Effect And Mechanism Of Caffeine-induced Autophagy On Inflammation In Experimental Autoimmune Encephalomyelitis Mice

BACKGROUND AND PURPOSEMultiple Sclerosis(MS)is an immune disease that occurs in the nervous system and is pathologically characterized by inflammatory cell activation.Activated microglia secretes inflammatory factors and aggravates the damage in MS.In the EAE model,autophagy can reduce oxidized protein aggregates and lesion enlargement,and improve the poor prognosis.Recent studies have shown that autophagy can reduce inflammation and improve the severity of MS or EAE.Autophagy induction may be an effective target for MS and EAE therapy.Caffeine is the most widely consumed psychostimulant in the world.It is mainly found in dietary sources such as coffee,tea and energy drinks.Caffeine can reduce inflammation and induce autophagy.Furthermore,ramamycin target protein(m TOR),the main regulator of autophagy,can be regulated by caffeine.This article aims to investigate the specific effects of caffeine on inflammation and autophagy,and its possible mechanisms in EAE,providing more possibilities for the treatment of MS patients.PART I: PROTECTIVE EFFECT OF CAFFEINE ON INFLAMMATORY CELL INFILTRATION AND DEMYELINATION IN EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS MICEMethods:In this part of the study,mice was treated with caffeine(10/20/30mg/kg/d)from day 0 after MOG35-55 immunization.The effect of caffeine on EAE was evaluated by HE,LFB and clinical scores.Results:1.CFA group: mice had no disease,normal diet,good spirits,and gradually increased body weight.EAE+water group: about 7 days after MOG35-55 immunization,mice showed reduced appetite,weight loss,poor mental state,sparse hair and decreased luster,and decreased activity,followed by various degrees of tail weakness and limb weakness or paralysis.These symptoms reached a peak around 16 days after immunization,and then the symptoms gradually eased.EAE+caffeine(10mg/kg/d)group: mice showed symptoms with no difference in severity compared with EAE group about 7 days after MOG35-55 immunization(P>0.05).EAE+caffeine(20mg/kg/d)group: the morbidity and weight loss of mice in the EAE+caffeine(20mg/kg/d)group were significantly lower than those in the EAE group(P<0.05),whereas the clinical scores were lower than those in the EAE group(P<0.05).EAE+caffeine(30mg/kg/d)group: the morbidity and weight loss of mice in the EAE+caffeine(30mg/kg/d)group were significantly lower than those in the EAE group(P<0.05),whereas the clinical scores were lower than those in the EAE group(P<0.05).2.HE showed that the HE scores and the number of inflammatory cells in the EAE group were higher than those in the CFA group.Additionally,there were obvious cell infiltration and "vascular-cuff-like" changes around small blood vessels in the EAE mice spinal cord.The HE scores in the EAE+caffeine(20mg/kg/d)and EAE+caffeine(30mg/kg/d)were significantly lower than those in the EAE group(P<0.05).3.LFB showed that the demyelination in the EAE group was more serious than that in the CFA group.The demyelination in the EAE+caffeine(10mg/kg/d)group was also serious.The demyelination was reduced in the EAE+caffeine(20mg/kg/d)group(P<0.05)and the EAE+caffeine(30mg/kg/d)group(P<0.05).PART II: EFFECT OF CAFFEINE ON AUTOPHAGY LEVEL AND ACTIVATION OF NLRP3 INFLAMMASOME IN EAE MICEMethods:Mice were divided into CFA group,EAE group,EAE+caffeine(20mg/kg/d)group and EAE+caffeine+3-MA group.The expression of Iba1 in the spinal cord microglia was observed by immunofluorescence.The levels of LC3,P62 and NLRP3 in the spinal cord of each group were detected by WB.The expression of inflammatory cytokines IL-1? and IL-18 in spinal cord tissues were detected by ELISA.Results:1.The mean immunofluorescence intensity of Iba1 was significantly lowerer in EAE+caffeine group than that in EAE group(P < 0.05).The mean immunofluorescence intensity of Iba1 in EAE+caffeine+3-MA group was significantly higher than that in EAE+caffeine group(P<0.05).These results suggest that caffeine may inhibit the activation of microglia in EAE via autophagy.2.The level of LC3II/LC3? was significantly increased in EAE+caffeine group than that in the EAE group(P<0.05).The levels of P62 and NLRP3 were significantly decreased in EAE+caffeine group than those in the EAE group(P<0.05).These results suggest that caffeine can up-regulate autophagy levels and decrease the level of NLRP3.The level of LC3II/LC3? in the EAE+caffeine+3-MA group was lower than that in the EAE+caffeine group(P<0.05).The level of NLRP3 in the EAE+caffeine+3-MA group was higher than that in the EAE+caffeine group(P<0.05).These results suggest that caffeine may reduce the level of NLRP3 via autophagy.3.Caffeine can reduce the levels of the NLRP3 inflammasome-related inflammatory cytokines IL-1?(P<0.05)and IL-18(P<0.05)in EAE mice,whereas autophagy inhibitor blocked this protective effect,suggesting that caffeine may reduce activation of the NLRP3 inflammasome via autophagy.PART III: EFFECT OF CAFFEINE ON MTOR SIGNALING PATHWAY IN PRIMARY MICROGLIA AND BV2 MICROGLIA CELL LINEMethods:In this section,mice primary microglia(PM)and BV2 cell line were cultured.WB was conducted to detect the effect of caffeine on LC3,P62 and m TOR pathway-related proteins p-m TOR,p-p70S6 K,p-4EBP1.Autophagy double-labeled adenovirus was conducted to detect autophagic flux in PM.Transmission electron microscopy was conducted to detect autophagosome formation in PM.ATG5 si RNA interference was conducted to block autophagy.ELISA was conducted to detect IL-18 and IL-1? levels.Results:1.The level of LC3II/LC3? in the caffeine group was higher than that in the control group(P<0.05).The levels of P62,p-m TOR/m TOR,p-p70S6K/p70S6 K and p-4EBP1 were lower in the caffeine group than those in the control group(P<0.05)in PM and BV2.2.Autophagy double-labeled adenovirus(m RFP-GFP-LC3)showed that GFP/m RFP/cell in the caffeine group was lower than that in the control group(P<0.05).3.Transmission electron microscopy revealed that caffeine promoted the formation of PM autophagosomes.4.The level of LC3-II/LC3-I in the Caffeine+Si-Atg5 group was lower than that in the Caffeine+Si-Con group(P<0.05).The level of NLRP3 in the Caffeine+Si-Atg5 group was higher than that in the Caffeine+Si-Con group(P<0.05).5.The effect of caffeine on reducing the release of inflammatory cytokines IL-I?(P<0.05)and IL-18(P<0.05)was weakened by ATG5 si RNA interference.Conclusions:1.Caffeine plays a neuroprotective effect,decreases inflammatory cell infiltration,and demyelination in EAE mice.The effect is dose-dependent,and the effect is most obvious at the peak of disease.2.Caffeine can enhance the autophagy level,decrease the level of NLRP3,inhibit the microglia activation in EAE mice.The protective effect of caffeine was attenuated after autophagy inhibitor treatment,indicating that the anti-inflammatory effects of caffeine are closely related to caffeine-induced autophagy in EAE mice.3.Caffeine reduce the phosphorylation level of m TOR and enhance the autophagic flux in microglia.Therefore,caffeine inhibits the activation of NLRP3 inflammasome by inducing autophagy via the m TOR pathway in microglia.