Capsaicin Enhances Antiviral Innate Immunity By Selectively Degrading STAT

BackgroundChilli peppers are traditional Chinese medicine used as both a medicine and a food that can warm channels to dispel coldness and promote digestion.Capsaicin(8-methyl-N-vanillyl6-nonenamide)is the main biological activities and spicy component in Chilli peppers,which is widely recognized as an agonist of transient receptor potential vanilloid subtype 1(TRPV1)and has been approved for the clinical treatment of post herpetic neuralgia(PHN),diabetic peripheral neuropathy(DPN)and HIV-associated neuropathic pain(HNP).Although the essential roles of its potencies in anticancer,anti-inflammatory,and metabolic diseases are emerging,the role of capsaicin in viral replication and antiviral immune response remains poorly studied.Type Ⅰ interferons(IFNs)play a crucial role in the innate immune defense against viral infection.Following the recognition of viral nucleic acids by pattern recognition receptors(PRRs)and subsequent IFN production,subsequently initiate type I IFN-mediated antiviral innate immune response by JAK/STAT,prompting the transcription of hundreds of ISGs with antiviral activities,thereby resisting viral infection.STAT3,the most promising target for cancer therapy,functions in innate antiviral immunity are largely underestimated.Several recent studies have shown that STAT3 functions as a transcriptional suppressor on the type I IFN pathway,inhibiting antiviral innate immune response.Therefore,it is a sensible strategy for broad-spectrum antiviral drugs to clear pathogen infection by improving ISGs and IFN expression via pharmacological inhibition of STAT3.A previous study suggested that capsaicin functions as a blocker of STAT3 constitutive activation in IL-6 stimulation,but its inhibitory mechanism is unclear.Based on the above problems,this study will focus on capsaicin’s ability to enhance the type I IFN-mediated antiviral innate immunity by inhibiting STAT3,aiming to elucidate the antiviral function and mechanism of capsaicin.Objective1.Evaluate the antiviral activity of capsaicin in vitro and in vivo.2.Explore the effect of capsaicin on type Ⅰ IFN-mediated antiviral immune responses and elucidate the specific mechanism by which capsaicin enhances the antiviral immune response mediated by type Ⅰ IFN by inhibiting STAT3.MethodsPart 1 Study on the antiviral activity of capsaicin1.The effect of capsaicin on viral infection in vitro:Flow cytometry,qRT-PCR,and Western Blot were used to detect the antiviral activity of capsaicin on vesicular stomatitis virus(VSV),encephalomyocarditis virus(EMCV),human influenza virus A(H1N1),and adenovirus(AdV).The time-of-addition assay and virus entry assay were used to measure the effect of capsaicin on virus attachment and entry in the viral life cycle.2.The effect of capsaicin on viral infection in vivo:We used VSV to construct a viral infection mouse model.Survival rates of mice infected with VSV were observed,and qRT-PCR was used to detect VSV replication and the expression levels of Tnf,Il1b,and Il6 in the liver,lung,and spleen.H&E examination of lung sections revealed infiltration of inflammatory cells and tissue damage.Part 2 Study on the antiviral mechanism of capsaicin1.The effect of TRPV1 on the antiviral function of capsaicin:Flow cytometry detected the effect of AMG517,a TRPV1 antagonist,on the antiviral function of capsaicin in VS V-eGFP infected A549 cells.Construct TRPV1 knockdown cells model by using siTRPV 1.On this basis,qRT-PCR was used to detect the antiviral effect of capsaicin in TRPV1 knockdown cells by measuring viral genome levels.2.The effect of capsaicin on antiviral innate immune response:qRT-PCR was used to detect the effect of capsaicin on Ifit1、Ift2、Ifi44、Oas2 mRNA levels in murine primary peritoneal macrophages.qRT-PCR was used to detect the effect of capsaicin on IFNB1 and ISGs mRNA levels in A549,MEF,iBMDM,and THP1 upon poly(I:C)or ISD stimulation.Western Blot was used to measure the effect of capsaicin on the protein level of ISGs and pSTAT1 triggered by poly(I:C).3.The effect of STAT3 on the antiviral function of capsaicin:Western Blot and qRT-PCR were used to detect the effects of STAT3 silencing on type Ⅰ IFN signaling.Western Blot and immunofluorescence were used to detect the effect of capsaicin on STAT3.qRT-PCR was used to detect the antiviral effect of capsaicin in STAT3 overexpression cells by measuring viral genome levels,IFNB1,and ISGs mRNA levels.4.The effect of capsaicin on the degradation of STAT3:qRT-PCR was used to detect the effect of capsaicin on STAT1,STAT2,and STAT3 mRNA.Inhibition of STAT3 protein biosynthesis using cyclohexide(CHX)and Western Blot detection of the effect of capsaicin on STAT3 protein degradation.Western Blot was used to detect the types of capsaicin affecting STAT3 degradation using proteasome inhibitor MG132,lysosomal inhibitor chloroquine(CQ),and bafilomycin A1(BafA1).The direct effects of capsaicin on STAT3 were analyzed using molecular docking,drug affinity response target stability(DARTS),and cellular thermal shift assay(CETSA)techniques.ResultsPart 1 Study on the antiviral activity of capsaicin1.In cells experiments,capsaicin effectively suppressed VSV,EMCV,H1N1,and AdV replications.Furthermore,the inhibitory effect of capsaicin on multiple viral infections was mainly acting at postviral entry steps.2.Higher survival rates and attenuation of VSV loads in the liver,lung,and spleen of capsaicin-treated mice were observed compared with model groups.Moreover,less lung tissue damage and the expression levels of Tnf,Il1b,and Il6 in the capsaicin-treated mice group.Part 2 Study on the antiviral mechanism of capsaicin1.AMG517 failed to antagonize the antiviral effect of capsaicin at different concentrations,and AMG517 at much higher concentrations alone inhibited VSV replication.Moreover,capsaicin still restricted viral replication,including that of VSV,HIN1,and EMCV in TRPV1 knockdown cells.2.Capsaicin alone significantly activated the transcript levels of ISGs in murine primary peritoneal macrophages.Moreover,capsaicin promoted Ifnb1 and ISG expression triggered by poly(I:C)or ISD.Furthermore,the protein levels of ISGs and p-STAT1 were markedly induced by capsaicin upon poly(I:C)stimulation.3.STAT3 knockdown greatly increased the p-TBK-1 and p-STAT1,the mRNA levels of IFNB1 and ISGs in poly(I:C)-challenged A549 cells.Capsaicin significantly restrained pSTAT3.STAT3 overexpression attenuated the antiviral effect of capsaicin and the induction of IFNB1 and ISGs mRNA.4.Capsaicin treatment did not affect STAT3 mRNA but slightly promoted STAT1 and STAT2 mRNA.Furthermore,capsaicin promoted the lysosomal degradation of STAT3 with direct interaction.ConclusionsOur study demonstrated that capsaicin is a promising drug candidate against viral infections,which effectively suppressed several viral replications after viral entry in a manner independent of TRPV1 in vitro,protecting mice from VSV infection in vivo.The findings provide scientific evidence to support wider clinical application of capsaicin.Mechanistically,we showed that capsaicin promoted the lysosomal degradation of STAT3 with direct interaction,thereby attenuating the negative regulation of STAT3 on the type Ⅰ IFN response and enhancing host resistance to viral infection.Overall,our research offers a feasible pharmacological strategy for improving host resistance to viral infection.