| Research backgroundLow back pain(LBP)affects the health of millions of people around the world,mainly due to disc degeneration.The treatment of intervertebral disc degeneration(IVDD)is mainly physical therapy,drug analgesia and other conservative symptomatic treatment.Some patients need surgical treatment several years after conservative treatment.Surgical treatment did not consider the pathophysiological factors of disc degeneration.Surgical treatment sacrificed the movement function of the spine,and there were anesthesia risks and the acceleration of disc degeneration in adjacent segments.The main characteristics of intervertebral disc degeneration are the reduction of the number of cells and the change of the content of extracellular matrix(ECM).The ECM is mainly composed of type I collagen and type II collagen.Therefore,it is very important to study the change process of different types of collagen content after intervertebral disc degeneration and find new biological treatment targets to improve the quality of life of patients with intervertebral disc degeneration.It is found that IL-13 plays an important role in disc degeneration,and sIL-13Rα2 is a decoy receptor,which can block the binding of IL-13 and IL-13Rα1/IL-4Rα,thus blocking the biological activity of IL-13.Therefore,it is speculated that sIL-13Rα2-Fc has the effect of inhibiting disc degeneration.Experimental purposesMasson staining was used to observe the pathological changes of the degenerative tissue of the intervertebral disc(IVD).In order to detect the change of collagen type in the degenerative tissue of intervertebral disc,Sirius red staining technique was used to stain collagen.RT-PCR and Western blot were used to analyze the expression of type I collagen,type II collagen and mRNA in the intervertebral disc tissue after the intervention of sIL-13Rα2-Fc at the transcription level and protein level,respectively,to explore the effect of sIL-13Rα2-Fc on the degeneration of intervertebral disc.Experimental methods72 S-D rats,weighing 200 g ± 20 g,6-8W,were randomly divided into group A,group B,group C,group D,group E and group F,with 12 rats in each group.Group A is the blank control group,group B is the sham operation group,group C is the model group,group D is the low-dose group of sIL-13Rα2-Fc fusion protein injection,group E is the middle dose group of sIL-13Rα2-Fc fusion protein injection,group F is the high-dose group of sIL-13Rα2-Fc fusion protein injection.The intervertebral disc degeneration model of rats was established by the way of annulus fibrosus(AF)puncture injury.Seven days later,rats in group D,E and F were injected with the same dose of sIL-13Rα2-Fc fusion protein(low dose 0.5mg/kg,medium dose 1mg /kg,high dose 2mg / kg)through the original operation route;rats in group B(routine feeding after modeling)and group C were injected with the same dose of normal saline at the same time,while rats in group A were not treated.According to the sampling requirements,rats were killed after 2,4 and 8 weeks.The intervertebral disc tissues of each group were extracted,paraffin sections were made,the pathological degeneration process of intervertebral disc was detected by Masson staining,the collagen type of intervertebral disc was detected by Sirius red staining technology,then the RNA and collagen in intervertebral disc were extracted,the mRNA expression of type I collagen and type II collagen in degenerative intervertebral disc was detected by RT-PCR and WB respectively Changes in levels and levels of type I and II collagen.Experimental results1.Masson staining showed that sIL-13Rα2-Fc fusion protein could significantly improve the histopathological staining of the intervertebral disc of rats.The specific manifestations of disc staining were uniform arrangement of AF,decrease of fracture,increase of nucleus pulposus cells,enlargement of nucleus pulposus area,uniform and bright collagen fibers without blue staining in nucleus pulposus area.The number of nucleus pulposus cells in group F was more than that in group D and group E,and the number of AF was less;2.Sirius red staining results: in group A,the nucleus pulposus(NP)was loose bright orange reticular structure,the inner layer of fibrous ring was sapphire blue,which showed that the nucleus pulposus and the inner layer of AF were rich in type II collagen,and the outer layer of AF was bright yellow,which was type I collagen;the inner and outer layers were clearly decomposed and arranged orderly;in group B,the NP was relatively dense yellow and green,It is suggested that the content of type II collagen is decreased,the structure of AF is disordered,the inner and outer layers are yellow or light green,the boundary is unclear,and they are basically fused together;the D group,e group and F group interfered by sIL-13Rα2-Fc fusion protein are between the two groups,suggesting that sIL-13Rα2-Fc can slow down the process of disc degeneration.3.RT-PCR was used to detect the mRNA expression level of type I and type II collagen in the intervertebral disc tissue.The results showed that the mRNA expression level of type II collagen in group D,group E and group F of sIL-13Rα2-Fc fusion protein intervention was significantly higher than that in group B of the intervertebral disc degeneration model,and was concentration dependent and time-dependent;The expression level of type I collagen mRNA was significantly lower than that of group B,which was also concentration dependent and time-dependent;4.Western blot analysis of the expression of type I collagen and type II collagen in the intervertebral disc showed that the sIL-13Rα2-Fc fusion protein could change the content of extracellular matrix of the intervertebral disc by up regulating the level of type II collagen and down regulating the level of type I collagen,and then slow down the degeneration of the intervertebral disc.Conclusion1.The degeneration of intervertebral disc is related to the change of collagen content,which is mainly manifested as the increase of type I collagen content and the decrease of type II collagen content;2.The sIL-13Rα2-Fc fusion protein can increase the level of II collagen mRNA expression,reduce the level of I collagen mRNA expression,and then reduce the content of I collagen,increase the content of II collagen to inhibit the degeneration of intervertebral disc,which is concentration dependent and time-dependent.The higher the concentration of sIL-13Rα2-Fc fusion protein,the longer the action time,the stronger the inhibition on the degeneration of intervertebral disc. |
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