Working Through Intestinal Microorganisms: The Nerve Repairing Effects Of Lappaconitine On Rats With Spinal Cord Injury

Objective: Spinal cord injury(SCI)is one of the most common disabling diseases in the world.In most cases,Spinal cord injury is caused by various direct or indirect violent external forces that act on the spine spinal cord and cause the spinal cord to compression or rupture.And it is also commonly seen in iatrogenic conditions,or damage caused by vascular origin.Spinal cord injury is notoriously difficult to cope for its high disability rate,high occurrences,high medical expenses,which brings huge economic and psychological pressure to the patient’s family,medical and health system,and the entire society.Lappaconitine(LA)is China’s first non-opioid,non-addictive analgesic,and current studies have shown that it can effectively inhibit the expression of inflammatory factors after spinal cord injury,the repair of spinal cord injury can be facilitated.Nevertheless,its working mechanism still needs further study.Methods:(1)Taking the rats with spinal cord injury as the research object,we firstly used BBB score and oblique pull test to verify the role of Lappaconitine in the functional repair of spinal cord injury in rats,and then used HE staining to evaluate the repairing effect of Lappaconitine by observing the tissues and its morphoplogical changes.The expression level of inflammatory factors(IL-6,IL-1β,TNF-α)in the serum was further detected by ELISA technology.(2)We collected the contents of the intestines(small intestine,cecum,and large intestine)of rats in different treatment groups,used Qualcomm sequencing technology,Venn analysis,PCo A analysis,and α diversity analysis to evaluate the effects of Lappaconitine on microbial diversity changes in the intestines(small intestine,cecum,and large intestine)of rats with spinal cord injury,and then applied species distribution analysis method to evaluate the changes of flora down to phylum and family level,thus the advantageous flora and specific bacteria after treatment with Lappaconitine can be screened.(3)The content of short-chain fatty acids(propionic acid,acetic acid,n-butyric acid,isobutyric acid,n-valeric acid,isovaleric acid)in the feces of rats in different treatment groups was measured by gas chromatography.Results:(1)The BBB scores and the angle of the inclined plate tests in the medium-dose group(4 mg/kg)and the high-dose group(8 mg/kg)were significantly improved(P<0.05)after treatment for 7 days(P<0.05);After treatment with medium dose(4 mg/kg)and high dose(8 mg/kg)of Lappaconitine,the morphology of spinal cord tissue was improved to a certain extent,the boundary of gray matter and white matter was clear,and the structure was relatively complete;it was further found that the serum levels of IL-6,IL-1β and TNF-α were significantly lower than those in the spinal cord injury group(P<0.05),but the levels of IL-10 were significantly higher than those in the spinal cord injury group(P<0.05).(2)The results of intestinal microbial diversity showed that the Chao1 index and Ace index of the small intestine,cecum,and large intestine after spinal cord injury were significantly higher than those in the Sham operation group(P<0.05),and Lappaconitine treatment could significantly reduce the index increase,Venn analysis and PCo A analysis found that the diversity of intestinal flora in the small intestine was significantly different from that in the cecum and large intestine as well.Further analysis of species distribution showed that the proportions of Ruminococcaceae,Muribaculaceae and Erysipelotrichaceae in the cecum and large intestine(rumen)in the injury group were lower than those in the Sham operation group.However,the proportions of Ruminococcaceae(Ruminococcaceae),Muribaculaceae and Erysipelotrichaceae(Weirongcoccaceae)were significantly raised after treatment with Lappaconitine.(3)High performance gas chromatography analysis results showed that spinal cord injury can significantly reduce the content of isovaleric acid in the excrement(P<0.05),and has no significant effect on propionic acid,acetic acid and butyric acid(P<0.05);treatment with Lappaconitine can significantly reduce the total acetic acid content of feces after spinal cord injury(p<0.05),but has no significant effect on the content of propionic acid(P<0.05);and Lappaconitine treatment can also significantly increase the content of butyric acid(n-butyric acid and isobutyric acid)and valeric acid(n-valeric acid and isovaleric acid)(P<0.05).Conclusion:(1)Lappaconitine can effectively promote nerve repair after spinal cord injury.(2)Lappaconitine can inhibit the complexity of microbes and restore the negative change to a balance in the intestine(small intestine,cecum and large intestine)after spinal cord injury,and at the same time promote the growth of Ruminococcaceae,Muribaculaceae and Erysipelotrichaceae in the cecum and large intestine.(3)Lappaconitine can significantly increase the content of butyric acid(n-butyric acid and isobutyric acid)and valeric acid(n-valeric acid and isovaleric acid)in the feces of rats after spinal cord injury.(4)It is speculated that Lappaconitine may change the diversity of intestinal microbes after spinal cord injury,promote the growth of butyric acid and valeric acid-producing bacteria,thereby increase the content of butyric acid and valeric acid;it may improve the level of intestinal immunity,regulate "the balance of flora-gut-brain axis",and ultimately promote nerve repair after spinal cord injury.