| Schistosomas japonicum is mainly epidemic in china.To the end of 2016,there are still 54454 schistosomiasis patients.Schistosomiasis is a chronic infectious immune disease.Schistosome soluble worm antigen(SWA)and soluble egg antigen(SEA)are two major antigens that may cause host immune response.Hepatic stellate cells(HSCs)are main effector cells of liver fibrosis.The induction factors and signaling pathways of continuous activation of HSCs has not been clarified.There are still no effective targets and methods for clinical treatment of schistosomiasis liver fibrosis.Recently,some studies have revealed that NLRP3 inflammasome,a multiprotein complex that is one of the components of the innate immune system,is involved in the pathogenesis of the inflammatory response in human liver disease by the processing of caspase-1 and IL-1β.It has been shown that NLRP3 inflammasome activation and IL-1β production play an important role in the development of liver inflammation and fibrosis.Our previous study has demonstrated that the activation of NLRP3 inflammasome and the expression of α-SMA began to upregulate at the early stage and sustain congtinuous high expression level at the acute stage and chronic stage of schistosome infection in mice.Meanwhile,a study has shown that NLRP3 inflammasome activation in liver is positively correlated with the extent of hepatic fobrosis in infected mice.Thus,we speculate that SWA and SEA can directly induce the activation of NLRP3 inflammasome signaling pathway and mediate the expression of α-SMA and ColI in HSCs.Besides of SEA and SWA,oxidative stress affects several cellular functions of the liver and plays a significant role in the progression of liver fibrosis and inflammation.The body produces ROS when it is subjected to various harmful stimuli through oxidative stress reaction.ROS may causes proliferation,activation and collagen generation in HSCs.Functionally,ROS is fully involved in the start-up phase of NLRP3 activation.Therefore,we speculate that ROS play an important role in the activation of NLRP3 inflammasome and the expression of α-SMA and ColI in HSCs.Our previous experiments showed that ROS mainly originates from macrophages and HSCs in the liver of schistosome-infected mice.So,we focused on the ROS from the HSCs and the macrophages and observed their effects on the formation and activation of NLRP3 inflammasome and the expression of α-SMA and ColI in HSCs.In this study,we mainly focused on the effect of SWA and SEA on activation of NLRP3 inflammasome and the expression of α-SMA and ColI in LX-2 cells in vitro.The effect of SWA on the apoptosis of LX-2 cells was observed.At the same time,we evaluated the influence of ROS from LX-2 cells and the human THP-1 macrophages cells on NLRP3 inflammasomes activation and fibrosis of LX-2 cells,in order to further confirme the pathogenesis of liver fibrosis of schistosomiasis infection.The main findings are as follows:1.The activation of NLRP3 inflammasomes and the expression of α-SMA and ColI in LX-2 cells could be induced by SWA and SEALX-2 cells were treated with different concentrations of SWA or SEA(25,50,100 μg/ml).The mRNA and protein levels of NLRP3,IL-1β,Caspase-1,α-SMA and ColI in LX-2 cells were analyzed by Real-time PCR and Western blotting.IL-1β was quantitated by ELISA analysis.The results showed SWA or SEA when reaches the concentration of 100 μg/ml could induced the expression of NLRP3,IL-β,Caspase-1,α-SMA and CoII.In addition,we mixed SEA and SWA to simulate LX-2 cells and compared them with separate stimuli of SEA or SWA in LX-2 cells.We found that 50μg/ml SWA+50μg/ml SEA could not induced the mRNA expression of NLRP3,Caspase-1,α-SMA and ColI.However,the mRNA expression of IL-β was significantly increased.When the concentration of one antigen reaches 100μg/ml,the expression of NLRP3,IL-1β,Caspase-1,α-SMA and ColI significantly increased.These results suggested that SWA and SEA are important factors for the activation of NLRP3 inflammasome and the expression of α-SMA and ColI in LX-2 in the liver disease caused by schistosomiasis infection.The effect of SWA and SEA on the activation of NLRP3 inflammasome is not simple additive effect.SWA and SEA may trigger other pathways to induce IL-1β,which needs further exploration.2.SWA may induce LX-2 cells pyroptosisOur previous studies have proved that SEA can induce pyroptosis through the activation of NLRP3 inflammasome in LX-2 cells.In this study,we evaluated the role of SWA on the apoptosis of LX-2 cells.Effects of SWA on cell viability and LDH release in LX-2 cells were detected by CCK-8 assay and LDH assay.The activity of Caspase-1 enzyme was detected by Western blotting.The results showed that the cell viability was significantly decreased,the LDH release and the expression of activated caspase-1 was significantly increased following the increase of SWA concentration.The above results indicated that LX-2 cell viability decreased in a dose-dependent manner under the stimulus of SWA,and SWA can induce cell pyroptosis.3.Inhibition of ROS downregulated the levels of the activation of NLRP3 and the expression of α-SMA and ColI induced by SWA and SEATo further evaluate the role of ROS in NLRP3 inflammasome activation and fibrosis in HSCs caused by schistosome infection,firstly,we analyzed the effects of SWA and SEA on the production of ROS in LX-2 cells by flow cytometry.The results showed that SEA and SWA could induce oxidative stress in LX-2 cells to generate ROS.Then,we used NAC to scavenge intracellular ROS.The results showed that the formation and activation of NLRP3 inflammasome were reduced and the expression of α-SMA and ColI was also suppressed in LX-2 cells when stimulated by SEA and SWA.This suggested that the increase of ROS is an important factor to induce NLRP3 inflammasome activation,activation and collagen deposition by SWA and SEA in LX-2 cells.4.Inhibition of macrophage ROS induced by worm-derived factors could inhibit the activation of NLRP3 inflammasome in LX-2 cellsMacrophages are also an important source of ROS during schistosome infection.In order to further confirm the effect of worm-derived factors on macrophage ROS,we analyzed the effects of SWA and SEA on the production of ROS in THP-1 macrophages by flow cytometry.The results showed that SWA and SEA could induce THP-1 macrophages to produce ROS.Macrophages were induced by SWA and SEA and co-cultured with LX-2 cells by using Transwell system.The results showed that the induced macrophages may cause the activation of NLRP3 inflammasome and the expression of α-SMA and ColI in LX-2 cells,In order to evaluate the role of ROS produced by macrophages in this process,we used NAC to inhibit ROS in THP-1 macrophages and found that the expression of NLRP3,Caspase-1,and IL-1β in LX-2 cells were suppressed.The results suggested that SWA and SEA can not only directly interact with HSCs,but also induce macrophage ROS to indirectly interact with HSCs to induce the formation and activation of NLRP3 inflammasomes.In summary,the present study found that SWA and SEA were important factors that may cause the activation of NLRP3 inflammasome,activation and collagen synthesis in LX-2 cells.The release of ROS from HSCs and macrophages induced by SWA and SEA may be one of the important mechanisms that cause these changes.We clarified the relationship between both PAMPs and DAMPs with the activation of NLRP3 inflammasome and collagen synthesis.These results will help us further understand the value of this pathway in activation and fibrosis of HSCs.At the same time,it also provides new drug targets for the treatment of fibrosis. |
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