Therapeutic Effects Of Ginsenoside Rg1 On Osteoarthritis

Background:Osteoarthritis(OA)is characterized by degenerative changes of articular cartilage.Chondrocyte apoptosis and degradation of extracellular matrix are closely related to the development and progression of osteoarthritis.Ginsenoside Rgl is a medicinal ingredient extracted from ginseng which shows anti-degenerative and anti-apoptotic properties in nervous system.However,the effects of Rgl on OA have not been reported.This study aimed to investigate the protective effect of Rgl on osteoarthritis.Experiment is divided into three parts:Part 1:The therapeutic effects of Rgl on rat OA modelObjective:Investigate the therapeutic effects of Rgl on rat OA model.Methods:For the in vivo study,a rat OA model was created by anterior cruciate ligament transaction(ACLT)or intraarticular injection of monosodium iodoacetate(MIA).Forty-eight rats were randomized into four groups of 12 rats each in ACLT or MIA OA model experiments.Four groups were defined as follows:Control group,non-osteoarthritic negative control rats.OA model group,rats underwent ACLT or injected with MIA but not treated with Rgl;R1 group,rats treated with 30 mg/kg Rgl by gavage once a day;R2 group,rats rats treated with 30 mg/kg Rgl by gavage once a day;All rats in ACLT or MIA model were killed 12 or 8 weeks after surgery respectively.The right knee samples were carefully dissected and the gross morphologic changes were observed by light microscope.The histologic joint damage was analyzed by HE staining,safranine-fast green staining and toluidine blue staining.The synthesize and expression of type II collagen,matrix metalloproteinase-13 and prostaglandin E2 were determined by immunohistochemistry.Pain was measured using the weight bearing test,paw-withdrawal latency(PWL)test and paw withdrawal threshold(PWT)test.Result:Administration of Rg1 to ACLT or MIA induced OA rats attenuated cartilage degeneration.The pathological results showed that the loss of chondrocytes,as well as type II collagen and aggrecan were remarkably reduced in Rg1 treatment groups compared with OA model group.Rgl also inhibited the expression of MMP-13 and PGE2.Moreover,administration of Rgl to the MIA-treated rats significantly increased the PWL and PWT and this resulted in recovery of hind paw weight distribution.Conclusion:This study demonstrates the ability of Rgl to inhibit inflammatory responses and reduce articular cartilage damage in OA,as well as relieving the pain.These results suggest that Rgl may exert a potential therapeutic value of Rgl in OA.Part 2:The protective effect of Rg1 on interleukin 1?-induced chondrocyteapoptosis and the underlying molecular mechanismsObjective:Investigate the protective effect of Rgl on interleukin 1?-induced chondrocyte apoptosis and the underlying molecular mechanisms.Methods:Chondrocytes were harvested from the joints of 1-week-old Sprague Dawley rats.Chondrocytes were treated with 10 ?g/mL Rgl or 10 ?g/mL Rgl +LY294002 for 2 h prior to treatment with 10 ng/mL IL-1?.Blank control group was left untreated except for a medium change.The MTT assay was performed to detect cell viability after 72 hours.The annexin V/propidium iodide staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling were used to detect chondrocyte apoptosis after 96 hours.The contents of total Akt,phosphorylated Akt(p-Akt),Bcl-2,Bax,cytochrome C,MMP-13 and Tissue inhibitor of metalloproteinase-1 were determined by Western blotting assay after 24 hours.A quantitative colorimetric assay was used to determine Caspase-3 activity after 24 hours.Result:Our present findings have shown that pre-treatment of chondrocytes with Rgl reduces IL-1? induced cytotoxicity/apoptosis.Rgl pretreatment also decreases the activity of IL-1? that reduces expression of Bcl-2 and level of p-Akt,and increases Bax activity,Cyt c release and caspase-3 activation.It also reverses the activity of IL-1? that reduces the expression of tissue inhibitor of metalloproteinase-1(TIMP-1)expression and increased the synthesis of matrix metalloproteinase(MMP)-13,with the net effect of inhibiting extracellular matrix degradation.Conclusion:These results indicate that Rgl may protect chondrocytes from IL-1?-induced apoptosis via the phosphatidylinositol 3-kinase/protein kinase B signaling pathway,through preventing Caspase-3 releasePart 3:The role of NF-?B signaling pathway in protective effects of Rg1 on rat osteoarthritic chondrocytesObjective:Investigate the protective effect of Rgl on interleukin 1?(IL-1?)-induced extracellular matrix degeneration in chondrocytes and the underlying molecular mechanisms.Methods:Chondrocytes were harvested from the joints of 1-week-old Sprague Dawley rats.Chondrocytes were cultured with IL-1?(10 ng/mL)alone or in combination with Rgl(10 ?g/mL)for 0,24,48 hours,the synthesize of type II collagen and aggrecan were determined by Western blotting assay.Chondrocytes were cultured with IL-1?(10 ng/mL)alone or in combination with Rgl(10 ?g/mL)for 0,15,30,45 minutes before harvest.The activities of I?B and NF-?B p65 signaling pathways were determined by Western blotting assay.Result:Treatment of chondrocytes with Rgl prevented IL-1?-induced degradation of type II collagen and aggrecan in a time-dependent manner.Rgl also inhibited the activation and translocation of NF-?B to the nucleus by suppressing the phosphorylation of I?B? in the cytoplasm.The inhibition of Rgl on phosphorylation of I?B? was not effected by a specific proteosome inhibitor ALLN.Conclusion:These results indicate that Rgl may protect chondrocytes from IL-1?-induced degradation of collagen II and aggrecan induced by IL-1?,as well as attenuating cartilage degeneration caused by OA.Rgl may exert these effects via inhibition of NF-?B signaling pathway.These results confirm the potential therapeutic value of Rg1 in OA.