Study On Efficacy And Mechanism Of Sweroside From Pterocephalus Hookeri To Ameliorate Bone Destruction In Rheumatoid Arthritis

Pterocephalus hookeri(P.hookeri)is a commonly used medicinal material of Tibetan,which has record in Sibu Medical Code and Jingzhu Materia Medica and other Tibetan medical classics.The Chinese Pharmacopoeia(2020 version)stipulates that drying whole plant of Pterocephalus hookeri(C.B.Clarke)H(?)eck is genuine source of Pterocephali Herba,which belongs to dipsacaceae and is treated various diseases containing cold,enteritis and dysentery.P.hookeri was used to treat "Zhenbu disease" for thousands of years.The symptoms of "Zhenbu disease " are like rheumatoid arthritis(RA)in modern medicine.The anti-inflammatory activity of the iridoid components isolated from P.hookeri were tested in our previous research,and sweroside could significantly inhibit inflammation in vitro.To further explore the anti-rheumatoid arthritis effect and mechanism of sweroside.This article was carried out following 4 parts of research:1.Study on anti-RA of P.hookeri based on network pharmacology.① Chemical components collection and target genes prediction: 92 chemical constituents of P.hookeri were collected by searching CNKI and Web of science literature databases.FAFDrugs4 database was used to screen the drug-like ingredients of P.hookeri,and 28 compounds were accepted.68 target genes were predictd by Pharm Mapper database.② RA-related genes collection: The RA disease target genes were obtained from the OMIM,Drug Bank,Dis Ge NET,TTD and TCMSP databases,and 1,895 RArelated genes were collected.③ Common target genes identification: 44 overlapping target genes were identified as common target genes of compounds and RA by Venn diagram,which were definedas anti-RA target genes of P.hookeri.○4 Network construction and potential mechanism prediction: The “compound-target gene” network was construct by Cytoscape 3.7.0 software,which indicated that 18 ingredients in P.hookeri related to 44 overlapping target genes of anti-RA,and among 18 components 14 were iridoids.To perform KEGG pathway enrichment analysis for target genes by David6.8 database,the target genes were enriched a total of 42 signaling pathways.Among them,TNF signaling pathway,VEGF signaling pathway and osteoclast differentiation pathway involved in the occurrence and development of RA.Combined with the results of previous researches and literature resaerches,sweroside was selected as the follow-up research compound to explore the therapeutic effect and mechanism aganist the bone destruction in RA.2.The pharmacodynamic research of sweroside ameliorated bone destruction in RA in vivo.Complete Freund’s adjuvant-induced arthritis(AIA)model in mice were used to study the anti-RA pharmacodynamic effect of sweroside.Sweroside was intragastric administration at the doses of 50,100 and 200 mg/kg for 31 days.① Body weight,paw swelling and arthritis score were measured.The results showed that sweroside could promote the normal growth of body weight and reduce the paw swelling and arthritis score in AIA mice.② The level of inflammatory cytokines in AIA mice serum were detected by ELISA assay,and it was found that sweroside decreased the levels of TNF-α,IL-1β and IL-6 in the serum.③ The pathologic status of synovium in AIA mice was observed by H&E staining analysis.The results indicated sweroside reduced the inflammtion of synovium in ankle joint.○4 TRAP staining analysis results showed sweroside suppressed TRAP staining positive area,inhibited osteocalst differentiation.⑤ Masson staining assay was used to detect the deposition and distribution of collagen fibers in the ankle of AIA mice,and the results displayed that sweroside could promote collagen fiber formation.⑥ The immunohistochemistry staining ananlysis results displayed sweroside reduced NFAT2 expression and increased RUNX2 expression,and Western blot results turned out that sweroside reduced the protein expressions of NFAT2,CTSK,MMP-9,MMP-13 and RANKL;increased the protein epressions of RUNX2,COL1A1,OCN and OPG.The above results indicated that sweroside could effectively alleviate RA symptoms of AIA mice through reducing inflammation and inhibiting bone destruction.Bone destruction is a pathological feature of RA,mainly caused by overactivated osteoclast differentiation and decreased osteoblast differentiation.Sweroside shown a prominent effect on inhibiting osteoclast differentiation and promoted bone formation in vivo,therefore,we further explored the effect and mechanism of sweroside on osteoclast differentiation and osteoblast differentiation in vitro.3.Study on sweroside reduced osteoclast differentiation in vitro.RAW 264.7 cells were turned into osteoclasts induced by 100 ng/m L RANKL and50 ng/m L M-CSF,and studied the effect and mechanism of sweroside reduced osteoclast differentiation.① MTT assay indicated that a concentration of 10 ～ 160 μmol/L,sweroside exhibited no cytotoxicity against at RAW 264.7 cells,indicating sweroside has a good safety.Combined with the previous anti-inflammatory study 20,40,80 μmol/L concerntration of sweroside were slected to treate osteoclasts for subsequent studies.②As determined by TRAP staining assay,a decreased activity of TRAP in sweroside treated group was observed compared with RANKL and M-CSF induced group.③ The osteoclast bone resorption was detected by specialised bone resorption plate,and significantly small bone resorption pit area was observed when treated with sweroside.○4 The expression of osteoclast specific markers and transcriptional factors(NFAT2,CTSK,MMP-9 and MMP-13)downregulated upon treatment with sweroside by Western blot analysis.