| Schistosomiasis japonica is still an important public health problem in China.Although the epidemic of schistosomiasis has declined year by year due to unremitting efforts during a long period,the prevention practice of schistosomiasis shows that after pathogen eliminate,the existing liver inflammatory environment still undergoes further deterioration,presented as the development of liver fibrosis to advanced schistosomiasis.It shows that understanding the pathogenesis of chronic schistosomiasis merits further exploration.Activation of hepatic stellate cells(HSCs)plays a vital role in liver fibrosis of chronic schistosomiasis,however,induction factors of continuous activation of HSCs,as well as signaling pathways in HSCs,has not been clarified.In addition,the lack of effective clinical targets restricts the prevention and control of schistosomiasis liver fibrosis.Former researches suggest that the formation of the early inflammatory microenvironment in liver plays an important role in the formation of egg granuloma.Our previous studies also demonstrated that the inflammatory response of schistosoma infection not only mediated acute egg granuloma formation,but also started the expression of fibrosis related genes.Therefore,in-depth study on the induction factors of continuous activation of HSCs in the process of hepatic fibrosis caused by Schistosoma infection will help us reveal the pathogenic mechanism of chronic schistosomiasis.Meanwhile,it will provide us with new drug targets in anti-fibrosis treatment for schistosomiasis.A large number of studies have shown that the formation of inflammatory microenvironment in liver play a crucial role in the development of fibrosis.Innate immunity,the body’s first line of defense,recognizes pathogen and harmful substances released from damaged cells by pattern recognition receptors(PRR).Then,it can activate the downstream signaling pathways and induce the inflammatory response for self-protection,which can also lead to tissue damage.The NLRP3 inflammasome is a multi-protein complex involved in instigating inflammation in many diseases.After sensing multiple kinds of dangerous signals,NLRP3 activates caspase-1,promotes the release of IL-1β/IL-18 and induced pyroptosis,which is a kind of programmed cell death.IL-1β can directly stimulate fibroblast to secrete collagen and regulate the expression of fibrosis related genes,such as MMP9,MMP13 and TIMP.Studies showed that the formation of inflammasomes in non-immune cells preceded immune cells at the site of infection or injury.Thus,we speculate that in the process of schistosoma japonicum infection,NLRP3 inflammasome activation exists in HSCs during different period(early,acute and chronic)of schistosomiasis.This study will dynamically analyze the activation of NLRP3 inflammasome signal pathways in activated HSCs and its function in the process of infection.1.The study on the expression of NLRP3 inflammasome in hepatic stellate cells of mice infected with schistosomas japonicumImmunohistochemistry technique was employed to analyze the expression of IL-1β,NLRP3 and Caspase-1 in the liver of mice infected with schistosoma japonicum at different stages.Meanwhile,the expression of some molecules related to NLRP3 pathway was further evaluated in the hepatic stellate cells(HSCs)isolated from mice infected with schistosoma japonicum at different stages.The results showed that IL-1β expression began to increase in the third weeks after infection,and reach expression peaks in the sixth and twelfth weeks after infection.Furthermore,the expression of NLRP3 and Caspase-1 significantly increased in the sixth week after infection and kept high expression level in the infection process.These results suggested that IL-1β was involved in the formation of early inflammatory microenvironment caused by infection.Meanwhile,NLRP3/Caspase-1/IL-1β signal pathway was activated in schistosome-caused liver pathologic changes as the inflammation and fibrosis gradually increased.This result illustrated that NLRP3 inflammasome activation contributed to the pathological process of liver inflammation and fibrosis caused by schistosome infection.In order to make sure the existence and effect of NLRP3 inflammasome signal pathway in the activation of HSCs,Human hepatic stellate cellcell line LX-2 was used to test the expression of NLRP3 inflammasome in HSCs.The results showed that a-SMA and NLRP3/IL-1β,the markers of HSCs activation,began to upregulate and sustain continuous high expression level in mice at the acute stage and chronic stage of infection.These results suggested that HSCs began to be activated in the early stages of the infectionbefore worm spawning.NLRP3 inflammasome,an important component of innate immune system,kept high expression level