| ObjectiveIn this study,replicating the severe model of cholestatic hepatitis by choledochal ligation(CBDL)in rats and observing the characteristics of hepatic injury caused by cholestatic hepatitis induced by this method.Through the study of the mechanism of drug efficacy,combined with physical signs and modern indicators to investigate the protective effect of Qingkailing injection on liver injury in rats with cholestatic hepatitis and the intervention of injecting on the corresponding syndromes,injection is related to anti-inflammatory and anti-endoplasmic reticulum stress.The effect of the route.Methods(1)Male Sprague-Dawley rats,SPF grade,35 animals,body weight 250-270 g,CBDL model was prepared by surgical operation.The free common bile duct was double-ligated and then cut in the intemodes and randomly divided into 4 groups.Blank control group(blank group),CBDL model group(model group),the above two groups were given an equal volume of saline,UDCA positive treatment group(UDCA group),the model was given by gavage at the same time 78mg/kg Medicine,Qingkailing injection treatment group(QingKaiLing group),the model was given by intraperitoneal injection of 3mL/kg dose,two weeks intervention time.At the same time,two weeks of observations were performed on each group of rats with cholestatic hepatitis,and basic sign information was collected and recorded(if there are 1 and 1 are not recorded),basic weight measurement and drinking water were performed once a week,dietary intake,basal body temperature,and ear micro-flow;collection of tongue and claws before sampling.(2)Analyze the corresponding information collected during the experiment,use the cluster analysis method and decision tree algorithm to summarize the model features on the information table of the survey,and perform RGB digital quantification on the collected tongue images and claw images of rats.Analyze statistical processing.(3)HE staining,Masson staining,and TUNEL apoptosis staining were performed on the liver tissue sections of rats.(4)Determination of hydroxyproline content(Hyp)and liver index in rat liver tissue.(5)Serum ALT(aspartate aminotransferase),ALT(glutamine transaminase),GGT(gamma glutamyl transpeptidase),TBIL(total bilirubin),and DBIL(direct bilirubin)in serum of rats IBIL(indirect bilirubin)was tested.(6)The inflammatory cytokines TNF-α,IL-6,IL-1β and NLRP3 in serum and rat liver tissue homogenate supernatants were detected by enzyme-linked immunosorbent assay(Elisa).(7)The expression of GRP78,Caspase-12,NLRP3,SLC10A1 in liver tissue of rats was detected by Western blotting.It was detected that Qingkailing injection played a therapeutic role by regulating endoplasmic reticulum stress and inhibiting inflammatory reaction.Results(1)ALT,AST,and GGT levels of liver function were significantly higher in rats with cholestatic hepatitis compared with those in normal rats,with statistically significant differences(P<0.001).The indexes of ALT,AST and GGT were lower,and the difference was significant(p<0.05).The hepatic index of rats in the model group was increased with significant statistical difference(P<0.001).The liver index of Qing Kai Ling group rats was decreased,with Significant statistical difference(P<0.001);Liver hydroxyproline content increased significantly statistically significant(P<0.05),Qingkailing group rats liver hydroxyproline content decreased,with significant Statistical differences(P<0.01);HE stained results showed that in the Qing Kai Ling group,the interstitial tubules were hyperplasia in the fibrous space,slight edema of cells and inflammatory cells infiltrated in the interstitial space,and the hepatocytes were arranged in disorder.MASSON staining prompted Qing Kai Ling group There were more collagen fibrils in the portal area and fibrotic septal tubule hyperplasia,but the hepatic parenchymal cells were not completely segmented and no bridging of fibers was evident.(2)The levels of TNF-a and IL-6 in the serum of rats with cholestatic hepatitis in the model group were higher than those in the normal group(P<0.001);TNF-α and IL-6 in the liver tissue the content of IL-1(3 and NLRP3 was significantly higher than that of the normal group,and the difference was statistically significant(P<0.01).The contents of TNF-a,IL-6,IL-1β and NLRP3 in the liver tissue of the Qingkailing group were significantly higher than those of the normal group.The contents of TNF-a,IL-6 and IL-1β in serum of the rats in the Qingkailing group were significantly lower than those in the model group(P<0.05);the difference was significant(P<0.05);The relative expression levels of GRP78,Caspase-12,NLRP3,and SLC10A1 protein were increased in rats and normal rats,and the difference was significant(P<0.001).The relative expression levels of GRP78,Caspase-12,NLRP3,and SLC10A1 proteins in the liver tissue of QingKaiLing group decreased significantly(P<0.01).The apoptotic staining results indicated that the QingKaiLing group liver the amount of apoptotic cells in the tissue was between the model group and the UDCA treatment group.(3)Signs collected combined with quantitative indicators Observed in rats with cholestatic hepatitis manifested as fever,thirst,irritability,epistaxis,furious bite,dark yellow urine,reddish tongue and other symptoms;and normal Compared with the normal rats,the model group had higher basal body temperature(P<0.05),and the Qing Kai Ling group had lower body temperature than the model group(P<0.05).0.001);Auricle micro-flow velocity increased in the model group,and the auricular micro-flow velocity was lower in the Qingkailing group than in the model group(P<0.05);The R+G+B)value was significantly higher(P<0.001).The Red value of tongue in the Qingkailing group was lower than that of the model group(P<0.01);the paw group in the model group was higher than the normal group R/(R+G+B)values were significantly higher(P<0.001).Qing Kai Ling group had lower Red value than normal group(P<0.01).Model group rats had low blood viscosity and low blood viscosity.The whole-blood viscosity of the high-cut group and the high-cut group was higher than that of the normal group(P<0.05).The whole blood viscosity of the Qingkailing group was lower than that of the normal group(P<0.05).The physical characteristics of the model group were as follows:Thin,bitter,bite,unsteady gait,less shiny hair,yellow hair,dark eyes and a senseless,red ears,nostrils blood marks,lip dark red,yellow urine,less,claw color red.Conclusions(1)The CBDL surgical model can better simulate the characteristics of hepatic injury in cholestatic hepatitis.Qingkailing injection can better protect the liver damage caused by cholestatic hepatitis,but the process of hepatic fibrosis in cholestatic hepatitis may be it cannot be reversed.(2)Cholestatic hepatitis caused an inflammatory response and may induce endoplasmic reticulum stress at the same time;Qingkailing injection can treat CBDL-induced bile by anti-inflammatory and anti-endoplasmic reticulum stress pathways.Sillous hepatitis,to a certain extent,can inhibit the absorption of sodium-dependent bile salts and relieve the progress of cholestasis.(3)Severe model of cholestatic hepatitis,the use of physical signs collected in combination with a variety of quantitative indicators validation method,the results found that CBDL cholestatic hepatitis in rats consistent with the characteristics of hot blood stasis syndrome,Qingkailing injection intervention on the syndrome effect. |
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