| Objective: To prepare the rat model of cirrhotic endotoxemia by using carbon tetrachloride(CCL4)combined modeling method to study the therapeutic effect of duxiao Ganqing Pill on cirrhotic endotoxemia.The changes of endotoxin content,serum tumor necrosis factor(TNF-?),interleukin-1?(IL-1?),interleukin-6(IL-6),TLR4,My D88,NF-?B protein and m RNA in liver and ileum,expression of TLR4,NF-?B,RIPI and TRAF6 in liver and ileum were observed.In order to provide a new direction for the treatment of endotoxemia in cirrhotic rats,the therapeutic effect and mechanism of duxiao Ganqing pill were studied.Methods: 66 rats were randomly divided into normal group and model group,6 in normal group and 60 in model-making group.The normal group was fed normally,and the model group was given 40% carbon tetrachloride olive oil solution in the back of the rat,at a first dose of 0.5 ml/100 g dose,followed by a dose of 0.3 ml/100 g twice a week.Simultaneously fed with 89.5% corn flour,0.5% cholesterol,10% lard mixed feed,and with 10% alcohol as its only beverage,the modeling cycle was 8 weeks.During the modeling process,there were 17 deaths in the model group.At the end of the model,3 rats were tested for endotoxin content and liver structure changes.The model group was randomly divided into model group,lactulose group(2.1 g/kg),high,medium and low dose duxiao Ganqing pill group(223.4 mg/kg,111.7 mg/kg,58.9 mg/kg),8 in each group.The normal group and the model group were given distilled water,the lactulose group was given lactulose solution once a day at the appeal dose,and the duxiao Ganqing pill group was given once a day,and each group was given continuously for 8 weeks.Hepatic and ileal tissues were used for HE staining to observe histopathological changes of liver and ileum;The levels of endotoxin were measured by limulus reagent and chromatin method;serum TNF-?,IL-1?,IL-6 were detected by enzyme-linked immunosorbent assay;TLR4,My D88,NF-?B were detected by RT-PCR;protein expression of TLR4,My D88 and NF-?B was detected by Western blot;TLR4,NF-?B,RIPI and TRAF6 were detected by immunohistochemical staining.Results: 1.Effect of duxiao Ganqing Pill on Endotoxin in Blood of Rats Compared with the normal group,the endotoxin content in the model group increased,and there was a significant difference(P<0.01);compared with the model group,the endotoxin content in the high,middle and low dose groups of duxiaoganqing pill and lactulose group decreased significantly(P<0.01);compared with the lactulose group,the endotoxin content in the high and middle dose groups had no significant difference(P > 0.05),and there was a significant difference in the low dose group(P<0.01);and Compared with the low dose group,the endotoxin content in the high and middle dose groups of duxiaoganqing pills decreased significantly(P< 0.01).2.Effect of duxiao Ganqing Pill on Serum TNF-?,IL-1? and IL-6 in Rats with Endotoxemia in Cirrhosis Compared with the normal group,the relative expression of TLR4 and NF-? B m RNA in the model group was significantly increased(P<0.01);compared with the model group,the TLR4 and NF-?B m RNA in the high,middle and low dose groups of lactulose group and duxiaoganqing pill were significantly increased(P<0.01)The relative expression of TLR4,NF-?B m RNA in the liver of rats in high and middle dose groups decreased significantly(P < 0.05),but there was no significant difference in the relative expression of TLR4,NF-?B m RNA between high and middle dose groups(P>0.05).Compared with the normal group,the level of IL-6 in the model group was significantly higher(P<0.01);compared with the model group,the level of IL-6 in the lactulose group,the high dose group,the middle dose group and the low dose group was significantly lower(P<0.01);compared with the lactulose group,the level of IL-6 in the low dose group was not significantly different(P>0.05),the level of IL-6 in the high dose group and the middle dose group was significantly higher(P<0.05);compared with the low dose group,There was a significant difference between high and middle dose of serum IL-6(P<0.05).3.Effect of duxiao Ganqing pill on expression of TLR4,My D88 and NF-?B m RNA in liver of rats with cirrhotic endotoxemia Compared with the normal group,the relative expression of TLR4?My D88?NF-?B m RNA in the liver of the model group was significantly increased(P<0.01);compared with the model group,TLR4?My D88?NF-?Bm RNA in the liver of the lactulose group,the high, middle and low dose groups were significantly increased(P<0.01)Compared with the low dose group,the relative expression of TLR4?My D88?NF-?Bm RNA in the liver of the high and middle dose groups decreased significantly(P<0.01),but there was no significant difference between the high and middle dose groups(P>0.05).Compared with the normal group,the relative expression of My D88 m RNA in the liver of the model group was significantly increased(P<0.01);compared with the model group,the relative expression of My D88 m RNA in the liver of the lactulose group,the high,middle and low dose groups of duxiaoganqing pill was significantly decreased(P<0.01);compared with the drug group,the high,middle and low dose groups of duxiaoganqing pill were significantly reduced(P<0.01);compared with the drug group There was no difference in the relative expression of m RNA(P>0.05).4.Effect of duxiao Ganqing pill on expression of TLR4,My D88 and NF-?B m RNA in ileal of rats with cirrhotic endotoxemia Compared with the normal group,the relative expression of TLR4 m RNA in the rat ileum in the model group was significantly increased(P<0.01).Compared with the model group,the relative expression of TLR4 m RNA in the ileum in lactulose group,high,medium and low dose duxiao Ganqing pill groups was significantly decreased(P<0.01).Compared with the lactulose group,there was no difference in the relative expression of TLR4 m RNA in the high dose,middle and low-dose duxiao Ganqing pill group in rat ileum(P>0.05).Compared with the normal group,the relative expression of TLR4,My D88 and NF-? B protein in the ileum of the model group was significantly increased(P<0.01);compared with the model group,the relative expression of TLR4,My D88 and NF-? B Protein in the