along with the development of infection.It indicated that the formation and activation of NLRP3 inflammasome in HSCs isimportance in the occurrence and development of liver fibrosis caused by schistosomiasis.2.The role of exogenous factors in the formation and activation ofNLRP3 inflammasome in HSCs from mice with schistosome-caused liver pathologic changesSoluble egg antigen(SEA)of schistosoma japonicum is the dominating factor of liver pathologic changes caused by schistosoma japonicum infection.This study found that SEA stimulation significantly enhanced the interaction between NLRP3 and ASC,while caspase-1 activity and IL-1β secretion increased.In addition,SEA could promote cell pyroptosis triggered by NLRP3 inflammasome activation.Pyroptosis is not only an important feature of inflammasome activation,but also causing cell damage and then releasing a lot of harmful substances.This may be an important factor for the continuous inflammation and liver fibrosis caused by Schistosoma infection.The results indicated that NLRP3 inflammasome could be activated by SEA and had important biological effects in HSCs.It suggested that SEA from deposited eggs in liver is an important exogenous factor in the formation and activation of NLRP3 inflammasome in HSCs.3.The effect of oxidative stress in the formation and activation ofNLRP3 inflammasome in HSCs from mice with liver pathologic changes caused by schistosomiasisPrevious studies have shown that a large number of reactive oxygen species(ROS)is generated during the process of hepatic fibrosis as excessive ROS promotes inflammation and fibrosis formation.Researches focusing on the mechanism of NLRP3 inflammasome activation showed that ROS may be the convergence of various factors leading to NLRP3 inflammasome activation.We isolated liver tissue and serum from mice infected with schistosoma japonicum at different stages of infection,and detected the markers of oxidative stress reaction.The levels of superoxide dismutase(SOD),glutathione(GSH)and catalase(CAT)were significantly decreased in sixth week after infection in liver tissue of mice,while kept low expression level in twelfth week after infection.However,the level of malondialdehyde(MDA)was significantly increased in sixth week after infection and persisted with high expression level in the twelfth week after infection.Meanwhile,immunofluorescence was employed to detect ROS level in the liver tissue.In the third week after infection,the level of ROS in liver began to upregulate.In the sixth and twelfth week after infection,ROS maintained a high expression level.These results showed that the expression of major antioxidant enzymes were significantly decreased,while products of oxidative stress increased in liver tissue with the exacerbation of liver pathologic changes caused by schistosoma infection.It is suggested that antioxidant capacity decreased in the process of schistosomiasis.In addition,further study was taken to evaluate the effect of ROS in NLRP3 inflammasome activation in HSCs after schistosome infection.We found that formation of NLRP3 inflammasome in HSCs induced by SEA was effectively prevented by the inhibition of ROS.ROS inhibition downregulated the levels of caspase-1 and IL-1β,thereby reduced proinflammatory cytokine in tissue microenvironment.These results indicated that the generation of extra ROS in HSCs was an important factor in NLRP3 inflammasome activation.NLRP3 inflammasome activation enhanced the activity of Caspase-1 and promoted proinflammatory cytokines releasing,which led to the formation of hepatic inflammatory microenvironment and resulted in liver fibrosis.In summary,this study showed that NLRP3 inflammasome activation is involved in early hepatic inflammatory microenvironment formation during the process of liver pathologic changes caused by schistosoma japonicum infection.NLRP3 inflammasome activation is initiated and persisted as the activation of HSCs.Moreover,SEA is an important exogenous factor in the formation and activation of NLRP3 inflammasome in HSCs.ESA-induced ROS releasing is an important mechanism in NLRP3 inflammasome activation.This study focused on the NLRP3 inflammasome.We verified the existence and activation of NLRP3/caspase-1 inflammasome signal pathway in HSCs in the pathogenesis of hepatic fibrosis caused by schistosomiasis japonicum infection.Meanwhile,we clarified the relationship between both PAMPs and DAMPs with the NLRP3/caspase-1 pathway during HSCs activation.Finally,we analyzed the effect of signal pathway in the activation of HSCs.These results will help us further understand the pathogenesisof schistosomiasis and provide the basis for further research on accurate targets foranti-fibrosis treatment. |
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