| Di(2-ethylhexyl)phthalate(DEHP)is a plasticizer that is widely added to polyvinyl chloride to increase its flexibility.DEHP is used in a variety of products,such as medical plastic hoses,food packaging bags and cosmetics.Studies have shown that DEHP and its metabolites can cause damage to the heart,liver,kidneys and reproductive system.Liver fibrosis is a chronic liver injury process with repeated injury and healing,including infectious diseases,toxin exposure,metabolic disorders and autoimmune diseases.It has become a worldwide clinical problem.Hepatic fibrosis is characterized by excessive deposition of extracellular matrix(ECM).Hepatic stellate cells(HSCs)are important sources of ECM.When activated,HSCs are transformed into myofibroblasts,causing an imbalance in ECM synthesis and degradation,resulting in excessive deposition of ECM.Liver fibrosis can be further aggravated into cirrhosis or even liver cancer if it is not prevented.Epidemiological studies have shown that daily consumption of phthalate-contaminated foods may be one of the risk factors for liver dysfunction.Prolonged exposure to DEHP can affect liver disease and exacerbate the progression of chronic liver injury.However,the effect of DEHP on liver fibrosis is unclear.In this study,we explored the effects of DEHP on carbon tetrachloride(CCl4)-induced liver fibrosis and further revealed its molecular mechanism.Objective: To determine the effect of DEHP on the pathologic progression of hepatic fibrosis in rats,and to reveal the role of TGF-?1/Smad and p38MAPK/NF-?B signaling pathways in hepatic fibrosis.Methods: In vivo experiments,we used a model of liver fibrosis induced by subcutaneous injection of CCl4.The model was given DEHP(0.05,5,500 mg/kg)and DEHP 500 mg/kg alone as a control.After nine weeks.Rats were sacrificed.HE staining and MASSON staining were used to observe the liver pathological damage of DEHP in rats with liver fibrosis induced by CCl4.The serum of aspartate aminotransferase(AST),glutamate aminotransferase(ALT)and liver tissue were detected by kit.Content of malondialdehyde(MDA),superoxide dismutase(SOD)and reduced glutathione(GSH)in serum;detection of tumor necrosis factor(TNF-?)and interleukin 6(IL-6)in serum by ELISA.The levels of ?-SMA,COL-I,COL-III,TGF-?1,P-Smad2,P-Smad3,P-p38 and P-p65 proteins were detected by Western blot.In vitro experiments,five groups were set up: normal group,TGF-?1,TGF-?1+DEHP(6.25,25,100 ?mol/L).After 24 hours of culture in vitro,CCK-8was used to detect the effect of DEHP on the proliferation of LX-2 cells.DCFH-DA probe was used to detect the production of ROS in LX-2 cells by DEHP.The expressions of ?-SMA,COL-I,P-Smad2,P-Smad3,P-p38 and P-p65 proteins were detected by Western blot.Results:1.Effect of DEHP exposure on the pathological damage process of CCl4-induced hepatic fibrosis in rats HE staining showed that there were a lot of fat vacuoles,hepatocyte necrosis and inflammatory cell infiltration in the liver tissue of CCl4 group compared with the normal group.DEHP exposure significantly aggravated the liver pathological damage of rats with liver fibrosis induced by CCl4.The liver tissue of DEHP(5 and 500mg/kg)showed cell necrosis,steatosis and inflammatory infiltration were significantly aggravated.MASSON staining found that normal and normal Compared with the group,there was significant collagen deposition around the hepatic wall of the CCl4 group,and a small amount of interstitial space appeared.DEHP exposure significantly promoted the deposition of collagen in the liver of rats with CCl4-induced hepatic fibrosis,from mild fibrosis and Steatosis is converted into severe fibrosis and a large amount of collagen fibrosis,especially in the DEHP500mg/kg dose exposure group.In the detection of serum transaminase levels,compared with the normal group,the serum levels of AST and ALT in the CCl4 group were significantly increased,and the DEHP 500 mg/kg dose significantly increased the levels of AST and ALT in the serum of CCl4-induced hepatic fibrosis rats.It is suggested that DEHP can significantly aggravate the pathological damage of liver in rats with liver fibrosis induced by CCl4.2.Effect of DEHP exposure on serum levels of inflammatory cytokines tumor necrosis factor(TNF-?)and interleukin-6(IL-6)in CCl4-induced hepatic fibrosis rats Compared with the normal group,CCl4 