| Purpose:A model of knee osteoarthritis was prepared by anterior cruciate ligament(ACL)cutting in rats.The IL-1??IL-6?TNF-? content of total saponins in knee joint was detected by observing the naked eye and pathological changes of knee joint in each group.To observe the effect of total saponins on the autophagy level of articular cartilage in osteoarthritis rats by detecting the expression of LC3?/? in knee joint cartilage tissue,to explore the mechanism of total saponins regulating the autophagy level of chondrocytes in osteoarthritis rats by detecting the expression of Beclin-1?atg3?4?7,to detect the expression level of PI3K?p-PI3K?AKT?p-AKT?mTOR?p-mTOR in knee joint cartilage tissue,and to explain the deeper mechanism of total saponins regulating the autophagy level of knee chondrocytes in knee osteoarthritis rats;the chondrocytes from osteoarthritis model rats were cultured in vitro and treated with dipsacus total saponins intervention,compared with autophagy inhibitor 3-MA and PI3K inhibitor ly294002.again,the mechanism of dipsacus total saponins regulating chondrocyte inflammation,apoptosis and autophagy in osteoarthritis rats was demonstrated.Material and method:Paper ?:SPF grade adult SD rats 60,weight 200±20 male and female half,purchased from Liaoning Changsheng Biotechnology Co.,Ltd.,experimental animal production license number:SCXK(Liao)2010-0001.Laboratory animals purchased,raised in Liaoning University of traditional Chinese Medicine Laboratory Animal Center SPF Animal Room,license No.:SYXK(Liao)2013-0009.conventional pellet feed feeding,free water,12 h circadian rhythm.After the rats were purchased,they were kept adaptively for a week,observed that they were in good condition,completely familiar with the surrounding environment and had good activity,and began the formal experiment.The adult rats were labeled 1-60 according to body weight,and then randomly divided into two groups:sham operation group(10)and model group(50).A model of knee osteoarthritis(KOA)was performed in the model rats by anterior cruciate ligament resection,After the model evaluation,the rats were randomly divided into three groups:model control group,high dose group,medium dose group,low dose group and positive control group.The intervention factors were given:sham operation group:no treatment after operation,normal feeding for 2 weeks.Model control group:no treatment after operation,normal feeding for 2 weeks.Dipsacus total saponins high dose group:high dose Dipsacus Zhuanggu capsule,orally administered once a day for 2 weeks.Dipsacus total saponins medium dose group:medium dose of Dipsacus Zhuanggu capsule,orally administered once a day for 2 weeks.Dipsacus total saponins low dose group:low dose Dipsacus Zhuanggu capsule,orally administered once a day for 2 weeks.Positive control group:Weigu Li capsule(produced by Rhoda Pharmaceutical Factory)was orally administered once a day for 2 weeks.after the last administration,the rats in each group were sacrificed by excessive anesthesia,and the PBS buffer 500 was injected into the knee joint cavity ?L,after repeated washing,the articular fluid was collected.the IL-1??IL-6?TNF-? content in the joint fluid of each group was detected by enzyme linked immunosorbent(ELISA)method.Then the right knee joint was intercepted,the skin was stripped,the articular cavity was dissected horizontally,the tibial articular surface and the femoral articular surface were separated,the naked eye changes of the articular surface were observed,and the pictures were taken.and then the knee joint was fixed in 4%paraformaldehyde solution.the pathological changes of the cartilage tissue of the knee joint of each group were observed by HE staining.Paper 2:SPF grade adult SD rats 40,weight 200±20 male and female half,purchased from Liaoning Changsheng Biotechnology Co.,Ltd.,experimental animal production license number:SCXK(Liao)2010-0001.Laboratory animals purchased,raised in Liaoning University of traditional Chinese Medicine Laboratory Animal Center SPF Animal Room,license No.:SYXK(Liao)2013-0009.conventional pellet feed feeding,free water,12 h circadian rhythm.After the rats were purchased,they were kept adaptively for a week,observed that they were in good condition,completely familiar with the surrounding environment and had good activity,and began the formal experiment.The adult rats were labeled 1-40 according to body weight,and then randomly divided into two groups:sham operation group(10)and model group(30).After model evaluation,the model rats were randomly divided into three groups:model control group,KOA group and positive control group.After successful model evaluation,rats were randomly divided into model control group,total saponins group and positive control group.The intervention factors were given:sham operation group:no treatment after operation,normal feeding for 2 weeks.Model control group:no treatment after operation,normal feeding for 2 weeks.Dipsacus total saponins group:Dipsacus Zhuanggu capsule,oral administration,once a day,2 weeks in a row.Positive control group:Weigu Li capsule(produced by Rhoda Pharmaceutical Factory)was orally administered once a day for 2 weeks.the rats in each group were sacrificed by excessive anesthesia after the last administration,then the right knee joint was intercepted,the skin was stripped,the articular cavity was transversely dissected,the tibial joint surface and the femoral joint surface were separated,and the femoral side was immersed in 4%paraformaldehyde solution for fixation;the tibial joint surface was stripped of cartilage tissue with a scalpel,collected in 2 ml cryopreservation tube,frozen in-80? refrigerator.western-blot and IHC methods were used to detect the expression level and position of LC3?