Triptolide Regulates Hepatotoxicity Caused By Oxidative Stress And Inflammation By Inducing CYP2E1

Triptolide(TP)is the main active ingredient extracted from the Tripterygium wilfordii plant and also the main ingredient that causes toxic and side effects.Xylem is the medicinal part.It has activating blood and dredging collaterals,reducing swelling and pain,good immune regulation and anti-tumor activity.In recent years,a large number of studies have shown that long-term exposure to TP may damage liver function and other functions.In view of its many toxic and side effects,it is also one of the Chinese herbal medicines that have been reported to have the most poisoning incidents in the past hundreds of years.However,because TP has its unique clinical effects on various diseases,it has almost not been completely similar to alternative Chinese medicines,the clinical use rate is still very high.Cytochrome P450 2E1(CYP2E1)is one of the main enzymes involved in liver metabolism.It is widely distributed in the liver,kidney,brain and lung.The liver is the main expressing organ of CYP2E1.Our results show that TP exposure accelerates the development of liver injury,and further triggers oxidative stress damage and inflammation;the pathway protein P-NF-κB related to liver inflammation and oxidative stress is significantly upregulated.Interestingly,the trend observed in the results of IHHA-1 cells is in line with the results of in vivo.In general,studies have shown that TP may aggravate the progression of liver injury in C57 mice through CYP2E1 and NF-κB signaling pathways.Clinicians should pay attention to understanding the characteristics of TP to maximize the therapeutic effect and reduce the toxic effects caused by TP.Objective: The C57 mice liver injury model was fed with TP to study the effect of TP on the pathological process of liver injury in C57 mice at different stages and the related pathways related to liver inflammation and oxidative stress.At the same time,human normal hepatocytes(IHHA-1)were used in in vitro experiments to further explore the role of CYP2E1,IL-6,TNF-α and NF-κB signaling pathways in the liver injury induced by TP.Methods: In the in vivo experiment,we used TP to feed C57 mice to induce liver injury model,and while inducing the model,we gave clomethiazole(75mg/kg)and pyrazole(120mg/kg)by gavage at the same time,and set TP 500μg/ kg as the control.Gavage the C57 mice with the prepared TP solution every afternoon for three weeks;the C57 mice were killed in strict accordance with the animal ethics method at one weekend,two weekends and three weekends.HE staining of liver tissue was used to observe the pathological changes of C57 mice at different stages.Serum ALT and AST activities;Western blot method(Western blot)to detect the expressions of proteins in tissues;In the in vitro cell experiment,four groups were also set up: control group and TP group(20nm),TP + CMZ(80μM),TP + PZ(60μM).After IHHA-1 cells were administered and cultured for 12 hours,the MTT kit was first used to detect the effect of TP on the viability of IHHA-1 cells;in the in vitro oxidative stress experiment,TP was used to stimulate 12 hours to establish an in vitro liver injury model.Using flow cytometry to accurately detect the ROS content in the cells after TP treatment.Cell protein was extracted,and the expression of CYP2E1,IL-6,TNF-α and P-NF-κB protein was detected by Western blot.Results: 1.Histopathological changes in C57 mice with liver injury caused by TP exposureIn the first week,from each group through macro observation,differences in liver samples were not significant.In the second week,HE staining of each group showed a small amount of inflammatory cells around the blood vessels in the TP group;slight inflammation in the TP+CMZ group;and obvious inflammation in the TP+PZ group.In the third week,HE staining in each group showed that there was severe inflammatory cells around the blood vessels in the TP group;a small amount of lymphocyte infiltration and mild inflammation in the TP+CMZ group;the TP+PZ group had obvious inflammation infiltration and massive cell necrosis.It shows that TP could accelerate the liver injury of C57 mice and even worse the condition of C57 mice.2.Effects of TP on liver function indexes and redox of C57 mice Liver index of C57 mice significantly increased after TP exposure,the;in the detection of biochemical indicators,the levels of ALT and AST in serum of mice were significantly increased.Homogenate in liver tissue,the content of TG and TC in the TP group and the TP+PZ group increased significantly;TP can reduce SOD activity in TP-induced liver injury,and simultaneously increase High content of MDA in tissues.It shows that the intake of TP can accelerate liver damage in C57 mice,and the oxidative damage is serious,which may be related to the breakdown of the balance between oxidation and oxidative defense systems in the liver.3.Effect of TP on the activity of normal liver cells(IHHA-1)Studies have shown that TP can alter IHHA-1 cell viability.Once the concentration of TP increased,the cell viability also decreased significantly.It shows that TP can promote cell apoptosis to a certain extent.4.Effects of TP exposure on ALT and AST and REDOX equilibrium system in supernatant of IHHA-1 cellsAfter TP exposure,the activities of alanine aminotransferase and aspartate aminotransferase in the cell supernatant were significantly increased.In the cell supernatant,the content of TG and TC in the TP group and the PZ group were significantly increased;TP can significantly inhibit the activity of SOD in TP-induced cell hepatotoxicity,while increasing the content of MDA in the tissue.It shows that the intake of TP can accelerate cell liver toxicity and severe oxidative damage may be closely related to the breakdown of the balance between oxidation and oxidative defense systems in cells.5.Effects of TP exposure on ROS levels in IHHA-1 cells and C57 liver With TP exposure,flow cytometer and slide scanner were used to detect the fluorescence intensity of reactive oxygen species.The results showed that compared with the TP group,the TP+CMZ group greatly reduced the content of reactive oxygen species;the TP+PZ group significantly increased the activity Oxygen content.6.The expression of CYP2E1,IL-6,TNF-α and P-NF-κB in liver tissues was analyzed by Western blot Compared with the control group,CYP2E1 was activated in the TP group,and the expressions of CYP2E1,IL-6,TNF-α and P-NF-κB changed significantly.Compared with the TP group,the TP+CMZ group showed a slight decrease in CYP2E1,IL-6,TNF-α,and P-NF-κB protein expression after CMZ treatment.Compared with the TP group,the TP+PZ group showed a increase in CYP2E1,IL-6,TNF-α,and P-NF-κB protein expression after CMZ treatment.It is suggested that the influence of TP on liver injury may be closely related to the CYP2E1,IL-6,TNF-α and NF-κB signaling pathways.7.Effects of TP on the protein contents of CYP2E1,IL-6,TNF-α and P-NF-κB in IHHA-1 cells The results showed that the trend of cell experiment was roughly the same as that of animal experiment.TP can increase the protein levels of CYP2E1,IL-6,TNF-α and P-NF-κB.Among them,TP combined with PZ histone increased most obviously,while TP combined with CMZ histone decreased slightly.8.Effects of transfection of CYP2E1 si RNA and PG-CMV-CYP2E1 on expression of related proteins in IHHA-1 cells The results of Western blot experiments showed that,compared with the control group,the expression of CYP2E1,IL-6,TNF-α and P-NF-κB in IHHA-1 cells was significantly increased after TP stimulation;compared with the TP group,CYP2E1 si RNA could significantly reduce the cells Compared with the TP group,PG-CMV-CYP2E1 can significantly increase the expression of CYP2E1,IL-6,TNF-α and P-NF-κB in the cells.Conclusions: Through experimental studies on the first 1,2 and 3 weeks,we found that high and prolonged intake of TP aggravated liver injury in C57 mice.This may be closely related to TP inducing liver cell damage and inflammatory response by promoting liver oxidative stress damage.According to cell and animal experiments,TP may aggravate liver injury by increasing the levels of CYP2E1,IL-6 and TNF-α,and activating the NF-κB signaling pathway.