Yinchenhao decoction attenuates obstructive jaundice-induced liver injury and hepatocyte apoptosis by suppressing protein kinase RNA-like endoplasmic reticulum kinase-induced pathway

BACKGROUND Chronic biliary obstruction results in ischemia and hypoxia of hepatocytes,and leads to apoptosis.Apoptosis is very important in regulating the homeostasis of the hepatobiliary system.Endoplasmic reticulum(ER)stress is one of the signaling pathways that induce apoptosis.Moreover,the protein kinase RNA-like endoplasmic reticulum kinase(PERK)-induced apoptotic pathway is the main way;but its role in liver injury remains unclear.Yinchenhao decoction(YCHD)is a traditional Chinese medicine formula that alleviates liver injury and apoptosis,yet its mechanism is unknown.We undertook this study to investigate the effects of YCHD on the expression of ER stress proteins and hepatocyte apoptosis in rats with obstructive jaundice(OJ).AIM To investigate whether YCHD can attenuate OJ-induced liver injury and hepatocyte apoptosis by inhibiting the PERK-CCAAT/enhancer-binding protein homologous protein(CHOP)-growth arrest and DNA damage-inducible protein 34(GADD34)pathway and B cell lymphoma/leukemia-2 related X protein(Bax)/B cell lymphoma/leukemia-2(Bcl-2)ratio.METHODS For in vivo experiments,30 rats were divided into three groups:control group,OJ model group,and YCHD-treated group.Blood was collected to detect the indicators of liver function,and liver tissues were used for histological analysis.For in vitro experiments,30 rats were divided into three groups:G1,G2,and G3.The rats in group G1 had their bile duct exposed without ligation,the rats in group G2 underwent total bile duct ligation,and the rats in group G3 were given a gavage of YCHD.According to the serum pharmacology,serum was extracted and centrifuged from the rat blood to cultivate the BRL-3A cells.Terminal deoxynucleotidyl transferase mediated dUTP nick end-labelling(TUNEL)assay was used to detect BRL-3A hepatocyte apoptosis.Alanine aminotransferase(ALT)and aspartate transaminase(AST)levels in the medium were detected.Western blot and quantitative real-time polymerase chain reaction(qRT-PCR)analyses were used to detect protein and gene expression levels of PERK,CHOP,GADD34,Bax,and Bcl-2 in the liver tissues and BRL-3A cells.RESULTS Biochemical assays and haematoxylin and eosin staining suggested severe liver function injury and liver tissue structure damage in the OJ model group.The TUNEL assay showed that massive BRL-3A rat hepatocyte apoptosis was induced by OJ.Elevated ALT and AST levels in the medium also demonstrated that hepatocytes could be destroyed by OJ.Western blot or qRT-PCR analyses showed that the protein and mRNA expression levels of PERK,CHOP,and GADD34 were significantly increased both in the rat liver tissue and BRL-3A rat hepatocytes by OJ.The Bax and Bcl-2 levels were increased,and the Bax/Bcl-2 ratio was also increased.When YCHD was used,the PERK,CHOP,GADD34,and Bax levels quickly decreased,while the Bcl-2 levels increased,and the Bax/Bcl-2 ratio decreased.CONCLUSION OJ-induced liver injury and hepatocyte apoptosis are associated with the activation of the PERK-CHOP-GADD34 pathway and increased Bax/Bcl-2 ratio.YCHD can attenuate these changes.

生物钟基因Period2对卵巢癌裸鼠移植瘤生长抑制作用的机制研究

目的:探讨生物钟基因Period2对卵巢癌裸鼠移植瘤生长抑制的作用机制。方法:采用卵巢癌SKVO3细胞株构建裸鼠卵巢癌移植瘤,利用基因转染技术外源性导入重组基因质粒Period2。采用Real-time PCR法和Western bolt法检测移植瘤中Period2表达,治疗期间测量移植瘤体积。治疗后2周处死裸鼠,称取瘤重,采用Real-time PCR和Western blot法检测肿瘤组织中Bcl-2和Bax基因的表达。结果:外源性导入Period2基因在裸鼠移植瘤肿瘤组织中成功稳定表达。与对照组比较,Period2基因过表达组的肿瘤生长速度减慢,肿瘤体积和重量降低,抑瘤率升高,凋亡基因Bax表达明显升高,凋亡抑制基因Bcl-2表达明显降低,差异均有统计学意义(P<0.05)。结论:Period2可能通过下调肿瘤凋亡抑制基因Bcl-2,促进凋亡基因Bax表达,促进肿瘤细胞凋亡,从而抑制卵巢癌的生长转移,发挥抑瘤作用。

