| Schistosomiasis is a zoonotic parasitic disease that is widespread in Asia,Latin America and Africa.It is a serious threat to human health and restricts economic development.The main epidemic in China is schistosomiasis japonica.With active prevention and treatment,new patients are becoming less and less,but the cumulative number of former patients is still large.By the end of 2016,there are still 54454 schistosomiasis patients in the country,and 30573 cases of advanced schistosomiasis patients remain.The pathological damage caused by schistosomiasis is mainly schistosomiasis eggs deposited in the liver and formed granulomas,followed by irreversible liver fibrosis or even cirrhosis,and severe clinical symptoms.Although the parasites in the body have been eliminated after the implementation of schistosomicidal treatment on patients,liver fibrosis will continue to develop.Therefore.after schistosomiasis patients are treated with schistosomicidal drug,the continuous development of liver fibrosis should not be neglected.Praziquantel(PZQ),as a traditional anti-parasitic drug,has a good helminthicide effect on worms such as trematodes and tapeworms,and is the preferred drug for treating schistosomes.It has the characteristics of safety,high efficiency,and less side effects.With the deepening of research on praziquantel,we have found that praziquantel not only has the role of schistosomacide,but also can directly act on the body's cells,with the role of regulating immunity and anti-fibrosis.Our previous experiments showed that praziquantel treatment for a prolonged period of time in mice infected with Schistosoma japonicum could suppress liver fibrosis.In the carbon tetrachloride-induced mouse liver fibrosis model,it was found that praziquantel inhibited the activation of hepatic stellate cells through the TGF-?/Smad signaling pathway.In the field of hepatic fibrosis,people often focus on the activation of myofibroblast-like cells,the synthesis and secretion of extracellular matrix,and the changes and effects of immune cells and cytokines.However,there are few studies on the source of raw materials required for collagen synthesis.Arginase is an enzyme that hydrolyzes L-arginine and eliminates ammonia in the liver.It was first found in the liver.Arginase has two isoenzymes:Arginase-1 and Arginase-2.Arginase 1 is a cytoplasmic enzyme expressed mainly in the liver and is lowly expressed in the extrahepatic tissue,whereas Arginase-2 as a mitochondrial enzyme is low in the liver and highly expressed in extrahepatic tissues.Arginase hydrolyzes L-arginine to produce urea and L-ornithine,which is a precursor to L-proline,a raw material for collagen synthesis.Therefore,arginase provides the raw material for collagen synthesis.Studies have shown that arginase can be regulated by the TGF-?/Smad signaling pathway,which in turn affects the progression of fibrosis.In this experiment,we mainly studied the praziquantel regulation of arginase via TGF-?/Smad signaling pathway in mice infected with Schistosoma japonicum,and further improved the anti-fibrosis mechanism of praziquantel.In vitro,the effect of praziquantel on arginase was observed on fibrosis-related cells-human hepatic stellate cell line(LX-2),mouse embryonic fibroblasts(NIH/3T3)and mouse mesangial cells(MES13).The knockdown of Smad7 using interfering RNA proved that praziquantel regulating arginase is mediated by the TGF-?/Smad signaling pathwayThe results of this study are as follows:1.Praziquantel reduces circulating arginase activity in mice infected with Schistosoma japonicumThe mice infected with Schistosoma japonicum for 12 weeks were treated with a praziquantel dose of 300 ?g/kg in a volume of 100 ?l.In order to observe the activity of arginase in the circulatory system of praziquantel-treated mice,the serum of mouse from the normal,infected,schistosomicidal and anti-fibrosis treatment groups was taken,and the ultrafiltration tube was used to filter the ammonia in the serum.Based on the principle of arginase hydrolysis of arginine to generate ornithine and ammonia,the amount of ammonia generated was measured to calculate the activity of arginase.The results showed that the arginase activity in the serum of mice infected with Schistosoma japonicum increased significantly.Serum arginase activity was not statistically different from that of the infected group after the mice were given schistosomicidal treatment,but there was still a tendency