The Mechanism Of P.acnes-induced NLRP3 Inflammasome Activation And Effects Of PPI In Inflammation

Acne vulgaris is an inflammatory disease of hair follicle-sebaceous glands,mainly involving adolescents and some adults,with a high incidence.Four factors have been identified to play critical roles in the pathogenesis of acne:sebum hyperproduction,altered follicular keratinization,Propionibacterium acnes(P.acnes)colonization and proliferation,and immune reaction and inflammation.P.acnes has been thought to participate in the pathogenesis of acne vulgaris for decades.In recent years,NLRP3 inflammasome has been received attention in acne inflammation.Researches have demonstrated that P.acnes induced inflammation in sebocytes and monocytes via NLRP3/caspase-1/IL-1 beta signaling pathways.Keratinocytes,as an important component of the pilosebaceous unit,have previously been shown to be associated with acne inflammation.However,whether P.acnes induces inflammation in keratinocytes by activating NLRP3 inflammasome remains to be clarified.Antibiotics and isotretinoin are frequently used therapeutic agents for acne inflammation,but they have potentially serious detrimental effects.Therefore,there is an increasing need to identify new anti-acne ingredients,especially natural products with relative safety.In 2015,Parispolyphylla was officially documented as an anti-inflammatory and hemostatic agent in the Chinese Pharmacopoeia.Polyphyllin I(PPI),a major steroidal saponin extracted from Paris polyphylla rhizomes,displays proapoptotic and anti-tumor effects.However,no studies have shown the role and underlying mechanism of PPI-mediated anti-inflammatory activity.We aimed to evaluate the effects of PPI in P.acnes induced inflammation in vitro.Our research includes the following two parts:Part 1:P.acnes induced NLRP3 inflammasome activation and PPI intervention studyObjective:To explore the mechanism of the activation of NLRP3 inflammasome by P.acnes and effect of PPI on it.Methods:(1)In HaCaT keratinocytes stimulated by P.acnes,we detected the secretion of IL-1β and IL-8 by ELISA method;(2)The expression of CD36,NOX1,NLRP1,NLRP3,NLRC4 and AIM2 in HaCaT keratinocytes stimulated by P.acnes were detected by qPCR and Western blot method;The expression of ASC,active caspase-1 and cleaved-1β were detected by Western blot method;Caspase-1 activity was detected by caspase-1 activity kit;The expression of CD36 and ROS were detected by flow cytometry method.(3)In cultured HaCaT keratinocytes,we interfered the expression of CD36,NOX1,and NLRP3 by siRNA respectively,and detected the P.acnes-induced expression of NLRP3,ASC,active caspase-1,and cleaved IL-β by Western blot method.(4)After CD36 was interfered by siRNA,the P.acnes-induced expression of NOX1 was observed by western blot method and ROS production was detected by flow cytometry method.(5)After NOX1 was interfered by siRNA,we obersved the P.acnes-induced expression of CD3 6 by western blot method,and ROS production by flow cytometry method;(6)In cultured HaCaT keratinocytes,after the addition of NAC and DPI respectively,we detected the P.acnes-induced expression of NLRP3 inflammasome protein components by western blot method and ROS production by flow cytometry method;(7)After CD36,NOX1 and NLRP3 were interfered by siRNA or pretreated with DPI,NAC or caspase-1 inhibitor,we detected the P.acnes-induced production of IL-1β and IL-8 by ELISA method.(8)After adding IL-1 neutralizing antibody and IL-lreceptor blocker,we performed ELISA method to detected the secretion of IL-8 in HaCaT keratinocytes stimulated by P.acnes;(9)After treated with PPI in different concentration gradients(0.3,0.6,0.9 g/ml),we detected the expression of CD36,NOX1,NLRP3,ASC,Active caspase-1,Cleaved IL-1β by western blot method,the expression of ROS by flow cytometry method,the secretion of IL-1β and IL-8 by ELISA method.Results:(1)P.acnes induced the secretion of IL-1β and IL-8 in HaCaT keratinocytes in a time-dependent manner within 24 hours.