/??Beclin-1?Atg3?4?7 in articular cartilage of rats in each group.Paper 3:Experimental animals,groups,intervention methods are the same as paper two.western-blot method was used to detect the expression of PI3K?p-PI3K?AKT?p-AKT?mTOR?p-mTOR in articular cartilage tissue of rats and immunofluorescence double standard method was used to detect the expression of PI3K/p-PI3K?AKT/p-AKT?mTOR/p-mTOR in articular cartilage tissue of rats.Paper 4:SPF grade adult SD rats 20,weight 200±20 male and female half,purchased from Liaoning Changsheng Biotechnology Co.,Ltd.,experimental animal production license number:SCXK(Liao)2010-0001.Laboratory animals purchased,raised in Liaoning University of traditional Chinese Medicine Laboratory Animal Center SPF Animal Room,license No.:SYXK(Liao)2013-0009.conventional pellet feed feeding,free water,12 h circadian rhythm.After the rats were purchased,they were kept adaptively for a week,observed that they were in good condition,completely familiar with the surrounding environment and had good activity,and began the formal experiment.20 SD rats were randomly divided into two groups:blank group(5)and model group(15).The rat model was duplicated by the method of anterior cruciate ligament cutting.Referring to the research method of Huang Yanping et al,the chondrocytes of knee joint of rats were isolated and cultured in vitro.The chondrocytes of rats from blank group were cultured to 6 bottles,chondrocytes from model group to 24 bottles,and the cells were grouped,The chondrocytes in the blank group were set as blank control group,and the chondrocytes in the model group were divided into 4 groups:model control group,3-MA group,total saponins group and ly294002 group.In each group,6 bottles of cells were set up for Western blotting detection,and 3 24 hole plates were inoculated.Anti-cover slide was added to the plate hole for cell inoculation.Each group set 3 multiple holes for immunofluorescence detection.Blank control group:replace fresh DMEM complete medium,do not do treatment,culture 48 h.Model control group:replace fresh DMEM complete medium,do not do treatment,culture 48 h.MA group:replace DMEM complete medium containing 3-MA,MA concentration of 5 mmol/L,48 h.culture Dipsacus total saponins group:replace the DMEM complete medium containing total saponins,Concentration of total saponin 50?g/ml,48 h.culture ly294002 group:replace DMEM complete medium containing LY294002,LY294002 concentration ?mo/L,75 48 h.culture ELISA method was used to detect the IL-1??TNF-??IL-6 content in the supernatant of cell culture;Using immunofluorescence,Detection of cell LC3??LC3??beclin-1 expression in each group,And count the double-positive cells,Statistical analysis;The expression level of cell PI3K?p-PI3K?AKT?p-AKT?mTOR?p-mTOR in each group was detected by western-blot method.Results:Paper 1:1.The rats in the sham operation group moved freely,the skin of the knee joint was intact,covered with hair completely,the rats had high activity,normal water,white and glossy hair color,the rats in the model control group were claudication,the right knee joint was deformed seriously,the rat activity was very low.2.The results of HE staining of cartilage tissue of knee joint in 2.groups showed that the cartilage tissue structure of rats in sham operation group was normal,the chondrocytes were arranged evenly,neatly and in large quantities,the cartilage tissue of rats in model control group was obviously reduced,the cartilage layer was thinner,the number of cells was reduced,the level was not clear,the cell arrangement was confused,and there was obvious inflammatory cell infiltration.The pathological changes of cartilage in Dipsacus total saponins and positive control group were significantly reduced compared with that in model control group.Although the cartilage layer was thinner and chondrocytes decreased,it was obviously more than that in model control group.The pathological changes of cartilage tissue in Dipsacus total saponins group decreased with the increase of dose.3.The results showed that the content of IL-1??IL-6?TNF-? in knee joint fluid in each group was significantly increased(p<0.01)compared with that in sham operation group.Compared with model control group,the IL-1??IL-6?TNF-? content in knee joint fluid in each dose group and positive control group decreased significantly(p<0.01).Compared with high dose group,the IL-1??IL-6?TNF-? content in middle and low dose group and positive control group increased significantly(p<0)The contents of TNF-? and IL-6 in articular fluid of rats in low dose group were significantly increased(p<0.01)compared with middle dose group.Paper 2:1.The expression and LC3?/? ratio of LC3??LC3? in articular cartilage of rats in 1.groups showed that the expression level of LC3? in cartilage tissue of model control group was significantly increased(p<0.01),the expression level was significantly decreased,and the LC3?/? ratio was significantly increased(p<0.01).Compared with model control group,the expression level of cartilage in total saponins group and positive control group was significantly decreased(p<0).The expression level of LC3? increased significantly,but the ratio of LC3?