to increase.Compared with the infected group and the schistosomicidal group,the activity of serum arginase significantly decreased in the anti-fibrosis treatment group.The results showed that praziquantel treatment can reduce the serum arginase activity in mice infected with Schistosoma japonicum.2.Praziquantel declines arginase-1 expression in hepatic stellate cells of mice infected with Schistosoma japonicumAt each time point,mice in each group were sacrificed and the primary hepatic stellate cells were isolated and purified to detect the expression of mRNA and proteins of arginase and Smad7.The results showed that the expression of mRNA and proteins of arginase 1 in hepatic stellate cells increased,and the expression of mRNA and proteins of Smad7 decreased in mice infected with S.japonicum.After giving schistosomicidal treatment,there was no statistical difference compared to mice infected in the same period.When praziquantel was given for 4 weaks,the expression of mRNA and proteins of Smad7 increased,and the expression of mRNA and proteins of arginase-1 decreased.These results suggested that regular administration of praziquantel treatment to chronic schistosomiasis mice can reduce the content of arginase-1 in hepatic stellate cells and improve hepatic fibrosis.3.Praziquantel reduces expression of arginase-1 and amino acid in exogenous TGF-?1 stimulated LX-2 cellsIn order to study the effect of praziquantel on arginase in vitro,we observed changes in arginase of TGF-?1-induced LX-2 cells,and effect of praziquantel on arginase.After induction and culture,cells were harvested and assayed for mRNA and protein analysis of Smad7 and arginase,as well as intracellular proline and hydroxyproline content.The results showed that exogenous TGF-?1 could induce the expression of arginase-1 in LX-2 cells and increase the content of proline and hydroxyproline.Praziquantel could increase the level of Smad7 and reduces the expression of Arginase-1 and its downstream amino acids.The results evidenced that praziquantel may inhibit the expression of Arginase-1 in hepatic stellate cell line through TGF-?/Smad signaling pathway.4.Praziquantel loses its inhibitory effect on Arginase-1 after knockdown of Smad7To verify that praziquantel suppressed Arginase-1 by upregulating Smad7 in the TGF-?/Smad signaling pathway,we use siRNA to knock down the expression of Smad7 and then observe the effect of praziquantel on LX-2 cells.The expression mRNA and protein of arginase-1 in LX-2 cells was detected by qRT-PCR and Western blot,respectively.The results showed that the expression of Arginase-1 in LX-2 cells was decreased after praziquantel stimulation,but the expression of arginase-1 was not decreased after knockdown of Smad7,and the expression of arginase-1 in LX-2 cells stimulated by praziquantel after NC-siRNA interference remains declining.The results suggested that praziquantel could inhibit the expression of Arginase-1 by targeting Smad7 to inhibit TGF-?/Smad signaling pathway,thereby inhibiting fibrosis.5.Praziquantel reduces Arginase-1 and its downstream amino acid expression in exogenous TGF-?1 stimulated fibrosis-related cellsTo further investigate the effect of praziquantel on arginase in other fibrosis-associated cell lines,we used exogenous TGF-?1 to stimulate MES13 cells and NIH/3T3 cells in vitro,and to detect levels of arginine and its downstream amino acids-proline and hydroxyproline-after praziquantel intervention.The results showed that exogenous TGF-?1 increased Arginase-1 and proline and hydroxyproline content in MES13 and NIH/3T3 cells,and the expression of Arginase-1 and the content of two amino acids decreased after praziquantel treatment.This indicates that praziquantel may inhibit the expression of Arginase-1 in MES13 and NIH/3T3 cells via TGF-?/Smad signaling pathway.In summary,the study found that praziquantel could reduce the expression of arginase by inhibiting the TGF-?/Smad signaling pathway in fibrosis-related cells,thereby reducing the source of collagen and inhibiting the formation of fibrosis.The results further revealed the mechanism of action of praziquantel against hepatic fibrosis,and it was suggested that praziquantel also has inhibitory effects on other fibrosis such as pulmonary fibrosis and renal fibrosis.These increase our deeper understanding of the old drug praziquantel and provide more evidence for the antifibrotic application of praziquantel. |
PDF Full Text Request