significantly increased TNF-? and IL-6 in the serum of rats.Compared with CCl4 group,medium and high dose of DEHP(5,500 mg/kg)significantly promoted the serum TNF-? level in rats,while serum IL-6level only changed significantly after high dose of DEHP exposure.It was suggested that the aggravating effect of DEHP on CCl4-induced hepatic fibrosis in rats was related to inflammatory factors.3.Effect of DEHP exposure on the balance of oxidation and antioxidant systems in liver tissues of CCl4-induced hepatic fibrosis in rats Compared with the normal group,MDA in the liver homogenate of CCl4 group significantly increased,and SOD and GSH significantly decreased.Compared with the CCl4 group,DEHP(5 and 500 mg/kg)significantly promoted MDA content in liver homogenate,and DEHP 500 mg/kg significantly decreased SOD and GSH activity.It is suggested that DEHP exposure aggravates liver injury induced by CCl4-induced liver fibrosis in rats,which may aggravate oxidative stress and destroy the balance between oxidation and antioxidant system.4.Effects of DEHP exposure on the expression and distribution of TGF-?1,?-SMA,COL-I and COL-III proteins in liver tissues of rats with hepatic fibrosis induced by CCl4 The expression and distribution of TGF-?1 and ?-SMA protein in liver tissue were detected by laser confocal method.Compared with the normal group,the expression of TGF-?1 and ?-SMA protein in liver tissue sections of the CCl4 group was significantly increased.Compared with CCl4 group,DEHP 500mg/kg significantly promoted the expression of TGF-?1 protein in liver tissue,while DEHP(5 and 500mg/kg)significantly promoted the expression of ?-SMA protein in liver tissue.The expression and distribution of COL-I and COL-III protein were detected by immunohistochemistry.Compared with the normal group,CCl4 group of liver biopsy in COL-? and COL-? expression increased significantly,compared with CCl4 group,DEHP5 and 500 mg/kg)exposure significantly promoted protein expression.5.Expression of ?-SMA,COL-I,COL-III,TGF-?1,P-Smad2,P-Smad3,P-p38 and P-p65 proteins in liver tissues Compared with the normal group,the CCl4 group was able to up-regulate the expression of ?-SMA,COL-I,COL-III,TGF-?1,P-Smad2,P-Smad3,P-p38,and P-p65 proteins;Compared with CCl4 group,DEHP significantly promoted the expression of ?-SMA,COL-I,COL-III,TGF-?1,P-Smad2,P-Smad3,P-p38 and P-p65 proteins in liver of rats with liver fibrosis induced by CCl4.Especially the effect of DEHP 500mg/kg was most obvious.It was suggested that DEHP could significantly aggravated the formation of liver fibrosis,which might be related to TGF-?1/Smad and P38MAPK/NF-?B signaling pathways.6.Effect of DEHP on the proliferation of human hepatic stellate cells LX-2Compared with the normal group,TGF-?1 significantly promoted the proliferation of LX-2 cells.Compared with the TGF-?1 group,the proliferation of LX-2 cells was significantly promoted by low-concentration DEHP exposure,while the proliferation of LX-2 cells was inhibited by high-concentration DEHP exposure.7.Effect of DEHP on ROS content in TGF-?1-stimulated LX-2 cells Compared with the normal group,TGF-?1 significantly increased the ROS content in LX-2 cells.Compared with the TGF-?1 group,DEHP(100?mol/L)significantly increased the ROS content in LX-2 cells.8.Effects of DEHP on the expression of ?-SMA,COL-I,P-Smad2,P-Smad3,P-p38 and P-p65 proteins in TGF-?1-stimulated LX-2 cells The study found that the overall trend of cell experiments was consistent with the body,compared with the normal group,TGF-?1 stimulation could significantly promoted ?-SMA,COL-I,P-Smad2,P-Smad3,P-p38,P-p65 Protein expression,compared with TGF-?1 group,DEHP could significantly promoted the expression of?-SMA,COL-I,P-Smad2,P-Smad3,P-p38,P-p65 protein,especially in high concentration DEHP 100?mol/L.Conclusion1.DEHP exposure can significantly aggravate the pathological damage of rats with liver fibrosis induced by CCl4.2.DEHP exposure can significantly promote the production of oxidative stress and destroy the dynamic balance between oxidase and antioxidant enzymes.It is suggested that DEHP aggravates liver fibrosis and may be related to oxidative stress.3.The mechanism of DEHP exposure aggravating liver fibrosis may be related to TGF-?1/Smad and p38MAPK/NF-?B signaling pathways. |
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