| Objective: To verify the regulation of miR-520 a on P65 and to explore the role of miR-520a/P65 in the regulation of the level of pro-inflammatory cytokines(IL-1β and TNF-ɑ)and anti-inflammatory cytokines(IL-10 and TGF-β1)under Mongolian medicine Naru-San Pill,the mechanism for the therapeutic effect of sciatica,and to provide a new idea and choice for the diagnosis and treatment of Mongolian medicine in inflammation and sciatica.Methods: The rat model of sciatica was firstly performed using a non-compressed lumbar disc herniation model.4 groups of rats were preoperatively,1day,3 days,5 days,7 days,10 days and 14 days after surgery administered.The threshold of the leg reflex under mechanical stimulation was verified on the success of the model.After 15 days of administration,using ELISA to determine the inflammatory factors in the serum of the rats after administration as indicators for evaluating the inflammatory response of sciatica.Subsequently,the differentially expressed miRNAs in the gene chip analysis model group and the administration group were performed.Fluorescence real-time quantitative PCR was used to detect miR-520 a,P65 and pro-inflammatory cytokines(IL-1β and TNF-ɑ)and anti-inflammatory cytokines(IL-10 and TGF-β1)at the mRNA level under Mongolian medicine Naru-San Pill.The expression of P65 at the protein level under Mongolian medicine Naru-San Pill was detected by Western blot.The ELISA assay was used to detect pro-inflammatory cytokines(IL-1β and TNF-ɑ)and anti-inflammatory cytokines(IL-10 and TGF-β1)expression at the protein level under the action of Mongolian medicine Naru-San Pill.HE staining and immunohistochemical analysis were used to detect the pathological changes of the sciatic nerve under the action of Mongolian medicine,Naru-San Pill,and the expression of P65 in the sciatic nerve.Finally,the dual luciferase reporter gene assay was used to detect the targeted regulation relationship between miR-520 a and P65.Results:The model was successfully tested by measuring the MWT value of the rats,and the ELISA showed that the serum levels of pro-inflammatory factors(IL-1β,TNF-ɑ)in therats before administration were significantly increased(P < 0.01).The content of anti-inflammatory factors(IL-10,TGF-β1)decreased significantly(P < 0.01).The levels of IL-1β and TNF-ɑ were significantly decreased after administration(P < 0.01),and the levels of anti-inflammatory factors IL-10 and TGF-β1 were significantly increased(P <0.01),suggesting that the above 4 inflammatory factors can be used as evaluation index of pain for sciatic nerve.The miRNAs with differential expression were screened by miR-520 a,and the downstream targeting of miR-520 a was detected by bioinformatics as P65.Real-time quantitative PCR confirmed the expression of miR-520 a at mRNA level and were consistent with the results screened by chip technology.The expression of miR-520 a in the mRNA level was negatively correlated with the pro-inflammatory factor,and the expression of miR-520 a was positively correlated with the anti-inflammatory factor,and negatively correlated with the expression of P65.The results of ELISA and Western blot proved that the expression of P65 was positively correlated with the pro-inflammatory factors and negative correlation with anti-inflammatory factors.HE staining analysis showed that the nerves in the sham operation group were arranged closely and orderly,and the degree of nerve damage in the model group was extremely obvious.The nerve fibers were severely deformed,and after administration,the nerve fibers were repaired,the vacuoles were obvious reduced,and the degree of nerve fiber damage has also improved.Immunohistochemical analysis showed that P65 was not expressed in the sham operation group,but P65 was severely expressed in the model group,while the expression of P65 was decreased after administration.The dual luciferase reporter assay again confirmed that luciferase signal was significantly decreased when co-transfected with the recombinant plasmid of miR-520 a mimics and P65 3’UTR compared with the other groups(P < 0.01).When miR-520 a mimics was expressed at high or low,there was no significant decrease in luciferase signal after co-transfection with the mutated P65 3’UTR recombinant plasmid,suggesting that miR-520 a had specific target effect on P65.Conclusion: In the rat model of non-compressed lumbar disc herniation,the administration of Mongolian medicine Naru-San Pill has a significant effect on analgesia.miR-520 a can specifically target P65 directly,and that Naru-San Pill can promote the expression of miR-520 a,thereby inhibiting the expression of P65.It can down-regulate pro-inflammatory factors IL-1β,TNF-ɑ and upregulate the effect of anti-inflammatory factor IL-10,TGF-β1,so as to treat sciatica. |
PDF Full Text Request