| Objective:By replicating the INH/RFP liver injury model in vivo and in vitro,we compared the liver protective effects of different active parts and component of Sagittaria Sagittifolia,and further study the protection mechanism of Sagittaria Sagittifolia extracts on hepatic injury induced by co-administration of isoniazid and rifampicin,to provide a scientific experimental basis for the development and utilization of Sagittaria Sagittifolia.Methods:1 HepG2 cells were incubated and randomly divided into 9 groups:blank control group,normal control group,INH/RFP model(injured by co-administration of isoniazid and rifampicin)group,positive control(vitamin E)group and different active parts of Sagittaria Sagittifolia,six parallel holes were set in each group.After giving different treatment,compared the liver protective effects by measuring the survival rate of liver cells and detecting the activities of LDH,ALT,AST and MDA in the cell culture media.2 According to the random number table method,90 mice were randomly divided into 9 groups,they were the normal control group,the INH/RFP model(replicated with isoniazid and rifampicin)group,the low,middle and high dose water extract from Sagittaria Sagittifolia group,the low,middle and high dose Sagittaria Sagittifolia polysaccharide(SSP)group and the positive control(silymarin)group,with 10 rats in each group.After continuous gavage of 30 d,through the detection of mouse general conditions,hepatic index,biochemical indexes of ALT,AST,LDH,MDA and GSH activity,as well as histopathological observation of liver tissue,we comparedthe liver protection effect of the water extract from Sagittaria Sagittifolia and SagittariaSagittifolia polysaccharide.3 Based on the Nrf2-ARE signaling pathway,the expression of Nrf2 and Keapl in liver tissues were detected by using RT-PCR and Western blot methods to assess the effect of Sagittaria Sagittifolia extract.Results:1 The cell viability results showed that compared with the normal control group,the cell survival rate of the model group was significantly reduced(P<0.01);compared with the model group,the viability of the cells of Sagittaria Sagittifolia different active parts and vitamin E were increased,from 56.0%to 57.9%,63.8%,61.4%,86.4%,83.3%,and 87.7%and the Sagitt butanol group had no statistically significant difference(P>0.05),the other groups had difference significantly(P<0.01).The results of biochemical indicators of cell culture fluid showed that the levels of LDH,ALT,AST and MDA of the model group were significantly higher than those in the normal group,the difference was statistically significant(P<0.01).Compared with the model group,the levels of LDH,ALT,AST and MDA in the cell culture supernatant decreased after Sagittaria Sagittifolia different active parts and vitamin E treatment.Among them,Sagittati oil ether group,Sagittaria mongolica extract group,and the water extract from Sagittaria Sagittifolia groups were statistically significant(P<0.05),especially the water extract from Sagittaria Sagittifolia group play a major role,with significant difference(P<0.01).2 The general conditions of mice in each group were observed:from the observations of body condition,weight gain,fur gloss and reaction sensitivity of the mice,we can found that the mice in the model group were not stronger than the control group,eating less,the coat was dull and bad-tempered,some showed yellow staining.Compared with the model group,the water extract from Sagittaria Sagittifolia groups and SSP groups with low,medium and high dose were improved,and the same dose of SSP group improvement is more pronounced than that the water extract from Sagittaria Sagittifolia group.The liver index results showed that compared with the control group,the hepatic index of the model group increased significantly(P<0.01);compared with the model group,the hepatic index of each dose of the water extract from Sagittaria Sagittifolia groups,each dose of SSP groups and silymarin group decreased(P<0.05),among which the high-dose of water extract from Sagittaria Sagittifolia group and the medium-dose,high-dose of SSP groups had significant differences(P<0.01).The activity of ALT,AST and LDH showed that compared with the control group,the activity of ALT,AST and LDH in the model group increased(P<0.01).Compared with the model group,the activities of ALT,AST and LDH of the medium-dose,high-dose of water extract from Sagittaria Sagittifolia groups and SSP groups as well as silymarin group all decreased(P<0.05).Among them,the high-dose groups and the silymarin group were significantly different(P<0.01).The content of MDA and GSH showed that compared with the control group,the MDA content increased and the GSH content decreased in the model group(P<0.01).Compared with the model group,the content of MDA of the high-dose of water extract from Sagittaria Sagittifolia group,the medium-dose,high-dose SSP groups and silymarin group decreased,however the content of GSH of these groups increased(P<0.05),among them,which the high-dose groups and the silymarin group were significantly different(P<0.01).The results of HE staining showed that compared with the control group,liver pathology of the model group showed obvious pathological changes and significant enlargement of liver cells,plasma osteoporosis,translucent;compared with the model group,the low-dose and medium-dose of water extract from Sagittaria Sagittifolia groups showed no obvious improvement,liver tissue inflammation of other groups improved significantly.3 The results of RT-PCR and Western blot showed that compared with the control group,Nrf2 gene relative expression and protein expression in the model group and the high-dose SSP group have increased(P<0.05,P<0.01,P<0.05,P<0.01),however Keap1 gene relative expression and protein expression were reduced(P<0.05,P<0.01,P<0.05,P<0.01);compared with the model group,the relative expression and protein expression of Nrf2 in high-dose SSP group increased significantly(P<0.05,P<0.05).The relative expression and protein expression of Keapl decreased,there was statistical difference(P<0.05,P<0.05).Conclusions:Through the comparison of different active parts of Sagittaria Sagittifolia and SSP,we found that SSP has stronger protective effects on hepatocytes injury induced by co-administration of isoniazid and rifampin,which may be related to the regulation of Nrf2 signaling pathway,by down-regulating expression of Keapl,up-regulating expression of Nrf2,activating antioxidant system in vivo to inhibite hepatocyte damage. |
PDF Full Text Request