP-ICE-I group （P 〈 0.05, vs SAP-S）.Caspase-1 inhibitors ameliorated the severit