Anti-inflammatory For Analgesic Mechanism Based On Autophagy And Efficacy Of Acupotomy Combined With Xiangbitong Granule On Wind-cold-dampness Cervical Spondylosis

ObjectiveWe aimed to observe clinical efficacy of acupotomy combined with Xiangbitong Granule(XBTG)on wind-cold-dampness cervical spondylosis by a randomized controlled study,and verify its mechanism through animal experiment,to find out the most suitable combination of acupuncture and TCM.MethodsClinical trial:Firstly,sample size was objectively estimated.126 wind-cold-dampness cervical spondylosis were averagely divided into three groups including acupotomy combined with XBTG(A&XBTG),acupotomy and XBTG.Pinknife acts on the posterior cervical muscle tenderness point or strip shape,parallel to the direction of muscle fiber,tendon and nerve vessel,and cut the fascia longitudinally to release muscle induration,once a week for 2 weeks.XBTG,5 g/bag,1 bag/time,3 times/day for 2 consecutive weeks.Cervical dysfunction was assessed by NDI,VAS was used to assess neck pain,ELISA detected serum TNF-α,IL-1β and PGE2,TCM syndromes were assessed with Quantitative Grading Criteria of Guidelines for Clinical Research of unprecedented TCM Drugs,recurrence rate was evaluated by telephone follow-up and adverse reactions of acupotomy and XBTG were evaluated by safety indicators.Animal experiment:56 SPF-male-SD rats were divided into control group(CG),model group(MG),XBTG,acupotomy group(AG),A&XBTG,3-MA and A&XBTG&3-MA on average.We fixed rats’ necks,kept them down at 45°,stimulated the wind,cold and dampness,and made the model for 2 hours each time for 2 times for 4 consecutive weeks.On the 2nd after mold-making(before intervention),serum speciens of 56 rats’ tail vein were collected and inflammation-related factors were assessed to verify mold-making success.Then,acupotomy was carried out.According to neck-muscle rigidity degree and hard knot position,a spinous process point in C2～6 and two points next to spinous process about 0.5 cm were determined as needle-knife insertion points.Cutting line was parallel to posterior median line,knife body was perpendicular to posterior coronal plane,and the lesion was cut longitudinally for 3 times and horizontally for 2 times,once a week for 2 weeks.TCM drug was diluted at a dose of g·kg-1 and 10 mL normal saline by body weight of kg-1 and the dose of XBTG was 4.5 g/kg·d-1 for 2 weeks.The injection of autophagic agents(3-MA)was performed in 3-MA group and A&XBTG&3-MA before intervention.After 14 days of intervention,6 rats in good overall condition were selected in each group and abdominal aorta serum and posterior cervical muscle tissue were collected.Rats’ general state,weight changes and inclined plate test were abserved.Cervical motor function was evaluated by inclined plate test,rats’neck pain was assessed with RGS,ELISA detected TNF-α,IL-1βand PGE2,myocyte morphological changes were observed by HE,WB checked relative expression level of PI3K,Akt,p-Akt,Beclinl,ATG5,LC3II and p62,and Real-Time PCR checked gene expression level of Beclinl and ATG5.ResultsClinical trial:The trial included 126 cases with wind-cold-dampness cervical spondylosis and 17 cases fell out from the three groups with a rate of 13.49%without significant adverse reaction after a follow-up of 3 months(A&XBTG 6 cases,acupotomy 5 cases and XBTG 6 cases).Compared with before treatment,NDI,VAS,TNF-α,IL-1β,PGE2 and TCM-symptom scores reduced after treatment for three groups(P<0.01).Compared with AG and XBTG,NDI,VAS,TNF-α,IL-1β,PGE2,TCM-symptom scores and recurrence rate reduced after treatment in A&XBTG(P<0.05).Animal experiment:On the 2nd after modeling(before intervention),TNF-α,IL-1β and PGE2 increased compared with CG for 6 model groups(P<0.01),between which there were no statistically significant difference(P>0.05).During the study,there was no statistical difference in rats’ weight at 5 different time points(P>0.05).After 14 days of intervention,the angle of oblique plate test decreased(P<0.01),cervical pain score increased(P<0.01),TNF-α,IL-β and PGE2 increased(P<0.01),the proteins of PI3K,p-Akt,Beclin-1,ATG5 and LC3Ⅱ and the mRNAs of Beclin-1 and ATG5 downregulated,p62 protein raised(P<0.05),myocyte nuclei decreased,structure was disordered,deformation and edema appeared and size was different opposite to CG for MG.In contrast to MG,the angle of oblique plate test increased in A&XBTG(P<0.01),cervical pain score decreased(P<0.01),TNF-α,IL-β and PGE2 reduced(P<0.05),myocyte structure was clear,degree of edema was reduced and shape was regular in A&XBTG,AG and XBTG,and there was the most obvious effect in A&XBTG;the proteins of PI3K,p-Akt,Beclin-1,ATG5 and LC3Ⅱ and the mRNAs of Beclin-1 and ATG5 downregulated,p62 protein raised(P<0.05)in 3-MA;the proteins of PI3K,p-Akt,Beclin-1,ATG5 and LC3II and the mRNAs of Beclin-1 and ATG5 raised,p62 protein downregulated(P<0.05)in A&XBTG and A&XBTG&3-MA.Relative to AG and XBTG,the angle ascended(P<0.05,P<0.01),cervical pain score decreased(P<0.05,P<0.01),TNF-α,IL-1β and PGE2 reduced(P<0.05)in A&XBTG.Relative to 3-MA group,the proteins of PI3K,p-Akt,Beclin-1,ATG5 and LC3II and the mRNAs of Beclin-1 and ATG5 upregulated,and p62 protein downregulated(P<0.05)in A&XBTG and A&XBTG&3-MA.Compared with A&XBTG,the proteins of PI3K,p-Akt,Beclin-1,ATG5 and LC3Ⅱ and the mRNAs of Beclin-1 and ATG5 reduced,p62 protein raised(P<0.05)in A&XBTG&3-MA.Conclusion1.The combination of acupotomy and XBTG with good synergy has good clinical efficacy and safety for patients with wind-cold-dampness cervical spondylosis,relieve neck pain,improve NDI and TCM symptoms,reduce serum inflammatory factors and recurrence rate,superior to acupotomy or XBTG.2.The combination of acupotomy and XBTG can alleviate neck pain in rats with wind-cold-dampness cervical spondylosis,which would contribute to synergistic activation for PI3K/Akt pathway,upregulation for cell autophagy,thereby reducing the number of inflammatory factors,inhibiting inflammatory reactions and alleviating inflammatory pain.