| ObjectiveIn this experiment, we take the theory of“ Bu yuan ”as the guiding principle, and take the concept of“disease prevention first ” as the principle of treatment.Then from four aspects,different levels which were cytokine levels、peripheral morphology、cell proliferation and apoptosis to study the preventive effects on rabbit knee osteoarthritis of ginsenoside Rg1 respectively,in order to provide scientific evidences for Traditional Chinese Medicine(TCM) for the prevention and treatment of osteoarthritis.MethodsFifty health adult New Zealand rabbits were equally divided into normal group、model group、Ginsenoside Rg1 low、medium and high dose group randomly.forty rabbits were established models with plaster cast unilateral hindlimb straighten place braking method,and intervention was used after molding on the second day.Ginsenoside Rg1 low(5mg/kg)、medium(10mg/kg) and high(20mg/kg)dose group were gastriced by 5 mL/kg,meanwile,the normal group、and model group were gastriced with isodose physiological saline,three times a week,6 weeks in all.1.Inflammatory cytokine detection:Each group were performed the operation of the left knee joint puncture in the strict aseptic operation condition respectively before model preparation and after the intervention,and injected normal saline 1ml.repeated aspiration in order to mix the synovial fluid fully,then aspirated it.The contents changes was measured before and after intervention in synovial fluid of NO、IL-1 and TNF-α.2.Cell morphological detection:All the experimental rabbits were killed by Air embolism under anesthesia after 6 weeks later.Morphological and histological changes in rabbit knee joint of each group were evaluated through macroscopic view,optical microscope,transmission electron microscopic(TEM) and Mankin’s core by examination the change of cartilage in histology and morphology.3.Cartilage cell proliferation experiment:Take out the weight-bearing area of articular cartilage,and use the Real-Time PCR technique to test the expression of Cyclin D1、P21 mRNA which were the key regulators of the cell cycle; Western-blot technique to test the expression of protein of the Cyclin D1、P21; Proliferating cell nuclear antigen(PCNA) and cell Proliferative Index to test the cartilage cell proliferation,so as to observe the impacts of ginsenoside Rg1 on chodrocyte proliferation.4.Cartilage cell apoptosis experiment:Take out the weightbearing area of articular cartilage,use the Real-Time PCR technique to test the expression of Caspase-3、Bax/bcl-2 mRNA which were the key regulators of the cell apoptosis; Western-blot technique to test the expression of protein of the Caspase-3、Bax/bcl-2;Proliferating cell nuclear antigen to observe the apoptosis of articular cartilage.So as to study the effect of ginsenoside Rg1 on chondrocyte apoptosis.Results1.Changes in content of inflammatory cytokines:Before the intervention,the content of NO、IL-1、TNF-αwere no significant difference(P>0.05),but after intervention,the content of NO、IL-1、TNF-αin model group were significantly higher(P<0.01),the content of Ginsenoside Rg1 low、medium and high dose group were lower than model group(P<0.01),meanwile, Ginsenoside Rg1 medium and high dose group’s content were lower than Ginsenoside Rg1 low dose group,but there were no significantly difference between the medium and high dose group.2.Morphological changes:Macroscopic view:the articular cartilage surface was smooth and elastic in normal group.but the articular cartilage of the model group was greyish yellow,wear-out and poor elasticity,ulceration was found in certain places.while the changs of articular cartilage in Ginsenoside Rg1 medium and high dose group were tiny than before.the articular cartilage in Ginsenoside Rg1 low dose group was yellow, slightly rough in the surface, no cartilage defect.Observation under optical microscope:The articular cartilage surface was smooth and the four layer structure was clear and orderly in normal group,the tidemark was integrity intact.Articular cartilage in model group became thin,the number of cartilage cells decreased and the morphological was changed,disorganized and crack depth into radiation layer, the tidemark was destructed,safranin O staining was severely weakening. while the changs of articular cartilage in Ginsenoside Rg1 medium and high dose group were tiny than before;The layer of articular cartilagein Ginsenoside Rg1 low dose group was basicly integrity, except few shallow cracks, the tidemark was complete, safranin O staining was moderate weakening.Mankin score:The model group was the highest,the difference was significant compared with orther groups(P<0.01).Ginsenoside Rg1 medium and high dose group’s score were lower than Ginsenoside Rg1 low dose group(P<0.01),but there were no significantly difference between the medium and high dose group(P>0.05).Observation under TEM:the ultrastructure of chondrocytes in group Ginsenoside Rg1 medium and high dose was similar to group normal.The cartilage cell in model group lost it’s contour and normal shape,the organelles was swelling or even disappeared,karyopyknosis,chromatin margination,even emergenced nuclear fission, apoptotic bodies formated.The cartilage cell in model group was mild atrophy,outline still clear,organelles was mild swelling,nucleuswas integrity,nuclear membrane slightly invagination,chromatin marginated slightly.3.Cartilage cell proliferation experiment:The expression of P21 mRNA and corresponding proten of Ginsenoside Rg1 low、medium and high dose group were lower than model group(P<0.05 or P<0.01),Cyclin D1 mRNA and corresponding proten were higher than model group(P<0.05 or P<0.01),PCNA showed that the proliferation rate of chondrocytes of Rg1 low、medium and high dose group were higher than model group(P<0.01).And there exist certain dose-effect relationship,of which the effect of medium dose group was best.4.Cartilage cell apoptosis experiment:The expression of Caspase-3、Bax mRNA and corresponding proten of Ginsenoside Rg1 low、medium and high dose group were lower thanmodel group(P<0.05 或 P<0.01),bcl-2 mRNA and corresponding proten were higher than model group(P<0.05 或 P<0.01),Tunel showed that the apoptosis rate of chondrocytes of Rg1 low、medium and high dose group were lower than model group(P<0.01).there were no signifycantly difference between the medium and high dose group,but lower than Ginsenoside Rg1 low dose group.all of which medium dose group was best.Conclusion1.The mode1 similar to human osteoarthritis can be successful established with immobilized in extension position for 6 weeks.2.A certain dose of ginsenoside Rg1 can effectively reduce the inflammatory cytokines content of NO、IL-1 and TNF-a in synovial fluid,so to relieve joint inflammation, improve the internal environment of the joint.3.A certain dose of ginsenoside Rg1 can effectively improve the expression of cyclinD1,meanwile inhibit the expression of P21,which are the key factors in G1 phase regulation.which could promote cartilage cells through the G1/S restriction point,and then entry into S phase to finish the cell cycle,so as to promote the proliferation and repair of cartilage cells effectively.4.A certain dose of ginsenoside Rg1 can regulate the ratio of Bax/Bcl-2 ratio,and down-regulation the expression of Caspase-3which are the key factors in apoptosis,to inhibit the apoptosis of cartilage cells and prevent cartilage progressive destruction. |
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