ileum of the lactulose group,the high and middle dose groups was significantly decreased(P<0.01);the expression of TLR4,My D88 and NF-?B m RNA in ileum of rats in high,middle compared with low dose groups were significantly different(P<0.05).5.Effect of duxiao Ganqing pill on expression of TLR4,My D88 and NF-?B protein in liver of rats with cirrhotic endotoxemia Compared with the normal group,the relative expression of TLR4 and NF-?B protein in the liver of the model group increased significantly(P<0.01);compared with the model group,the relative expression of TLR4 and NF-?B protein in the liver of the high,middle and low dose groups of lactulose group and duxiaoganqing pill decreased significantly(P<0.01);compared with the low dose group of duxiaoganqing pill,the relative expression of TLR4 and NF-?B protein in the liver of the high and middle dose groups decreased significantly(P<0.01);compared with the drug group,compared with the low dose group of duxiaoganqing pill The relative expression of TLR4 and NF-?B protein in the liver of rats in the high and middle dosages of duxiaoganqing pill group was not significantly different(P>0.05).Compared with the normal group,the relative expression of My D88 protein in the liver of the model group was significantly increased(P<0.01);compared with the model group,the rats in the lactulose group and the duxiaoganqing pill high,middle and low dose group had a significant difference(P<0.01)There was no significant difference in the relative expression of My D88 protein between the drug groups(P>0.05).6.Effect of duxiao Ganqing pill on expression of TLR4,My D88 and NF-?B protein in ileal of rats with cirrhotic endotoxemia Compared with the normal group,the relative expression of TLR4 and My D88 protein in the ileum of the model group was significantly increased(P<0.01);compared with the model group,the relative expression of TLR4 and My D88 protein in the ileum of the lactulose group and the high,middle and low dose group of duxiaoganqing pill was significantly decreased(P<0.01);compared with the lactulose group,the relative expression of TLR4 and myd8 protein in the ileum of the high and medium rats was no difference(P>0.05).The expression of TLR4 and My D88 protein in the ileum of high and middle group was significantly higher than that of low dose group(P<0.05).Compared with the normal group,the relative expression of NF-?B protein in the ileum of the model group was significantly increased(P<0.01);compared with the model group,the rats in the lactulose group,the high,middle and low dose groups had significant difference(P<0.01)Compared with lactulose group,there was no significant difference in the relative expression of NF-?B in ileum between high and low dose groups(P>0.05),and there was significant difference in the relative expression of NF-?B in middle dose group (P<0.01).Compared with the low dose group,there was no difference in the expression of NF-?B protein in the high dose group(P>0.05),and there was significant difference in the expression of NF-?B protein in the middle dose group(P<0.01).7.Effect of duxiao Ganqing pill on expression of TLR4,NF-?B,RIPI and TRAF6 immunohistochemical staining in liver of cirrhotic rats with endotoxemia Compared with the normal group,the number of NF-?B positive cells in the liver of the model group increased significantly(P<0.01);compared with the model group,the number of NF-?B positive cells in the lactulose group and the high,medium and low dose groups of duxiaoganqing pills decreased significantly(P<0.01);compared with the low dose ratio of duxiaoganqing pills,the number of NF-?B positive cells in the high and medium dose groups decreased significantly(P<0.05).Compared with the normal group,the number of TLR4,RIP1 and TRAF6 positive cells in the liver of the model group increased significantly(P<0.01);compared with the model group,the number of positive cells in the lactulose group and duxiaoganqing pill high,middle and low dose groups decreased significantly(P<0.01);compared with the drug group,the number of positive cells in the liver of the high and middle dose groups had no significant difference(P>0.05).8.Effect of duxiao Ganqing pill on expression of TLR4,NF-?B,RIPI and TRAF6 immunohistochemical staining in ileum of cirrhotic rats with endotoxemia Compared with the normal group,the number of NF-?B and RIP1 positive cells in ileum of the model group increased significantly(P<0.01);compared with the model group,the number of positive cells in lactulose group and duxiaoganqing pill high,middle and low dose group decreased significantly(P<0.01);compared with the lactulose group,the number of positive cells in duxiaoganqing pill high,middle group had no difference(P > 0.05).Compared with the low dose group,the number of ileal positive cells in the high and middle dose groups of duxiaoganqing pills was different(P<0.05).There was significant difference in the number of NF-?B and RIP1 positive cells in the ileum between the high dose group and the middle dose group(P<0.05).Compared with the normal group,the number of TLR4 and TRAF6 positive cells in the ileum of the model group increased significantly(P<0.01);compared with the model group,the number of TLR4 and TRAF6 positive cells in the high dose,middle dose and low dose groups of lactulose group and duxiaoganqing pill decreased significantly(P<0.01);compared with the low dose group,the number of TLR4 and TRAF6 positive cells in the high dose and middle dose groups of duxiaoganqing pill decreased significantly(P<0.01),especially in the lactulose group There was no difference in the number of ileal positive cells between the high and middle dose groups(P>0.05).Conclusion: 1.Duxiao Ganqing pill group can reduce the plasma endotoxin content of cirrhotic endotoxin rats.2.Duxiao Ganqing pill group can reduced effectively the expression of TNF-?,IL-1? and IL-6 in serum.3.Duxiao Ganqing pill group can reduce the expression of TLR4,My D88,NF-?B m RNA and protein in liver tissue and ileum.4.The mechanism by which duxiao Ganqing Pill protects against endotoxemia in cirrhosis may be to inhibit the TLR4/NF-?B signaling pathway by inhibiting the release of inflammatory factors. |
PDF Full Text Request