The Molecular Mechanism Of Panax Notoginseng Saponins(PNS) Ameliorating Intervertebral Disc Degeneration Based On The Theory Of “Dispelling Stasis To Promote Regeneration”from Traditional Chinese Medicine

Background:“Stasis syndrom”is one of the most common pathogenesis for Low back pain,which is revealed with oxidative stress micro-environment and inflammatory infiltration as critical pathological products by molecular and biological technology.Meanwhile,intervertebral disc degeneration（IDD）which is induced mainly by oxidative stress and inflammatory stimulation and characterized with apoptosis of nucleus pulposus（NP）cells and degradation of extracellular matrix（ECM）contributes most to low back pain.“Dispelling stasis to promote regeneration”is one of the main methods to drive away“stasis syndrom”,protecting cells and tissues against oxidative stress and inflammation,decreasing apoptosis of cells and promoting regeneration of tissues.“Sanqi（Panax notoginseng）”as one of the most popular Chinese medicine for back pain treatment,excels at invigorating blood and disrupting stasis.The active compounds,Panax Notoginseng saponins（PNS）has been proved anti-oxidant and anti-inflammatory.Theoretically,according to“Dispelling stasis to promote regeneration”method,PNS may have effect on IDD treatment,however,very few literature reports and no enough molecular and biological evidences support.Aim of the study:This study is designed to explore the effect and mechanism of PNS on IDD under the theory of“Dispelling stasis to promote regeneration”from biological information technology,in vitro and in vivo experiments as follows:1.To predict the theoretical targets of IDD by PNS from network pharmacology;2.To explore the effect and mechanism of PNS on NPC under oxidative stress;3.To investigate the effect and mechanism of PNS on NPC under inflammatory stimulation;4.To verify the effect of PNS on IDD in animal models.Methods:1.PNS compound information was acquired by searching pharmacological database and analyzing platform,and predicted PNS targets were assured by Swiss Target prediction database.Targets of IDD were collected from Gene Cards database.Overlapped targets of PNS and IDD were summed by Venny 2.1 and active ingredient-targets network was constructed by Cytoscape 3.7.2.GO function and KEGG pathway Enrichment analysis were conducted by Metascape database.2.Human NP（HNP）cells were separated from degenerated intervertebral disc（IVD）specimens from patients who received surgeries.Oxidative stress to HNPC was induced by H₂O₂.The experiments carried out were as followings:（1）Cell viability was detected by CCK8.（2）Cell apoptosis was detected by TUNNEL test and Cleaved-caspase3 protein expression.（3）Gene expression of MMP13,ADAMTS-5,COL2A1 and ACAN were detected by real-time PCR.（4）Protein expression level of Cleaved-caspase3,Bax,Bcl-2,MMP13,ADAMTS-5,COL2A1,ACAN,p-m TOR,p-Akt,LC3Ⅰ/Ⅱ,P62 and Atg7 were detected by WB.（5）Expression level of Cleaved-caspase3,MMP13,COL2A1 and ACAN were detected by IF,meanwhile,lysosomal formation was tracked by IF,too.3.Inflammatory effect was induced by IL-1β,and the following experiments were carried out:（1）Gene expression levels of i NOS,COX-2,MMP1,MMP3,MMP13,ADAMTS-5,COL2A1 and ACAN were detected by PCR;（2）Protein expression levels of COX-2,MMP13,ADAMTS-5,COL2A1 and ACAN under IL-1βstimulation were detected by WB.（3）P65phosphorylation was detected by IF.4.3-month SD male rats were punctured percutaneously into discs at Co6/7 and Co8/9 by needles to induce IDD,and were intraperitoneally injected with PNS（160mg/Kg、200mg/Kg）every other day starting at the day after surgery.Coccygeal vertebral MRI scan and H&E stain were conducted at 4^th and 8^th week postoperatively.Results:Study I:the main compounds of PNS include 5 monomers:notoginsenoside R1, ginsenoside Rg1,ginsenoside Re,ginsenoside Rb1 and ginsenoside Rd.Except ginsenoside Rb1,the rest compounds show high DL values,especially ginsenoside Rg1 and ginsenoside Rd qt with DL value>0.18.Regarding to MW,TPSA and RBN,ginsenoside Rd qt shows superiorly druggable.2.100 predicted targets from Swiss Target Prediction were collected for each of five compounds.For IDD,3652 targets were found using key words“disc degeneration”in Gene Cards,and 891 uniprot codes were converted.3.Forty-two overlapped targets were summed by Cytoscape.GO enrichment analysis included mocular function,biological process and cell constitution.Pathway analysis indicated that p-Akt,apoptosis pathway,autophagy signaling pathway were the probable mechanisms.4.Topological analysis marked Caspase3,Akt1,STAT3,EGFR,JUN,MAPK1,VEGF,MMP1/9/13 and PLG as the key nodes.Caspase3,Akt1,MAPK1,and MMP1/9/13 were components of importance of sub-network.Study II:1.Cytotoxicity of PNS and H₂O₂:（1）There were no cytotoxic differences after 24-hour treatment between different concentrations of PNS（0.01,0.1,1,5,10,100μg/ml,resolved in 1%FBS-DMEM/F12）and control group（P>0.05）.