脊髓伤方抑制RIP1/RIP3/MLKL信号通路保护脊髓型颈椎病大鼠神经元OA

Objective To observe the effect of Jisuishang Formula(JSSF)on the expression of receptor interacting protein kinase 1(RIP1)/receptor interacting protein kinase 3(RIP3)/mixed-lineage kinase domain-like protein(MLKL)signalling pathway related mRNAs and proteins in cervical spondylotic myelopathy(CSM)rats,and to explore the potential mechanisms of its protective effects on neurons in CSM rats by mediating necrotic apoptosis.Methods The modelling rats were modelled using the spinal cord compression method,and the compression materials were removed from the sham-operated group of rats using the same procedure without compression.The model rats were divided into model group,positive control group,and JSSF group of low,medium and high dose of JSSF(n=8).The sham-operated group and the model group were given saline by gavage at a dose of 10 mL/kg,the positive control group was injected intraperitoneally with 2 mg/kg Necrostatin-1 solution,and the JSSF low-,medium-,and high-dose JSSF groups were all given JSSF at doses of 0.49,0.97,and 1.94 g/mL(10 mL/kg)by gavage,respectively.The treatments were administered twice daily for 4 weeks.The Basso,Beattie,and Bresnahan(BBB)score and modified Rivlin inclined plane score were assessed at 2 and 4 weeks after surgery.The histopathological condition of cervical spinal cord in each group was observed by HE staining at 4 weeks after surgery,and the changes of Nissl bodies were observed by Nissl staining.The expression of tumour necrosis factor-α(TNF-α)in tissues was observed by immunofluorescence,the concentrations of serum interleukin(IL)-6 and IL-1β were detected by ELISA,and the expression levels of RIP1/RIP3/MLK signalling pathway related mRNA and protein were detected by qRT-PCR and Western Blot.Results Compared with the sham-operated group,the BBB score and modified Rivlin inclined plane score reduced in the model group,the cervical spinal cord tissue was sparse,the vacuole-like structures increased,some neurons ruptured,the nuclei of the cells became smaller,the number of Nissl bodies decreased,the expression of TNF-α proteins in the tissues and the concentrations of serum IL-6 and IL-1β increased,and mRNAs such as RIP1,RIP3,p-MLKL and protein expression levels of RIP1,RIP3 and p-MLKL/MLKL increased(P＜0.05).Compared with the model group,BBB scores and modified Rivlin slant board scores of the positive control group and the JSSF low,medium,and high dose JSSF groups were significantly higher,the cervical spinal cord tissue structure was slightly tighter,the vacuole-like structure reduced,the neuron damage was less,the Nissl bodies increased,and the tissue TNF-α protein expression decreased,serum IL-6 and IL-1βconcentrations decreased,mRNA such as RIP1,RIP3 and MLKL and RIP1,RIP3 and p-MLKL/MLKL protein expression levels increased(P＜0.05).The effect of JSSF was better in the medium-dose group(P＜0.05).Conclusion JSSF can inhibit neuronal necrotic apoptosis by interfering with the RIP1/RIP3/MLKL signalling pathway,thus improving limb motor function in cervical spinal cord compressed rats.