Study On Anti-hepatic Fibrosis Of Praziquantel With Different Optical Rotation

Schistosomiasis japonica is a zoonotic parasitic disease that seriously harms the society.Its pathological damage is mainly caused by the deposition of eggs excreted by adult schistosome in the liver,which leads to chronic inflammation.The continuous chronic infection leads to excessive damage repair,which induces the formation of egg granuloma and liver fibrosis.Praziquantel（PZQ）has been recommended as the first choice for clinical treatment of Schistosomiasis japonica due to its advantages of safety,low cost and high insecticide efficiency.After the patients received praziquantel insecticide treatment,the egg excretion was effectively inhibited,but the egg granuloma that had been formed would still continue to develop which causes liver fibrosis.Therefore,it has always been a difficult problem in the medical field to alleviate liver fibrosis in the advanced stage of Schistosomiasis japonica.Liver fibrosis is directly caused by excessive deposition and insufficient degradation of extracellularmatrix（ECM）,which is a common pathological process in the development of many chronic liver diseases.Hepatic stellatecell（HSC）is the main source of ECM generation and excessive proliferation and activation of HSC are the direct promoter of liver fibrosis.Studies have confirmed that targeted activation inhibitory drugs for HSC can inhibit liver fibrosis,so targeted drugs for HSC have become a research hotspot.In recent years,in the uptrend of"new use of old drugs",praziquantel has been found to affect the activation of hepatic stellate cells by up-regulating the expression of Smad7 to inhibiting the TGF-β/Smad signaling pathway,thus it plays a direct anti-fibrosis role in liver fibrosis,which will provide a possible choice for the clinical treatment of hepatic fibrosis.The current commercially available praziquantel is a racemic praziquantel（DL-PZQ）which is composed of about 50%levopraziquantel（L-PZQ）and 50%dexpraziquantel（D-PZQ）.Studies have shown that levopraziquantel is an effective component to kill schistosoma,and its efficacy is better than racemic praziquantel,while dexopraziquantel has almost no effect.In view of the significant differences in insecticidal efficacy between L-PZQ and D-PZQ,exploring whether their anti-fibrosis effects also have obvious differences can provide experimental evidence for the use of praziquantel in the treatment of clinical liver fibrosis.So this experiment is divided into two parts:in vivo experiment and in vitro experiment:1.Effect of praziquantel with different optical rotation on hepatic fibrosis induced by CCl₄ in miceThe model mice with liver fibrosis was established by intraperitoneal injection of25%CCl₄ for 6 weeks.At the end of modeling,Masson staining of liver sections and m RNA level of liver fibrosis indicators were used to evaluate the success of model building.Low-dose（L-PZQ,150 mg/kg）and high-dose（h-PZQ,300 mg/kg）levorotatory,dextrorotatory,and racemic praziquantel were intragastrically administered every 12 hours after molding,and 2.5%EL was used as control.After four weeks,mice are weighed and sacrificed after anesthesia to obtain serum,liver,etc.Masson staining of liver tissue sections,liver fibrosis indexes in serum and the m RNA and protein levels of liver fibrosis indexes were detected to evaluate the anti-fibrosis effects of different optical rotation praziquantel.Masson staining of liver tissue sections showed a significant decrease in fiber spacer and blue Collagen fibers in l-D-PZQ,h-D-PZQ and h-DL-PZQ groups,but no significant change in L-PZQ of high and low concentration groups;Compared with the CCl₄ model group,the serum COLIV,HA,LN,and PCIII levels of mice in the l-D-PZQ,h-D-PZQ and h-DL-PZQ groups all decreased to varying degrees,and the difference was statistically significant（P<0.05）.For L-PZQ group,only the contents of COLIV and PCⅢin the h-L-PZQ group were significantly reduced（P<0.05）,no significant difference was found between HA and LN（P>0.05）,and no significant changes were found in the four indicators of the l-L-PZQ group（P>0.05）;RT-PCR results showed that compared with the CCl₄ model group,the m RNA levels of CollagenⅠandα-SMA gene in mouse liver in l-D-PZQ,h-D-PZQ and h-DL-PZQ groups were significantly decreased（P<0.05）.However,there were no significant differences in the m RNA levels of CollagenⅠandα-SMA gene in l-L-PZQ,h-L-PZQ and l-DL-PZQ groups（P>0.05）.Compared with l-L-PZQ,l-D-PZQ can significantly reduce the m RNA expression levels