Effect Of Non-Neuropathic Cholinergic System On Hepatitis C Virus Infection

BackgroundAccording to the statistics of the world health organization,there are 130 million to 150 million people in the world who are infected with chronic hepatitis c virus.HCV infection does not cause obvious symptoms for a long time,and the disease progresses slowly.Many infected people cannot receive diagnosis and treatment in time,so they have developed cirrhosis and liver cancer at the time of diagnosis.High degree of chronic HCV is one of the important causes of liver cirrhosis and liver cancer.In China,there are about 10 million people infected with hepatitis c virus,and the number of new reported cases has been increasing steadily in recent years,which has become a considerable medical burden.As a new generation of direct acting antiviral agents(DAAs)coming into the Chinese market,its higher cure rate brings hepatitis c patients more hope,but its long-term efficacy and safety still need further observation and evaluation based on a amount of real clinical data.Cholinergic system is not only closely related to the central nervous memory,learning,but more and more studies found the expression of cholinergic system components also exists in the non-neurogenic cells,and participate in the basic function such as cell proliferation differentiation,even with angiogenesis,inflammation,immune regulation and other biological function,we calls this part of the cholinergic system the non-neurological cholinergic system(NNCS).ObjectiveThis study seeks to clarify the combination between the neurological cholinergic system(NNCS)and hepatitis c virus(HCV),trying to better understand the HCV infection and immune escape mechanism,reveal more complex mechanism function between HCV infection and host cells,and provide important clues for development of new HCV antiviral drugs from a new angle,which may also provide reference for development in other chain RNA virus infection mechanism.MethodsAcetylcholine test kit was used to detect the changes in the intracellular and extracellular acetylcholine(Ach)solubility after HCV infection on Huh7 cell line.The AchE enzyme activity was inhibited by inhibitors and the expression level of AchE protein was changed for purpose of clarifying the effect of intracellular Ach on HCV infected cells under the catalysis of acetylcholine esterase(AchE).Moreover,the expression of the corresponding apoptosis marker molecule caspase3 and inflammatory factors McP-1,TNF-? and IL1? were detected by western blot and qPCR when the cells were treated with extracellular Ach,so as to clarify the effect of extracellular Ach on cell apoptosis,inflammation and other biological functions by acting on the acetylcholine receptor(AchR)on the cell membrane.Furthermore,the lipid raft was extracted by sucrose gradient centrifugation,and the expression of Caveolin2,the marker molecule of lipid raft,was detected by cholera toxin staining to explore how AchE changed the positive rate of HCV infection on Huh7 cells.results and conclusionsWhen the AchE enzyme activity was inhibited and the AchE protein expression was down-regulated,the positive rate of HCV infection was decreased.After 36 h of HCV infection on Huh7 cell,the expression of AchE,ChAT and VACHT were increased in the cells,and the intracellular Ach solubility was low and unchanged,while the extracellular Ach solubility was up-regulated after HCV infection.When Huh7 cells was directly treated with extracellular Ach,the apoptosis caspase3 did not cleave,but the expression levels of intracellular inflammatory factors MCP-1 and TNF-? were elevated.But when treated with ?7nAchR receptor antagonist before dealing with extracellular Ach,the mRNA expression levels of inflammatory factors MCP-1 and TNF-? were not changed at this time.Further,after extracting lipid rafts by treating Huh7 cells with an AchE inhibitor and extracellular Ach,the expression level of lipid rafts was observed unaltered.At the same time,it was also found that the infection of Vero cells was also reduced by Zika and WNV flavivirus after the inhibition of AchE enzyme activity.Taken together,the results show that changing the activity and expression of AchE enzyme can reduce the infection rate of HCV on Huh7 cell.AchE not only plays a role when HCV infected cells,but also when similar flaviviruses infected cells.AchE does not regulate the HCV infection by altering the expression of lipid rafts.The combination of extracellular Ach and AchR receptor does not change the apoptosis,but it inhibits the expression of some inflammatory factors to some extent.When the inflammatory reaction is inhibited,the cells will be a good state for HCV infection on cells.