Experimental Study On The Effect Of Huoxue Bushen Recipe On Autophagy Mediated By MTOR Pathway In Degenerated Intervertebral Discs Of Rats

Purpose: To observe the effects of Huoxue Bushen decoction on autophagy mediated by mTOR signaling pathway in intervertebral disc degeneration of rats,and to elucidate the mechanism of Huoxue-Bushen decoction.Material and method: 60 SD rats are randomly divide into 5 groups: Control group,Model group,Rapamycin group,Huoxue Bushen decoction group,Huoxue Bushen decoction&Rapamycin group.In addition to the control group,the other 4 groups use amputate both forelimbs to prolonged upright posture induces intervertebral discs degeneration rats model.After successful modeling,each group is treated as their names with Huoxue Bushen decoction gavage,rapamycin intraperitoneal injection,saline gavage,and dimethyl sulfoxide intraperitoneal injection.After 6 weeks of administration,the rats are sacrificed,L5/6 intervertebral discs are taken as samples.The samples are separated taken HE staining,alcian blue-nuclear fast red staining for of intervertebral disc degeneration morphological observation.And western-blot is used to detect the situation of protein expression: 4E-BP1 and p70S6K-downstream proteins of mTOR signaling pathway;beclin-1 and lc-3b-autophagy related proteins.Results: 1.Morphological observation of the degree of intervertebral disc degeneration shows that: the Model group has the most degenerative changes,and the Control group has the least degenerative changes.The Rapamycin group,Huoxue Bushen decoction group and Huoxue Bushen decoction&Rapamyc group are in similar level,between the other 2 groups above.The content of proteoglycan in the extracellular matrix shows that: the Control group has the most.Huoxue Bushen decoction&Rapamyc group is the second.And then comes the Rapamycin group and Huoxue Bushen decoction group which in similar level.The Model group content the least.2.The downstream protein detection of mTOR signaling pathway shows that: the expression level of p70S6 K from high to low is: Model group > Rapamycin group and Huoxue Bushen decoction group > Huoxue Bushen decoction&Rapamycin group.The expression level of 4E-BP1 from high to low is: Model group > Huoxue Bushen decoction group > Rapamycin group and Huoxue Bushen decoction&Rapamyc group.Both Huoxue Bushen decoction and Rapamycin can inhibite the synthesis of p70S6 K and 4E-BP1,the downstream protein of the mTOR signaling pathway,in the intervertebral disc.Autophagy-related protein detection shows that: the expression level of beclin-1 from high to low is: Huoxue Bushen decoction&Rapamyc group > Huoxue Bushen decoction group > Rapamycin group > Model group > Control group.The expression level of lc-3b from high to low is: Huoxue Bushen decoction&Rapamyc group group > Huoxue Bushen decoction group > Rapamycin group > Model group > Control group.Both Huoxue Bushen decoction and Rapamycin can increase the expression of autophagy-related proteins in the intervertebral disc of model rats.Conclusion: Huoxue Bushen decoction can increase the level of autophagy in intervertebral disccan via inhibit the activity of mTOR signaling pathway,to delay the intervertebral discs degenaration of amputate both forelimbs rats model.