⑤ The immunofluorescence and Western blot assay also were used to detect the protein expression of NF-κB pathway in osteoclasts.The results showed sweroside decreased fluorescence intensity of NF-κB,reduced proteins expression of the IKKα and p-NF-κB,and enhanced expression of IκB.Meanwhlie sweroside reduced inflammatory factors lever included TNF-α,IL-1β,and IL-6 in osteoclast.The results illustrated that sweroside could alleviate inflammation in osteoclasts.⑥ an enhanced expression of β-catenin was observed by immunofluorescence analysis.Besides,sweroside reduced RANKL and M-CSF induced expression of PI3 K,p-AKT,p-GSK-3βand β-catenin in RAW 264.7 cells by Western blot analysis.These results illustrated sweroside could active PI3K/AKT/GSK-3β/β-catenin pathway in osteoclasts.Further to validate the activation of sweroside on protein expression of PI3K/AKT/GSK-3β/β-catenin signaling pathway in osteoclast,we blocked the activation of PI3K/AKT/GSK-3β/β-catenin signaling pathway with LY294002,MK2206 and XAV-939,they were,respectively,an inhibitor of PI3 K,AKT and β-catenin.sweroside treatment failed to activate the expression of p-AKT,p-GSK-3β and β-catenin in LY294002,MK2206 pretreated RAW 264.7 cells.At the other hand,a markedly increased in the expression of osteoclast specific markers and transcriptional factors were observed in LY294002 or MK2206 treated group compared with sweroside stimulated group.Also,similar results were observed when β-catenin activation was abrogated by XAV-939 in RAW 264.7 cells Cumulatively,these results elucidated that sweroside prevented osteoclast differentiation of RAW 264.7 via activating PI3K/AKT/GSK-3β/β-catenin signaling pathway.4.Study on sweroside promoted osteogenic differentiation in vitro.MC3T3-E1 cell osteogenic differentiation basal medium was used to differentiate MC3T3-E1 cell into osteoblast,and a model of osteogenic differentiation inhibition induced by LPS was established to investigate the effect and mechanism of whether sweroside could promote osteogenic differentiation.① 1,10 and 100 μg/ m L LPS were used to treat osteoblasts cells.ARS staining result indicated 10 μg/ m L LPS could significantly reduce the number of mineralized nodules in osteoblasts.What’s more,and there was no significant cytotoxicity against osteoblast cells of LPS at this concentration.Therefore,osteoblasts induced by 10 μg/ m L LPS were selected as the study model.②The cytotoxicity of sweroside on MC3T3-E1 cells was examine through MTT assay,and we found sweroside had no obvious cytotoxicity at concentrations of 10 ～ 160 μmol/L,indicating sweroside had a better safety range.Thus,LPS-induced osteoblasts were treated with sweroside at concentrations of 20,40,and 80 μmol/L for subsequent study.③ The differentiation degree of osteoblasts was detected by ALP staining analysis and activity test,and sweroside could significantly increase the ALP in LPS-induced osteoblasts.○4 ARS staining results showed that sweroside could boost the number of mineralized nodules.⑤ Western blot analysis results found sweroside could up-regulate the expression of RUNX2,COL1A1,OCN and OPG,down-regulated the RANKL expression.⑥ The immunofluorescence and Western blot method were applied to measure the protein expression of NF-κB pathway.The results showed sweroside reduced fluorescence intensity of NF-κB in osteoblasts,decreased IKKα and p-NF-κB protein expression,increased IκB protein expression,and reduced the inflammatory factor levels of TNF-α,IL-1β,and IL-6,compare with those in mode group.The results suggested that sweroside alleviated the inflammation of LPS-induced osteoblasts via inhibited NF-κB pathway.⑦ The PI3K/AKT/GSK-3β/β-catenin pathway was tested by immunofluorescence and Western blot assay.The results indicated sweroside heightened the β-catenin fluorescence intensity,and up-regualtedthe expression levels of PI3 K,pAKT,p-GSK-3β and β-catenin,when compared with those in LPS-induced osteoblasts.To verified if sweroside could promote osteoblast differentiation via activing PI3K/AKT/GSK-3β/β-catenin pathway,we treated LPS-induced osteoblasts with PI3 K,AKT and β-catenin inhibitors of LY294002,MK2206 and XAV-939 respectively.And the Western blot results showed PI3 K,p-AKT,p-GSK-3β and β-catenin proteins expression levels were reduced cpmpared with sweroside group,and the RUNX2,COL1A1,OCN proteins expression decreased accordingly.Taken together,sweroside could promote LPS-induced osteoblast differentiation,the mechanism might be active PI3K/AKT/GSK-3β/β-catenin pathway.In this study,we used network pharmacological methods to speculate that iridoids may be the key effective constituentsof P.hookeri to treat RA,and the mechanism may be related to treat bone destruction in RA.Combined with our previous study,sweroside was considered as the representative iridoid active constituent of P.hookeri.We further studied the therapeutical effect and mechanism of sweroside in the treatment of RA bone destruction.that the result showed that sweroside can significantly inhibit osteoclast differentiation and promote osteoblast differentiation to ameliorate the RA bone destruction in vivo and vitro,and the mechanism may be related to the activation of PI3K/AKT/GSK-3β/β-catenin signaling pathway.The above results illuminated that pharmacodynamic and mechanism of sweroside in the treatment of RA bone destruction,which layed a foundation for the discouvery of anti-RA innovative drugs and provided a theoretical basis for the modern development of Tibetan medicine P.hookeri.