| Objective: After isolating and culturing nucleus pulposus cells of human lumbar disc herniation in vitro,add different concentrations of Bugan Jianyao direction medicated serum for culture,detect the expression of MMP-3,aggrecan protein and m RNA,and then analyze whether "Bugan Jianyao direction " can delay the degeneration of nucleus pulposus cells through the regulation of "nf-k B p65" signal pathway.To more accurately explore the possible action target and utility mechanism of "Bugan Jianyao direction " in improving pain and other discomfort symptoms of patients with lumbar disc herniation,in order to find the action mechanism and effect target of traditional Chinese medicine formula in the treatment of lumbar disc herniation.This kind of research provides objective experimental basis,development enlightenment and exploration direction.Methods:The number of liver cells from the lumbar nucleus of rats was determined by logarithmic fluorescence staining,and the number of liver cells from the lumbar nucleus of rats in the first stage of bone marrow humidification was determined by the method of increasing the number of blood cells from the lumbar nucleus of rats in the first stage of digestionWhen in a long state,they were immediately grouped and intervened.The experiment was divided into nine groups: blank group,si RNA NC group and NF group-κ In the bp65 si RNA blocking group,one group of 5%,10% and 20% normal serum group(serum concentration 5,10 and 20%)and one group of drug containing serum group corresponding to the same percentage(drug concentration 5,10 and 20%).Five parallel holes are set in each group,with each hole 200 μ 50.After the culture time was set at 24 h and 48 h,the expressions of MMP-3 and aggrecan m RNA in nucleus pulposus cells of each group were detected by RT q PCR,and the expressions of MMP-3 protein and aggrecan protein in nine groups were detected by WB(Western blot).Results: The results of blank group and si RNA NC group were almost the same,and there was no significant difference in Statistics(P > 0.05);Compared with the other groups(except the blank group),the effect of si RNA NC group on MMP-3 m RNA transcription and its protein expression increased significantly.In aggrecan,according to the results of RT q PCR and WB(Western blot),both of them decreased significantly,and the value-added of nucleus pulposus cells also decreased significantly;NF-κ Compared with the normal serum group,the m RNA transcription and protein expression of MMP-3 in the bp65 si RNA blocking group were significantly down regulated,the aggrecan m RNA transcription and protein expression were significantly increased,and the value-added ability of nucleus pulposus cells was significantly improved.There was no significant difference compared with the 20% medicated serum group(P > 0.05);Compared with the normal serum group,there was no significant difference among the three groups.The difference between the three different(increasing)concentrations of normal serum did not show statistical significance(P > 0.05).Compared with the drug containing serum group,the drug containing serum group with the same volume integral number compared with the normal serum group,MMP-3,According to the results of RT q PCR detection of m RNA transcription and WB(Western blot)detection of protein expression,both of them decreased significantly,the expression of aggrecan protein m RNA and protein increased significantly,and the appreciation ability of nucleus pulposus cells increased(P < 0.05).Three different concentrations of drug containing serum were compared between groups(5,10,20%).The m RNA transcription and protein expression of MMP-3 in blood at 20% concentration decreased the most.Aggrecan,According to the results of RT q PCR detection of m RNA transcription and WB(Western blot)detection of protein expression,both of them increased significantly,and the proliferation of nucleus pulposus cells also increased significantly.The difference between the three groups was statistically significant(P <0.05)In terms of time: The effect of Bugan Jianyao direction on Aggrecan m RNA transcription,Aggrecan,MMP3 protein expression level and nucleus pulposus cell proliferation has no obvious difference between 24 h and 48 h.The effect on MMP-3m RNA expression was different at 24 h and 48 h,and the degree of regulation in 48 h was higher than that in 24 h.Conclusion: "Bugan Jianyao direction " can promote the proliferation and slow down the degeneration of nucleus pulposus cells.Its mechanism may be by inhibiting the transcription of MMP-3 m RNA and the expression of its protein,promoting the transcription of aggregan m RNA and then promoting the expression of its protein.It works together from two aspects to further clarify the precise targets and special signal pathways that "Bugan Jianyao direction " may affect,This mechanism can improve the clinical symptoms of lumbar disc herniation;It enlightens us that we can more finely explore the detailed targets of traditional Chinese medicine in the treatment of lumbar disc herniation and the key pathways affecting its role,so as to contribute objective experimental results,exploration basis and derived development ideas to such research in the future. |
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