of CollagenⅠandα-SMA genes in mouse liver（P<0.05）.There was no statistically significant difference in the m RNA expression levels of CollagenⅠandα-SMA genes in the liver between h-L-PZQ and h-D-PZQ groups（P>0.05）;Compared with the CCl₄ model group,liver hydroxyproline content in l-D-PZQ,h-L-PZQ,h-D-PZQ and h-DL-PZQ groups was significantly decreased（P<0.05）.There was no statistically significant difference in the level of hydroxyproline in l-L-PZQ and l-DL-PZQ groups（P>0.05）.In this part,mice with liver fibrosis induced by CCL₄ were used to explore the anti-liver fibrosis effect of different optical rotation praziquantel.The results showed that compared with the CCl₄ model group,dexpraziquantel and racemic praziquantel can improve the deposition of collagen fibers in the liver of mice and significantly reduce the contents of the four serum indicators（COLⅣ,HA,LN,and PCⅢ）of liver fibrosis in mice,CollagenⅠandα-SMA m RNA levels of liver fibrosis markers,and the content of hydroxyproline in liver.Levopraziquantel had a certain anti-fibrosis effect only at high doses.In addition,compared with the same dose of levopraziquantel,dexopraziquantel showed stronger anti-fibrosis effect.In conclusion,dexpraziquantel is more effective than levopraziquantel in the treatment of CCl₄-induced hepatic fibrosis mice.2.Effects of different optically active praziquantel on hepatic stellate cells in vitroHuman hepatic stellate cells（LX-2）in logarithmic growth period were precultured in culture plate,and blank control group was set up.Starved for 12 hours and stimulated with TGF-β（2.5ng/m L）.After 24 hours,L-PZQ,D-PZQ and DL-PZQ were added to each well and incubated in the incubator.CCK-8 assay was used to detect cell proliferation ability,scratch assay was used to detect cell migration ability,RT-PCR was used to detect the m RNA expression level of activation indicator genes,Cellular Immunofluorescence and Western Blot were used to detect the proteins expression level of activation indicator.The results showed that when the concentrations of L-PZQ,D-PZQ and DL-PZQ were lower than 30μg/m L,the proliferation ability of activated LX-2 cells was not significantly affected.When the drug concentration was higher than 30μg/m L,L-PZQ,D-PZQ and DL-PZQ began to inhibit the proliferation ability of activated LX-2 cells,and showed a dose-dependent effect.When the drug concentration was lower than 30μg/m L,there was no statistically significant difference among the three groups（P>0.05）.When the drug concentration was higher than 40μg/m L,the inhibitory effect of D-PZQ was significantly greater than that of L-PZQ and DL-PZQ,the difference was statistically significant（P<0.05）.There was no significant difference between L-PZQ group and DL-PZQ group（P>0.05）;Compared with blank control group,low-dose L-PZQ（15μg/m L）can promote the migration of LX-2,the difference is statistically significant（P<0.05）,while the same dose of D-PZQ and DL-PZQ did not find significant effect（P>0.05）.High-dose D-PZQ and DL-PZQ（30μg/m L）can significantly inhibit the migration ability of activated LX-2（P<0.05）,while the same dose of L-PZQ has no obvious effect（P>0.05）;As for the m RNA level of liver fibrosis activation index,except that L-PZQ has no obvious inhibitory effect on the m RNA expression level of CollagenⅠgene（P>0.05）,L-PZQ,D-PZQ and D-PZQ all have obvious inhibitory effects on the m RNA of LX-2 activation index genes（P<0.05）,and the inhibitory effects of D-PZQ are significantly stronger than L-PZQ,and the difference is statistically significant（P<0.05）;The immunofluorescence results showed that D-PZQ could reduce the immunofluorescence intensity of CollagenⅠandα-SMA,while L-PZQ had no obvious effect on CollagenⅠandα-SMA;Western Blot results showed that D-PZQ and DL-PZQ could significantly reduce the protein expression levels of CollagenⅠ,CollagenⅢandα-SMA（P<0.05）,but no inhibitory effect of L-PZQ was found（P>0.05）.Compared with L-PZQ group,the three index proteins in D-PZQ group were significantly decreased（P<0.05）.In this part of the experiment,it was found that dexopraziquantel could significantly inhibit the proliferation,activation and migration of LX-2 cells in vitro which showed dexopraziquantel had significant anti-hepatic fibrosis effects,while levopraziquantel only had a partial inhibitory effect.Compared with levpraziquantel,dexpraziquantel inhibited the proliferation,activation and migration of LX-2 cells.These results suggest that the new anti-fibrosis effect of praziquantel in vitro may be dominated by dexpraziquantel.