/? decreased significantly(p<0.01),the difference was statistically significant.2.The expression of Beclin-1?Atg3?4?7 in cartilage tissue of knee joint of rats by 2.Western-blot method showed that the expression level of Beclin-1?Atg3?4?7 in cartilage tissue of model control group was significantly decreased(p<0.01)compared with that of model control group.The expression of Beclin-1?Atg3?4?7 in cartilage tissue of Dipsacus total saponins group and positive control group was significantly higher(p<0.01),the difference was statistically significant.Paper 3:the expression of PI3K?p-PI3K?AKT?p-AKT?mTOR?p-mTOR in knee cartilage tissue.The results showed that the expression level of PI3K?AKT?mTOR in knee cartilage tissue of rats in each group had no significant difference(p>0.05).The expression level of p-PI3K?p-AKT?p-mTOR in cartilage tissue of rats in model control group increased significantly(p<0.01)compared with that in model control group.The expression level of p-PI3K?p-AKT?p-mTOR in cartilage tissue of rats in Dipsacus total saponins group decreased significantly(p<0.01)significantly.Paper 4:1.The content of IL-1?,IL-6 and TNF-? in cell culture supernatant of each group showed as follows:Compared with blank control group,the contents of IL-1?,IL-6 and TNF-? in cell culture supernatant of model control group,total saponin group,3-MA group and LY294002 group were significantly increased(P<0.01),and the differences were statistically significant.Compared with the model control group,the contents of IL-1?,IL-6 and TNF-? in cell culture supernatant of the total saponins group and 3-MA group were significantly decreased(P<0.01),while the contents of IL-1?,IL-6 and TNF-? in cell culture supernatant of the LY294002 group were significantly increased(P<0.01),with statistical significance.Compared with the total saponins group,the contents of IL-1?,IL-6 and TNF-? in cell culture supernatant of 3-MA group were significantly increased(P<0.01),while the contents of IL-1?,IL-6 and TNF-? in cell culture supernatant of 3-MA group were significantly decreased(P<0.01),the difference was statistically significant.2.LC3?,? and Beclin-1 expression in each group were detected as follows:Compared with blank control group,the number of LC3? positive cells in model control group,continuum total saponin group,3-MA group and LY294002 group was significantly increased(P<0.01),while the number of LC3? and Beclin-1 positive cells was significantly decreased(P<0.01),the difference was statistically significant.Compared with model control group,the number of LC3? positive cells in total saponin continuum group and LY294002 group was significantly decreased(P<0.01),and the number of LC3? and Beclin-1 positive cells was significantly increased(P<0.01).The number of LC3? positive cells in 3-MA group was significantly increased(P<0.01),and the number of LC3? and Beclin-1 positive cells in 3-MA group was significantly decreased(P<0.01),with statistical significance.Compared with the total saponin continuum group,the number of LC3? positive cells in 3-MA group was significantly increased(P<0.01),and the number of LC3? and Beclin-1 positive cells in 3-MA group was significantly decreased(P<0.01).The number of LC3? positive cells in LY294002 group was significantly decreased(P<0.01),and the number of LC3? and Beclin-1 positive cells in LY294002 group was significantly increased(P<0.01),the difference was statistically significant.3.The expression levels of PI3K,p-PI3K,Akt,p-Akt,mTOR and p-mTOR in each group were detected by Western-blot method,and the results showed that there was no significant change in the expression levels of PI3K,Akt and mTOR in each group compared with the other groups,with no statistical significance(P>0.05).Compared with blank control group,the expression levels of p-PI3K,p-Akt and p-mTOR in model control group,total saponin group,3-MA group and LY294002 group were significantly increased(P<0.01),and the difference was statistically significant.Compared with the model control group,the expression levels of p-PI3K,p-Akt and p-mTOR in 3-MA group were significantly increased,while the expression levels of p-PI3K,p-Akt and p-mTOR in the total saponins group and LY294002 group were significantly decreased(P<0.01),with statistical significance.The expression levels of p-PI3K,p-Akt and p-mTOR in 3-MA group were significantly increased(P<0.01),while the expression levels of p-PI3K,p-Akt and p-mTOR in LY294002 group were significantly decreased(P<0.01),the difference was statistically significant.Conclusion:1.Total saponins of dips exerted anti-inflammatory effects by inhibiting the levels of inflammatory factors in articular fluid and secretion of inflammatory factors in chondrocytes of rats with knee osteoarthritis.2.Diptyl total saponins promoted autophagy by regulating the expression of autophagy-related proteins LC3II/I,Beclin-1,Atg3,4,and 7.3.Autophagy was inhibited and apoptosis was increased in rat chondrocytes with osteoarthritis,which was closely related to the overactivation of PI3K/Akt/mTOR signaling pathway.4.Dendal total saponins can regulate the expression of autophagy-related proteins,which may promote autophagy in rat chondrocytes through inhibiting the over-activation of PI3K/Akt/mTOR pathway. |
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