Expression Of ATF6?GRP78 And CHOP In Vitro Liver Cell Models With Serum Of Rats With Obstructive Jaundice And The Mechanism Of Traditional Chinese Medicine

Objective: In this study,a rat model of obstructive jaundice liver injury was established to observe the expression changes of ATF6,GRP78 and CHOP after intervention of rat normal hepatocytes by different serums.The mechanism of hepatic injury of obstructive jaundice and the hepatoprotective effect of Chinese medicine artemisia capillaris decoction were explored.The mechanism of action provides a theoretical basis for the rational use of heat-clearing and dampness-removing Chinese medicine during the clinical perioperative period.Methods: 1.45 adult male Sprague-Dawley rats(250-300 g)were selected and animal grade was clean grade.They were purchased from Animal Center of Nankai Hospital of Tianjin.Rats were randomized into groups: Sham-operated control group(S group,15 rats);Yinchenhao decoction group(Y group,15 rats);Obstructive jaundice group(O group,15 rats);In group O,the xiphoid ventral midline incision was taken into the abdomen of a rat to separate the bile ducts and double bile ducts were aseptically ligated to establish a rat model of obstructive jaundice.The S group was only involved in abdominal exploration and the common bile duct was not ligated;the Y group was fixed daily.Time,according to each dose of 1ml/100 g with Yinchenhao soup gavage.All rats in the 3 groups were bled from the abdominal aorta 1 week later and serum was collected by centrifugation.Part of serum in three groups were used to detect aspartate aminotransferase(AST),alanine aminotransferase(ALT),and serum total bilirubin((TBIL),serum direct bilirubin(DBIL)content,and the remaining serum is kept for future use.2.The rat normal hepatocyte BRL-3A line was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences and the cells were divided into groups A,B and C.Group A cells were cultured with normal rat serum,group B cells were cultured with obstructive jaundice serum,and group C cells were cultured with obstructive jaundice serum plus medium containing serum.After 6 hours,24 hours,and 48 hours after culturing,the cells were harvested,and the hepatocytes and supernatant were collected after centrifugation.The protein expression of ATF6,GRP78 and CHOP in hepatocytes of each group was detected by Western-blot,and the gene expression of ATF6,GRP78 and CHOP was detected by reverse transcription polymerase chain reaction(RT-PCR).The supernatant was measured for ALT and AST content.Results: 1.Changes in serum biochemical indicators: 1 Animal experiment: The serum levels of ALT,AST,TBIL and DBIL in group O were significantly higher than those in group S,and the difference was statistically significant(P<0.05).2 Cellular experiment: Compared with group A,the levels of ALT and AST in cell culture fluid of group B and group C at various time points were higher than that of group A,the difference was statistically significant(P<0.05).Compared with group B,C,the levels of ALT and AST in the group cell culture fluid were lower than those in group B at each time point,but higher than that in group A,statistically significant difference(P < 0.05).2.The expression of ATF6,GRP78 and CHOP in hepatocytes of each group was detected by Western-blot method: actin was taken as the internal reference,group A was the control group,and the expression levels of ATF6,GRP78 and CHOP protein in groups B and C at various time points were higher than that of group A.Compared with group B,group C at various time points ATF6,GRP78 and CHOP protein expression levels were lower than group B,but higher than those in group A,statistically significant difference(P < 0.05).Compared with group B,,but higher than group A,statistically significant difference(P < 0.05).3.Real-time PCR method was used to detect the expression of ATF6,GRP78 and CHOP mRNA in hepatocytes of each group: GAPDH was taken as the internal reference,group A was the control group,and the expression levels of ATF6,GRP78 and CHOP mRNA were detected at each time point in groups B and C.All of them were higher than group A,and the difference was statistically significant(P<0.05).Compared with group B,the expression levels of ATF6,GRP78,and CHOP mRNA in group C were lower than those in group B at each time point,but higher than those in group A,statistical significance(P<0.05).Conclusions: 1.The obstructive jaundice rat serum in vitro interventional rat normal liver cells can successfully establish obstructive jaundice liver injury cell model.In this cell model,the ATF6 signaling pathway in ERS and the CHOP-induced apoptosis pathway are activated,which may be the mechanism of liver injury caused by obstructive jaundice.2.The blood serum of Yinchenhao decoction can improve the liver function of the obstructive jaundice liver injury cell model.The mechanism of action may be through the regulation of ATF6 expression at the molecular level,and to reduce the degree of ERS mediated by ATF6 when obstructive jaundice is happened to reduce hepatocyte apoptosis,thereby improving liver function.3.In clinical treatment of patients with obstructive jaundice,on the basis of a reasonable choice of surgical methods,can be differentiated using Yin Chen Hao Tang,can improve the abnormal liver function caused by obstructive jaundice,integrated traditional Chinese and Western medicine treatment may obtain better clinical efficacy.