Anti-inflammatory Mechansim Of Sargentodoxa Cuneata And Patrinia Villosa Extract Through FAK/PI3K/Akt Pathway Regulation And Glucose Metabolism Reprogramming

Objective:Pelvic inflammation disease(PID)refers to the inflammation of the pelvic peritoneum,internal genitalia and its surrounding connective tissue,which can lead to infertility,ectopic pregnancy,chronic pelvic pain and other diseases.As present,antibiotics are often used in clinical treatment,the current treatment strategies have limited efficacy due to side effects and drug resistance.Emerging studies suggest that regulation of the FAK/PI3K/Akt pathway and glucose metabolism reprogramming are novel strategies for the treatment of inflammation-related diseases.Our previous studies have found that the combination of S.cuneata and P.scabiosifolia exerts the anti-inflammatory effect in PID with dampness-heat stasis syndrome in silico study,and the major signaling pathway involves AGE-RAGE,Focal adhesion and PI3K/Akt and NF-κB pathway.However,the anti-inflammatory mechanism of S.cuneata and P.villosa need to be elucidated.Therefore,this study aims to elucidate the mechanism of S.cuneata and P.villosa for PID through in vitro and in vivo experiments to provide theoretical support for clinical application and transformation.Contents and Methods:The active components of S.cuneata and P.villosa extract(S&P extract)were analyzed through liquid chromatography-mass spectrometry(LC-MS/MS).The effect of S&P extract on PID in mice is firstly validated by in vivo experiments.CCK8,LDH,adhesion and transwell assays were performed to investigate the effects of S&P extract on the proliferation,adhesion and migration ability of macrophages.The effect of S&P extract on the release of nitric oxide(NO)in macrophages was detected by Griess reagent.Cytometric bead array assay was used to detect the effects of S&P extract on expression of interleukin-6(IL-6),interleukin-10(IL-10),monocyte chemoattractant protein-1(MCP-1),interferon gamma(IFN-γ),tumor necrosis factor α(TNF-α)and interleukin-12 p70(IL-12p70)of LPS-induced macrophages.Flow cytometry assay was conducted to observe the effect of S&P extract on the polarization of macrophages.RNAseq assay and western blot were used to detect the effect of S&P extract on the transcriptome and protein expression of macrophages;The effect of S&P extract on the metabolic spectrum of macrophages was detected by metabolomics assay.Results:The results of LC-MS/MS analysis showed that the active components in the S.cuneata were(+)-epicatechin,chlorogenic acid,procyanidin B1 and procyanidin B2;the active components in P.villosa were kaempferol,cynaroside,quercitrin,genistin,homoorientin,isoquercitrin,rutin,oleanolic acid,ursolic acid and palmitic acid.Animal experiments showed that in the model group,congestion and edema were evident in the uterine tissue,and the infiltration of lymphocytes,neutrophils,and plasma cells were observed in tissues,further endometrial hyperplasia and shedding;the congestion and edema of uterine tissue in the treatment group was less than that in the model group,only a few inflammatory cells were seen,and the endometrial epithelium was more intact.S&P extract(100 μg/mL,200 μg/mL and 400 μg/mL)inhibited the proliferation and migration of macrophages and LPS-induced macrophages,changed the morphology of macrophages,and inhibited the release of NO and iNOS expression of LPS-induced.Further,after adding the S&P extract,the expression of IL-6,MCP-1 and TNF-α is significantly inhibited and the expression of IL-10,IFN-y and IL-12p70 were increased in a dose-dependent manner.Different concentrations of S&P extract could significantly inhibit the LPS-induced CD11c expression and enhance the expression of CD206 and Argl of macrophages.FAK inhibitor(Defactinib)significantly inhibited the LPS-induced