| Objective: To investigate the intervention effect and mechanism of shaoyao gancao decoction on myospasm rats after spinal cord injury(SCI)by inhibiting neuronal apoptosis through plasma exosome miR-125a-5p.Methods: Eighty SPF-grade male SD rats were numbered by the random number table method,and 15 rats were randomly selected as the blank control group.Except for the blank control group,all rats were modeled by the spinal cord halftransection method,and spasticity was assessed in the surviving rats by the slantplate test and the modified Ashworth table after feeding for 5 days.The surviving rats with successful modeling assessment were again randomly divided into model group,drug group The surviving rats with successful modeling were again randomly divided into a model group,a drug group,and a positive control group.The model group was fed with deionized water,the medicated group was gavaged with peony and licorice soup,and the positive control group was gavaged with baclofen solution,and the motor function of each group was recorded by BBB score at 7 and 14 days of gavage,respectively.14 days later,blood was collected from the heart under anesthesia,and the spinal cord tissue of the injured segment was removed.Then,PCR was used to verify the differential expression of miR-125a-5p in the plasma of the model group and the drug-administered group.The dual-luciferase reporter gene assay was performed to verify the regulatory relationship between miR-125a-5p and the apoptosis-inhibiting regulator Bax.The effect of drug treatment and overexpression and silencing of miR-125a-5p on the expression of Bax in the cells was observed;and then the protein expression of Bax was measured by WB again after in vivo administration of drug and silencing or overexpression of the screened miRNAs in rats,demonstrating that peony and licorice soup can inhibit the expression of the pro-apoptotic gene Bax by regulating the expression of miR-125a-5p in plasma exosomes,which has a positive effect on positive therapeutic effect of myospasm after spinal cord injury.Results: As observed by BBB score analysis,the scores of the blank control group were higher than t he other intervention groups at 5 days of modeling,and the differences were statistically significant(P < 0.05).There was no statistically significant difference between the medicated group and the positive control group,indicating that the interventio ns of Shaoyao Gancao Decoction were effective at 7 days of gavage;the difference between the blank control group and the model group,the medicated group and the positive control group at 14 days of gavage was statistically significant(P < 0.05),indicating that the rats with spinal cord injury could recover some functions without interventions,but the BBB scores of rats with spinal cord injury were reduced after gavage with shaoyao gancao decoction.However,after gavage with baclofen and shaoyao gancao decoction,the BBB scores improved compared with the model group,and the mean scores of shaoyao gancao decoction were higher,indicating that the intervention effect of shaoyao gancao decoction was slightly better;comparing the BBB scores of the same groups at different time points,the differences in BBB scores were not statistically significant in the blank control group with uninjured spinal cord,and the BBB scores of the model group,drug group,and positive control group at 5 days of modeling compared with 7 days of gavage and 14 days of gavage were all statistically The BBB scores of the group with drug intervention were higher than those of the model group,and at 14 days,the BBB scores of the shaoyao gancao decoction group were the highest,indicating that the effect of the drug was better with the extension of time;the Westernblot results of Bax protein in the spinal cord tissues of the four groups of rats suggested that the expression of Bax in the model group After extraction of plasma exosomes,the lipid bilayer structure with a biconcave morphology and a particle size of about 70-120 nm was observed by electron microscopy in both groups,and the expression of proteins TSG101 and CD9 sho wed positive,which proved that the samples contained exosomes.Differentially expressed miRNAs were screened by microarray,and the bioinformatics website predicted that miR-125a-5p had 6-7 binding sites to Bax.After drug administration intervention,the expression of miR-125a-5p in plasma exocytosis was elevated in the drug-administered group compared to the model group.Dual luciferase reporter gene assay confirmed that the binding activity of miR-125a-5p to Bax was reduced after mutation,demonstrating a regulatory relationship between the two.Both in vitro cultured neuronal cells,and in vivo administration of the drug demonstrated that the drug could affect miR-125a-5p expression in plasma exosomes by.Conclusion: shaoyao gancao deboction had positive intervention effects on rats with myospasm after spinal cord injury,and the mechanism may be that the expression of miR-125a-5p was influenced by plasma exosomes,which inversely regulated the expression of pro-apoptotic factor Bax and thus inhibited neuronal apoptosis. |
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