表没食子儿茶素没食子酸酯对人宫颈癌细胞株CaSki增殖、凋亡及凋亡相关基因表达的影响观察

目的观察表没食子儿茶素没食子酸酯(EGCG)对人宫颈癌细胞株CaSki增殖、凋亡及凋亡相关基因表达的影响。方法取对数生长期CaSki细胞分为实验组和对照组,实验组分别加入不同浓度(5 mg/L、10 mg/L、20 mg/L和40 mg/L)的EGCG溶液和5%胎牛血清培养液,记为5 mg/L组、10 mg/L组、20 mg/L组、40 mg/L组;对照组加入等体积的5%胎牛血清完全培养液。各组继续培养48 h后,采用MTT法测算各组细胞增殖抑制率,使用流式细胞仪检测各组细胞凋亡率,采用荧光实时定量PCR法检测各组细胞凋亡相关基因半胱天冬氨酸酶-3(Caspase-3)、P53、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)mRNA。结果5 mg/L组、10 mg/L组、20 mg/L组、40 mg/L组、对照组细胞增殖抑制率分别为5.14%±0.54%、15.42%±1.22%、27.16%±2.11%、40.51%±1.57%、0,细胞凋亡率分别为1.95%±0.21%、5.39%±1.54%、15.14%±2.12%、27.46%±3.50%、0.74%±0.21%,组间相比,P均<0.05。实验组Caspase-3、P53、Bax mRNA相对表达量均升高,Bcl-2 mRNA相对表达量均降低,且呈一定的浓度依赖性。结论EGCG可抑制CaSki细胞增殖,促进其凋亡;EGCG促进CaSki细胞凋亡的作用可能与其上调凋亡相关基因Caspase-3、P53、Bax、下调凋亡相关基因Bcl-2表达有关。

工频电磁场对作业人员淋巴细胞凋亡相关基因mRNA水平影响研究

目的探讨工频电磁场对作业人员周围血淋巴细胞凋亡相关基因P53、B细胞淋巴瘤/白血病基因2(Bcl-2)及其相关X蛋白基因(Bax)的mRNA水平及血浆天冬氨酸特异性半胱氨酸蛋白酶3(Caspase-3)蛋白水平的影响。方法随机选择某500 kV变电站工龄5年以上的45名工频电磁场巡检作业工人作为接触组,并按照1∶1配对选取该变电站45名行政管理及后勤人员作为对照组。2组研究对象均采集周围静脉血,提取淋巴细胞,采用实时荧光定量聚合酶链式反应测定其淋巴细胞中P53、Bcl-2和Bax的mRNA水平,采用酶联免疫吸附实验法测定血浆Caspase-3蛋白表达水平。数据采用中位数与四分位数间距[M(P25,P75)]描述。结果接触组和对照组人群P53mRNA相对表达量(RQ)分别为4.00(3.60,4.37)和4.21(3.70,4.64),Bcl-2 mRNA RQ为4.07(3.47,4.50)和4.04(3.48,4.35),Bax mRNA RQ为4.37(4.02,4.89)和4.53(4.11,4.85),Bcl-2与Bax的mRNA RQ比值为0.89(0.81,1.01)和0.87(0.80,0.95),Caspase-3蛋白表达水平分别为7.56(4.05,15.32)和9.70(6.40,21.13)pmol/L。2组上述5个指标比较,差异均无统计学意义(P>0.05)。结论长期接触工频电磁场对机体淋巴细胞凋亡相关基因P53、Bcl-2、Bax的mRNA水平及血浆Caspase-3蛋白水平影响不明显。