CD16/32 expression,when combined with S&P extract(400 μg/mL),this effect is more pronounced.However,Defactinib had little effect on LPS-induced CD206 expression,whereas it decreased after combined with the extract(400 μg/mL),but the expression of Argl was not affected.The above results suggest that S&P extract may participate in M2 macrophage polarization by targeting FAK signaling pathway.RNA sequencing shows that S&P extract(400 μg/mL)was regulated LPS-induced genes,among them,2747 genes are upregulated,and 3811 genes are downregulated.The up-regulated genes are involved in M2 macrophage:Il10,Ccl17,Ccl22 and Cd68;the down-regulated genes are involved in M1,FAK/PI3K/Akt pathway and glycolysis process:Stat1,Il18,Cd80,Cd86,Nos2,Il1β,Vegfa,Map2k1,Vegf,116,Pik3ap1,Rafl and Pdhb.The results indicates that S&P extract could regulate the M1/M2 macrophages phenotype polarization and the glycolysis metabolism in genetic level.Western Blot showed that the S&P extract significantly inhibited the phosphorylation of FAK,PI3K and Akt in a dose-dependent manner after 24 h or 15 min of treatment.Defactinib alone exerts a strong inhibitory effect on the phosphorylation of FAK,PI3K and Akt,when combined with S&P extract(400 μg/mL),it exerts a strong synergy inhibitory effect.The results indicated that FAK/PI3K/Akt signaling cascade could be a potential target of S&P extract.After S&P extract treatment 24 h,the expression of PD,PKM2,PKM1/2,HK Ⅰ and HK Ⅱ was remarkably inhibited,this result is consistent with the above RNA sequencing results,these results indicated that S&P extract can regulate the glycolysis related protein and redirect the metabolism programming.Metabolomics analysis showed that the S&P extract exerts a strong amelioration of LPS-induced metabolic disturbance.By heatmap and KEGG module pathway enrichment analysis,most of these metabolites were glucose metabolism,including UDP-alpha-D-glucose,pyruvic acid,cytarabine,guanosine and glutathione.KEGG analysis showed that HIF-1,PI3K/Akt,Glycolysis,MAPK,Pyruvate metabolism,Carbon metabolism,Focal adhesion,and TNF pathway were the main pathways.Conclusion:(1)The active components in S&P extract were(+)-epicatechin,chlorogenic acid,cynaroside,quercitrin,kaempferol,procyanidin B1,procyanidin B2,genistin,homoorientin,isoquercitrin,rutin,oleanolic acid,ursolic acid and palmitic acid.(2)S&P extract can significantly reduce the inflammatory response of LPS-induced PID in vivo.(3)S&P extract significantly changed the morphology of macrophages and inhibit the proliferation,adhesion and migration ability of LPS-induced macrophages in a dosedependent manner.S&P extract remarkably inhibited the release of NO and expression of iNOS in LPS-induced macrophages,inhibited the expression of LPS-induced IL-6,MCP-1 and TNF-α,and enhanced the expression of IL-10,IFN-γ and IL-12p70.(4)S&P extract inhibited the expression of M1 markers CD11c and CD 16/32,and promoted the expression of M2 markers CD206 and Arg1.(5)S&P extract regulated the M1/M2 macrophages phenotype polarization,glycolysis metabolism and FAK/PI3K/Akt pathway in genetic level.(6)S&P extract significantly inhibited the phosphorylation of FAK,PI3K and Akt in a dose-dependent manner,and the expression of PD,PKM2,PKM1/2,HK Ⅰ and HK Ⅱ was remarkably inhibited.These results indicated that FAK/PI3K/Akt signaling cascade was a be a potential target of S&P extract,and the extract can regulate the glycolysis related protein and redirect the metabolism programming.(7)S&P extract exerts a strong amelioration of LPS-induced metabolic disturbance,most of these metabolites were glucose metabolism,including UDP-alpha-D-glucose,pyruvic acid,cytarabine,guanosine,glutathione;KEGG analysis showed that HIF-1,PI3K/Akt,Glycolysis,MAPK,Pyruvate metabolism,Carbon metabolism,Focal adhesion,TNF pathway were the main pathways.