（2）Cell viability treated with H₂O₂was significantly lower than the control group（P<0.05）,especially for 400μM group（P<0.01）at 6^th hour,furtherly decreased at 12^th hour（100μM,200μM,P<0.01;300μM,400μM,P<0.001）,and sharply fell down at 24^th hour（P<0.0001）.（3）Cell viability in H₂O₂（+）/PNS（0.01μg/ml）,H₂O₂（+）/PNS（0.1μg/ml）groups were significantly lower than the control group（P<0.0001）.The cell viability in the group of H₂O₂（+）/PNS（1μg/ml）and H₂O₂（+）/PNS（10μg/ml）were also lower than the control group（P<0.05）;Compared to the groups of PNS（-）/H₂O₂（+）and H₂O₂（+）/PNS（0.1μg/ml）,the cell viability of H₂O₂（+）/PNS（1μg/ml）and H₂O₂（+）/PNS（10μg/ml）were higher（P<0.05）.2.TUNNEL test:the apoptosis rate was increased in H₂O₂-treat groups compared to control group（P<0.01）,however,it’s less apoptotic with PNS treatment（P<0.01）,which was as well as confirmed by IF of Cleaved-caspase3（P<0.05）and Bax/Bcl-2 by WB（P<0.05）;3.Gene and protein expression of MMP13、ADAMTS-5by PCR,WB and IF were both decreased in PNS（+）/H₂O₂（+）group,however,only MMP13showed significant difference compared to PNS（-）/H₂O₂（+）（P<0.05）.Besides,COL2A1 and ACAN showed less gene and protein expression in PNS（-）/H₂O₂（+）compared to control（COL2A1,P<0.001;ACAN,P<0.0001）,but with PNS more COL2A1 and ACAN were preserved（COL2A1,P<0.01,ACAN,P<0.05）;4.Compared to PNS（-）/H₂O₂（+）group,protein expression of p-Akt and p-m TOR by WB were activated（P<0.05）,and autophagy was inhibited characterized by decrease of LC3Ⅰ/Ⅱconversion（P<0.05）,less P62 consumption（P<0.05）,increase trend of Atg7 expression P>0.05)and stronger intensity of lysotracker（P<0.05）in PNS（+）/H₂O₂（+）group.With rapamycin（50n M）treatment,striking autophagy level was detected combined with H₂O₂（P<0.0001）,meanwhile,higher Bax/Bcl-2 protein expressed（P<0.0001）.But PNS still showed inhibiting autophagy effect with rapamycin and H₂O₂ treatment,and less Bax/Bcl-2 protein expressed（P<0.05）.Study III:IL-1βwas proved to significantly strike the gene expression of inflammatory cytokines（i NOS,COX-2）and MMP13（P<0.01）and promote the decreased expression of COL2A1 and ACAN（P<0.05）.However,PNS treatment showed a failure in down-regulation of gene expression of i NOS,COX-2,IL-6,MMP1,MMP3,MMP13,ADATMS-5（P>0.05）by PCR and protein expression of COX-2,MMP13,ADATMS-5by WB（P>0.05）,neither increased the gene and protein expression of COL2A1 and ACAN（P>0.05）.IF stain of p-P65 showed strong intense by IL-1βstimulation compared to control（P<0.001）,however,no difference observed with or without PNS treatment（P>0.05）.Study IV:4 and 8 weeks after operation,MRI was taken to scan Co6/7 and Co8/9 of rat tail,presenting degenerative disc formed in Co8/9 as early as at 4^th week（Grade 3～4 by Pfirrmann classification）in IDD-saline group,less degenerated in PNS treatment groups（Grade 1～2）and deteriorated at 8^th week（Grade 4）in IDD-saline group,better in PNS treatment groups（Grade 3～4）（P<0.05）.But Co6/7 didn’t manifest degeneration even as late as at 8^th week（Grade 1～2）;From histological analysis,destruction of NP tissue was more severe in IDD-saline group than in PNS treatment group（P<0.05）.Conclusion:Our study revealed the effect and mechanism of PNS on IDD treatment,and the results were mostly satisfactory.It improves IDD in rat’tail IDD model,and for“Stasis syndrom”characteristics,it prefers inhibiting apoptosis of NP cells and degradation of ECM under oxidative stress to inflammatory stimulation in in vitro experiments.Inhibiting the autophagy which may be regulated by Akt/m TOR signaling pathway is the mechanism for PNS protecting NP cells under oxidative stress.Interestingly,the different results for PNS treating“Stasis syndrome“of IDD show the potential mechanism of“Therapy with syndrome differentiation”and further investigation is required.