Anti-inflammatory Effect And Mechanism Of Hederasaponin C In Vitro And In Vivo

Objective:Pulsatilla chinensis（Bge.）Regel is a plant of the buttercup family and is widely used in the treatment of diseases such as enteritis,dysentery and cancer.Hederasaponin C（B5）is one of the important active ingredients in the triterpene saponins of Hederasaponin C,which belongs to the zidonolide-type triterpene saponins.At present,some studies have found that Hederasaponin C has the effect of relieving colitis,but other biological activities are not yet known.In vitro Lipopolysaccharide（LPS）/LPS+ATP macrophages and in vivo acute lung injury（ALI）and acute kidney injury（AKI）models were used to investigate the pharmacodynamic evaluation and mechanism of anti-inflammatory activity of B5.Methods:In vitro,using LPS-induced RAW264.7,J774A.1 and THP-1 cells to detect the release of pro-inflammatory factors by ELISA;intracellular calcium ion(Ca²⁺)levels by immunofluorescence and flow cytometry.The expression of NF-κB,NLRP3 and PIP2 signalling pathway related proteins was detected by western blotting,immunofluorescence and co-IP.In vivo experiments,the ALI model was simulated by tracheal drip injection of LPS,ALI-induced mice death,lung aspiration function tests,the levels of leukocytes,neutrophils and lymphocytes in whole blood were detected by blood routine,the expression of TNF-α,IL-6 and IL-1βin serum,alveolar lavage fluid（BALF）and lung tissues were detected by ELISA,the pathological changes in lung tissues were observed by HE staining,and the expression of NF-κB,NLRP3 and PIP2 signaling pathway-related proteins in lung tissues were detected by western blotting.The AKI model was simulated by intraperitoneal injection of LPS.The expression of TNF-α,IL-6 and IL-1βin serum and kidney tissues,the levels of creatinine and urea nitrogen in serum were detected by ELISA,the pathological changes in kidney tissues were observed by HE staining,and the expression of NF-κB,NLRP3 and PIP2 signaling pathway-related proteins in kidney tissues were detected by western blotting.Evaluation of the in vitro and in vivo anti-inflammatory activity and mechanism of Hederasaponin C.Results:In vitro,in LPS-stimulated macrophages,our data showed that B5decreased the release of inflammatory factors such as TNF-α,IL-6 and modulated the TAK1/PKCαpathway to inhibit the nuclear translocation of NF-κB/P65.CO-IP results indicated that B5 inhibited the formation of the PIP2-TAK1 protein complex and suppressed the activation of TKA1;in addition,B5 reduced Ca²⁺production via the PIP2 signaling pathway,which in turn inhibited NLRP3 inflammasome activation.In vivo,using an LPS-induced ALI mouse model to evaluate the therapeutic effect of B5 on ALI.The results showed that B5 increased the survival rate,improved respiratory function and significantly reduced the release of inflammatory cytokines such as neutrophils,lymphocytes,leukocytes,TNF-α,IL-6,IL-1βand lesions in lung tissues.In addition,western blotting results showed that B5 inhibited NF-κB,NLRP3 inflammasome in mouse lung tissues and PIP2 signaling pathway activation in mouse lung tissue.A mouse model of LPS-induced AKI was used to evaluate the therapeutic effect of B5 on AKI.The results showed that B5 inhibited the release of creatinine,urea nitrogen,TNF-α,IL-6 and IL-1βand kidney histopathology.In addition,western blotting results showed that B5 inhibited the activation of NF-κB,NLRP3 inflammasome and PIP2signaling pathway in mouse kidney tissues.Conclusion:In vivo,B5 inhibited the activation of PIP2 signaling pathway and maintained the intracellular Ca²⁺balance,thus inhibiting the activation of NLRP3 inflammasome;in addition,the reduced expression of PIP2 inhibited the formation of PIP2-TAK1 complex and suppressed the activation of TAK1.In vitro,B5 had a protective effect on LPS-induced ALI and AKI in mice,and the mechanism may be through NF-κB,NLRP3,and PIP2 signaling pathways.