Experimental Study On The Effect Of Icariin On Inflammatory Factors Of Degenerative Nucleus Pulposus Cells In Rats

Objective:Through in vivo experiments,a rat model of intervertebral disc degeneration was established and administered to high,middle and low concentrations of icariin.The expression of inflammatory factors IL-1? and IL-6 was detected by Western blot and Real-time PCR according to the experimental time.MRI was used to observe the degree of intervertebral disc degeneration,HE staining to observe the morphology of the intervertebral disc and immunohistochemical staining to detect the expression of extracellular matrix Agg and co? in nucleus pulposus(NP).In vitro experiments were conducted to detect the effects of icariin on the proliferation of inflammatory factors IL-6,IL-1? and nucleus pulposus cells and the extracellular matrix Agg and co? in rat degenerative nucleus pulposus cells.Through in vivo and in vitro experiments,it is clear whether icariin affects the secretion of inflammatory factors in rat degenerative nucleus pulposus cells,thereby delaying the degeneration of intervertebral discs.Methods:1.In vivo experimentSixty SD rats were randomly divided into 5 groups:sham operation group(Sham),intervertebral disc degeneration group(IDD)and low,medium and high concentration icariin group(ICAL,ICAM,ICAIH),with 12 rats in each group.The IDD model was prepared by puncture rat tail disc method.Rats in each group were intragastrically administered until the rats died.The ICA group was intragastrically administered with ICA(25,50,100 mg/kg.d),and the Sham and IDD groups were given the same amount of normal saline.Rats were anesthetized for MRI at 2w and 6w after surgery.The samples were taken at 2w and 6w after operation.The expressions of inflammatory factors IL-1? and IL-6 were detected by Western blot and Real-time PCR.The morphology of intervertebral discs was observed by HE staining.The extracellular matrix Agg in NP was observed by immunohistochemical staining.Co? expression.2.In vitro experimentThe NP cells extracted from Sham were labeled as control group(Control),and the NPs extracted from IDD group were randomly divided into 4 groups(IDD,ICAL,ICAM and ICAM).The ICA group was treated with different concentrations of ICA(10,20,40 ?M)for 24 hours.In the Control and IDD groups,an equal amount of medium was added.The value-adding ability of NP cells was determ:ined using CCK-8.Cell fluorescence imaging system were used to observe the acquired images.The NP cells of each group were collected for Western blot and Real-time PCR to detect the expression of IL-1? and IL-6.The IL-1? and IL-6 levels in the cell supernatant were determined by the ELSIA method.Result:1.ICA can inhibit IL-1?,IL-6 expression and promote Aggrecan,Collagen? expressionThe expression of IL-1? and IL-6 in NP tissues of IDD group was higher than that in Sham and ICA groups,while the expression of Aggrecan and Collagen? was lower than other groups,indicating the expression of inflammatory factors IL-1? and IL-6 during IDD.Up-regulation and extracellular matrix Aggrecan,Collagen? expression down-regulation;ICA can inhibit IL-1?,IL-6 expression,promote Aggrecan,Collagen? expression.2.ICA can alleviate degeneration caused by rat disc punctureThe T2 weighted signal intensity of the intervertebral discs in the IDD group and the ICAM group was compared in the 7.0T MRI image:the ICAM group signal was stronger than the IDD group at 2w postoperatively;the IDD group disappeared at 6w after surgery,and the ICAM group showed no significant change.The Pfirrmann MRI grade score of the ICAM group(indicating the degree of disc degeneration)was significantly lower than that of the IDD group.The HE staining at 6w after operation showed that the IDD group had severe degeneration of the intervertebral disc,the rupture of the annulus fibrosis,the interstitial space was widened,the NP tissue volume in the intervertebral disc was small,and the irregular arrangement was disordered.The number of cells was small and the morphology was cartilage-like cell-like changes.The ICA group was superior to the IDD group in terms of extracellular matrix content,annulus fibrosus and NP cell morphology.3.ICA promotes the proliferation of degenerative NP cellsAccording to the literature,different concentrations of ICA(10,20 and 40?M)were administered to treat NP cells in ICA group.The proliferation activity of NP cells was determined by the CCK-8 method.The results showed that the proliferation of NP cells in IDD group was significantly lower than that in control group and ICA group,indicating that ICA promoted the proliferation of degenerative NP cells.4.ICA inhibits IL-1?,IL-6 expression in NP cellsIn vitro,Real-time PCR and ELSIA results showed that IL-1? and IL-6 expression in NP cells of IDD group were significantly up-regulated compared with Control group.Compared with the IDD group,the expression of IL-1? and IL-6 in NP cells was significantly down-regulated after ICA treatment(P<0.05).Immunofluorescence showed that the fluorescence intensity of IL-1?and IL-6 accumulated in nucleus and cytoplasm of NP group was weaker than that of IDD group.5.ICA inhibits the degradation of extracellular matrix Aggrecan,CollagenIIAccording to Western blot and Real-time PCR results,compared with the Control group,the mRNA levels of NP extracellular matrix Aggrecan and Collagen II were significantly decreased in the IDD group,while the expression levels of Aggrecan and Collagen II were increased after ICA treatment.Conclusion:Icariin can reduce the degeneration of rat intervertebral disc,promote the proliferation of degenerative nucleus pulposus cells,inhibit the expression of inflammatory factors and the degradation of extracellular matrix,so it may become a drug for the treatment of intervertebral disc degeneration.