(2)The expression of CD36mRNA,NOX1 mRNA,NLRP3 mRNA,ASC mRNA and active-caspase-1 mRNA were up-regulated in P.acnes stimulated HaCaT keratinocytes.The expression of CD36,NOX1,NLRP3 inflammasome protein components and ROS were significantly up-regulated.(3)After the expression of CD36,NOX1 and NLRP3 were interfered with siRNA respectively,or pretreatment with NAC or DPI,flow cytometry results domostreated that ROS expression was significantly decreased;Western blot results showed that NLRP3 inflammasome componotes were significantly down-regulated,and the expression of NOX1 was significantly decreased after CD36 siRNA,while there was no significant changes in CD36 expression after NOX1 siRNA.After pretreatment with caspase-1 inhibitor,the expression of active caspase-1 and cleaved IL-1β protein components were significantly down-regulated.(4)CD36 siRNA,NOX1 siRNA,NLRP3 siRNA,NAC,DPI and capase-1 inhibitors all significantly inhibited the P.acnes-induced production of IL-1β and IL-8.Both anti-IL-1β-Ab and IL-1 Ra inhibited the production of IL-8.(5)In P.acnes stimulated HaCaT keratinocytes,PPI significantly inhibited the secretion of IL-1β,IL-8,production of ROS,and the expression of CD36,NOX1,NLRP3,ASC,Active caspase-I,and Cleaved IL-1β proteins.All of these results were in a concentration-dependent manner.Conclusions:In HaCaT keratinocytes,P.acnes might activate NLRP3 inflammasome via CD36-NOX1-ROS signal pathway.The production of IL-1β in P.acnes-treated HaCaT keratinocytes mediated the production of IL-8.P.acnes induced the production of IL-8 at least partially regulated by the IL-1β/IL-1R signal pathway.PPI inhibited the activation of NLRP3 inflammasome and the secretion of inflammatory cytokines induced by P.acnes.Part 2:PPI inhibited the inflammatory response via the TLR2-p38MAPK-NF-κB signaling pathwayObjective:To investigate the effects of PPI in P.acnes induced inflammation viaTLR2-p38MAPK-NF-κB signaling pathway.Methods:(1)we detected the expression of inflammatory cytokines involved TLR2.In this part,we used different concentrations of P.acnes(105-107CFU/ml)to stimulate HaCaT keratinocytes to establish an in vitro inflammatory model.The secretion of IL-6,IL-8,and TNF-α were detected by ELISA method,and the expression of TLR2 and TLR4 were detected by flow cytometry and western blot method.In the subsequent experi1ents,we used P.acnes(107CFU/ml)to stimulate HaCaT keratinocytes.We detected the expressions of TLR2 mRNA,IL-1 mRNA and K16 mRNA by qPCR mehod,and TLR2,IL-1,K16 proteins,NF-κB and MAPK by western blot method.(2)To investigate the inhibitory effects of PPI on the inflammatory response mediated by TLR2-p38MAPK-NF-κB:In this part,we add PPI to inflammatory models with different concertrations.According to the results of cck-8,we selected different safe concentrations of PPI for subsequent experiments.The production of IL-6,IL-8 and TNF-α were detected by ELISA method.The expressions of TLR2,IL-1 and K16 were detected by qPCR and western blot method.We analysed NF-κB and MAPK activation related proteins by Western blot method.The translocation of NF-κB was detected by immunofluorescence method.In addition,the effects of PPI,NF-B inhibitor and p38 MAPK inhibitor on IL-8 secretion were detected by ELISA method.Results:(1)P.acnes stimulated the expression of TLR2 and TLR4 in HaCaT keratinocytes and secretion of IL-6,IL-8 and TNF-α,and the expressions and secretion were in a concentration-dependent manner.(2)P.acnes significantly up-regulated the expression of TLR2 mRNA,IL-1 mRNA,K16 mRNA and TLR2,IL-1,K16 protein.The expression levels of NF-κB and MAPK associated proteins were significantly increased.(3)PPI inhibited the production of IL-6,IL-8,TNF-αand the expression of TLR2,L1-1α,and K16 in a concertration-dependent manner.With the concertration increased,the inhibitory effects of PPI on NF-κB and MAPK related proteins were enhanced.(4)The secretion of IL-8 was significantly inhibited by PPI,NF-κB inhibitor and p38 MAPK inhibitor.Conclusion:PPI can inhibit the activation of TLR2-p38MAPK-NF-κB signaling pathway in HaCaT keratinocytes stimulated by Pacnes.