Regulation Of The Protective Mechanism Of Spinal CXCL13/CXCR5 Signaling Against Motor Neurons In ALS^SOD1-G93A Mice

Objective:In this topic,we observed the effects of early electroacupuncture（Electro-acupuncture）intervention on the behavior of amyotrophic lateral sclerosis（Amyotrophic lateral sclerosis,ALS）transgenic mice,and then investigated the therapeutic effect of electroacupuncture targeting ALS and the possible mechanism of action.It aims to clarify whether early electroacupuncture intervention protects impaired motor neurons by regulating spinal cord CXCL13/CXCR 5 signaling in mice,and thus plays a role in improving ALS.This study illustrates that early electroacupuncture intervention reduces the spinal cord chemokine（C-X-C motif）ligand 13（Chemokine（C-X-C motif）ligand 13 in ALSSOD1-G93A mice,Upregulation of CXCL13）,Thus inhibiting astrocytes with their receptor chemotactic factor（C-X-C motif）ligand 5（Chemokine（C-X-C motif）ligand 5,CXCL5）activation further attenuated astrocyte activation,Correct the abnormal release of the pro-inflammatory cytokines IL-1β,IL-6,and TNF-α,Ultimately protecting against the damaged motor neurons,Mechanism of action to improve the motor function in ALS^SOD1-G93Amice,To provide a new way to explore the mechanism of electroacupuncture treatment in ALS.Methods:1.After identification of offspring by PCR,ALS^SOD1-G93Amice were divided into ALS model group,electroacupuncture group and inhibitor group by random number table,and wild littermates were used as blank control group,12 in each group.In the electric injection group,"Zusanli"and"suspension bell"were selected,2 Hz,1 m A,continuous wave,15min,5 times/7 days,7 days for a course of treatment,a total of 8 courses.Meanwhile,in order to further explore whether early electroacupuncture improved the motor function of ALS^SOD1-G93Amice by regulating the CXCL13/CXCR 5 signaling pathway,mi R-186-5p（250μL/only/d,i.v.）,an inhibitor of CXCL13 administered by the tail vein,2 times/7days,7 days for a course of treatment,a total of 8 courses,served as a positive control group,and the expression of proteins related to CXCL13/CXCR 5 signaling pathway was observed.The intervention was the same as the electroacupuncture group.2.The motor function of experimental animals was evaluated by climbing,rotating and open-field experiments to observe whether early electroacupuncture intervention could modulate the motor function of ALS^SOD1-G93Amice.3.The morphology and structure of ALS^SOD1-G93Amice were used to observe the effect of early electroacupuncture intervention on Nissin spinal motor neurons of ALSSOD1-G93A mice.4.The levels of IL-1β,IL-6,and TNF-αm RNA in ALS^SOD1-G93Amice were determined by RT-q PCR for the effects of early electroacupuncture intervention on IL-1β,IL-6,and TNF-αm RNA levels in the spinal cord of SOD1-G93A transgenic mice.5.Using immunoblot（Western blot,WB）,CXCL13,CXCR 5 were in the spinal cord of ALS^SOD1-G93Amice to observe the effect of early electroacupuncture intervention on the expression levels of CXCL13,CXCR 5 proteins in the spinal cord of ALS^SOD1-G93Amice.6.Immunofluorescent double staining was used to observe the coexpression of CXCL13 and Neu N relative fluorescence intensity and CXCR 5 and GFAP relative fluorescence intensity in SOD1-G93A transgenic mice,and to analyze the effect of early electroacupuncture intervention on the level of CXCL13 and Neu N and CXCR 5 and GFAP relative fluorescence intensity in the spinal cord of ALS^SOD1-G93Amice.Rezults:1.Electroacupuncture intervention could improve the morphology and motor function of ALSSOD1-G93A miceALS^SOD1-G93Amice lost weight and had motor dysfunction in pole climbing,stick transfer and absenteeism experiments;after electroacupuncture intervention,ALS^SOD1-G93Amice gained weight and improved transport function.The results in the inhibitor group were similar to those in the electroacupuncture group.It suggested that electroacupuncture intervention could improve the morphology and motor function of ALS^SOD1-G93Amice.2.Electroacupuncture intervention protected spinal motor neurons in SOD1-G93A transgenic micePartition,cell breakdown,and decreased quantity in ALS^SOD1-G93Amice in spinal cord motor neurons in ALS^SOD1-G93Amice.3.Electroacupuncture intervention inhibited the levels of IL-1β,IL-6,and TNF-αm RNA in the spinal cord of ALS^SOD1-G93AmiceIL-1β,IL-6 and TNF-αm RNA levels were increased in the spinal cord of ALS^SOD1-G93Amice;electroacupuncture intervention suppressed IL-1β,IL-6 and TNF-αm RNA levels in the spinal cord of ALS^SOD1-G93Amice.4.Electroacupuncture intervention regulated the abnormal expression of CXCL13,CXCR5 in the spinal cord of ALS^SOD1-G93AmiceALS^SOD1-G93Amice increased CXCL13,CXCR 5 protein expression;electroacupuncture intervention regulated the abnormal expression of CXCL13,CXCR 5 in ALSSOD1-G93A mice.5.Electroacupuncture intervention regulated the relative fluorescence intensity of CXCL13 and Neu N,and CXCR 5 and GFAP in the spinal cord of ALS^SOD1-G93AmiceThe relative fluorescence intensity of CXCL13 and Neu N and CXCR 5 and GFAP were significantly increased in the spinal cord of ALS^SOD1-G93Amice;the abnormal expression of CXCL13 and Neu N and CXCR 5 and GFAP in the spinal cord of ALS^SOD1-G93Amice was regulated by electroacupuncture intervention.Research conclusions:This study confirmed that the motor function of ALSSOD1-G93A mice was significantly improved after electroacupuncture intervention,and further studies found that electroacupuncture intervention inhibited the CXCL13 and CXCR 5 protein expression in the spinal cord of mice.The results of this study indicate that electroacupuncture intervention may inhibit the expression of inflammatory factors by inhibiting the activation of its receptor CXCR 5,and improve the mechanism of motor function in ALS mice.