Study On The Mechanism Of Ginsenoside Rg1 Inhibiting LPS-induced Pyroptosis In Periodontal Ligament Cells By Regulating The AMPK/Drp1 Pathway

Objective: Periodontitis is a common inflammatory disease of the oral cavity accompanied by resorption and destruction of the alveolar bone.Severe periodontitis can cause tooth loss,and increase the risk of diabetes and cardiovascular disease,which compromises systemic health.The classical pyroptosis is a Caspase-1-dependent programmed cell death mediated by NLRP3 inflammasome,followed by the release of large amounts of inflammatory mediators.Recent studies have shown that NLRP3-mediated pyroptosis is involved in the development and progression of periodontitis,but its upstream related regulatory mechanism needs to be further elucidated.Mitochondria are the power plant of cellular energy and a key hub for regulating cell death.Stimulated by pathological factors,Drp1 mediates excessive mitochondrial division,causing mitochondrial dysfunction and initiating multiple death pathways by activating the caspase system,suggesting that mitochondria may be involved in mediating pyroptosis and playing a regulatory role in periodontitis.Additionally,numerous studies have shown that ginsenoside Rg1 plays a protective role in various inflammatory diseases by inhibiting NLRP3 activation,such as myocarditis,sepsis,and acute liver injury.However,whether it can regulate periodontal inflammation and whether its related protective mechanism is related to mitochondrial function are unclear.The present study was based on Drp1-mediated mitochondrial fission and function dysfunction,to investigate the effect of Ginsenoside Rg1 on LPS-induced pyroptosis in HPDLCs and the underlying mechanism.Methods: Human periodontal ligament cells(HPDLCs)of the 2nd-4th generation with good growth status were randomly divided into six groups,except for the blank control group,the other five groups were treated with different concentrations of LPS(5,10,25,50,100 μg/m L)for 24 and 48 hours,and the cell proliferation viability was measured using the CCK-8 assay to determine the LPS concentration and treatment time,and to simulate the inflammatory microenvironment of periodontitis in vitro.In addition,to investigate the effect of Ginsenoside Rg1 on the viability of HPDLCs in the inflammatory environment,cells in good growth condition were randomly grouped and pretreated with corresponding concentrations of Ginsenoside Rg1(50,100,200 μM)for two hours,then co-cultured with LPS for 24 hours,and set up a blank control group(complete medium culture for 24 hours)and a positive control group(complete medium with LPS treatment for 24 h),then cell viability was determined by CCK-8 assay.To explore the effect of Ginsenoside Rg1 on LPS-induced pyroptosis,the pyroptosis ratio was measured by flow cytometry analysis and the RT-q PCR was applied to determine the expression levels of pyroptosis-related genes(NLRP3,ASC,Caspase-1).Besides,to assess mitochondrial function,mitochondrial fluorescent probes were used to visualize changes in mitochondrial morphology,analyze mitochondrial reactive oxygen species(mt ROS)and mitochondrial membrane potential(MMP)levels,and ATP production was tested using the ATP assay kit.To further explore the underlying mechanism of Ginsenoside Rg1 on HPDLCs pyroptosis,the cells were randomly divided into seven groups as follows: blank control group,LPS group,LPS+Ginsenoside Rg1 group,LPS+Ginsenoside Rg1+Compound C(AMPK inhibitor)group,LPS+AICAR(AMPK activator)group,LPS+Mdivi-1(Drp1inhibitor)group,and LPS+MCC950(NLRP3 inhibitor)group.The secretion levels of pro-inflammatory cytokines(IL-1β,IL-18)and the release of lactate dehydrogenase(LDH)were detected by ELISA.The expression levels of signaling pathway-related proteins p-AMPK/AMPK,p-Drp1/Drp1,NLRP3,ASC,Caspase-1 and GSDMD-NT were detected by Western blot.Results: 1.CCK-8 assay results showed no significant difference in cell viability after 5 μg/m L LPS treatment compared to the control group(P>0.05),while treated with LPS(10,25,50,100 μg/m L)inhibited cell viability in a dosedependent manner.Additionally,the Ginsenoside Rg1 treatment increased cell viability compared with the LPS group,and the best effect was detected at a Ginsenoside Rg1 concentration of 100 μM,with a statistically significant difference(P<0.05).2.ELISA results showed that LPS stimulation promoted the secretion of IL-1β and IL-18 and increased the release of LDH,while ginsenoside Rg1 treatment significantly reduced the release of IL-1β,IL-18 and LDH(P<0.05).3.Flow cytometry analysis and RT-q PCR results indicated that LPS stimulation increased pyroptosis ratio and the expression levels of pyroptosisrelated genes NLRP3,ASC,and Caspase-1.Ginsenoside Rg1 treatment effectively reduced the pyroptosis rate and suppressed the expression of the above pyroptosis-related genes(P<0.05).4.The results of the mitochondrial function assay revealed that LPS treatment increased abnormal mitochondrial morphology and mt ROS generation while decreasing MMP levels and ATP content.Ginsenoside Rg1 up-regulated ATP content and MMP levels while decreasing mitochondrial fission and mt ROS production(P<0.05).5.Western blot results demonstrated that Ginsenoside Rg1 upregulated the expression levels of p-Drp1(Ser 637)and downregulated the expression levels of p-Drp1(Ser 616)in an AMPK-dependent manner,and reduced the expression levels of pyroptosisrelated proteins NLRP3,ASC,Caspase-1,and GSDMD-NT(P<0.05).Conclusion: Ginsenoside Rg1 attenuated LPS-induced pyroptosis and had a protective effect on HPDLCs.Mechanistically,Ginsenoside Rg1 alleviated mitochondrial dysfunction by activating AMPK and inhibiting Drp1-mediated mitochondrial fission,further reducing mt ROS generation and NLRP3 inflammasome expression,thereby attenuating pyroptosis in HPDLCs.