| Objective1.The study is to make differentially expressed analysis to plasm miRNA in patients with lumbar intervertebral disc degeneration(LIDD),and then to verify the specific miRNA in the LIDD population.At the same time,the diagnostic value of specific miRNA for LIDD will be evaluated.2.The animal model of rat LIDD was established to further confirm the relationship between differentially expressed miRNA expression and LIDD,and to detect the expression changes of signal transduction pathway mediated by the differentially expressed miRNA target gene in lumbar intervertebral disc.3.To investigate the effect of Bushen Zhuangdu recipe on the expression of differentially expressed miRNA and its target gene-mediated signal transduction pathways in LIDD rats,and to further reveal the mechanism of Bushen Zhuangdu recipe in delaying LIDD.Methods1.The clinical research? Between December 2015 and April 2016,three patients with LIDD in the Third Affiliated Hospital of Guangzhou University of Chinese Medicine Department of spine inpatient and outpatient were selected as the experimental group.At the same time,age and sex matched three volunteers without LIDD were selected as the control group.The plasma samples of patients with LIDD and volunteers without LIDD were screened by MicroRNA microarray.? According to the microRNA microarray's probe detectable flag(?1 undetected case),differential miRNAs were confirmed by an independent real-time quantitative RT-PCR in these 6 samples for selecting the specific miRNAs of LIDD population.? Between December 2015 and August 2016,thirty patients with LIDD in the Third Affiliated Hospital of Guangzhou University of Chinese Medicine Department of spine inpatient and outpatient were selected as the experimental group.At the same time,age and sex matched twenty volunteers without LIDD were selected as the control group.According to the results of the situ validation and the literature study,the differentially expressed miRNA of LIDD were further validated by an independent real-time quantitative RT-PCR in the plasma samples of 30 patients with LIDD and 20 volunteers without LIDD.The specificity of differentially expressed miRNA expression of LIDD was verified.In order to evaluate the predictive value of the specific expression miRNA,receiver operating characteristic(ROC)curve analysis was carried out and area under curve(AUC)was applied.2.The experimental study60 male SD rats(3 months old)were randomly divided into sham operation group,model group,low-dose group of Chinese medicine,medium-dose group of Chinese medicine and high-dose group of Chinese medicine.Twelve SD rats were treated in each group.Rats of sham operation group were just exposed the L4 to the L6 intervertebral discs from the incision in the middle of the right side of the abdomen,and then the wound was routinely sutured.In other groups,the rats were exposed to the L4 to the L6 intervertebral discs from the incision in the middle of the right side of the abdomen,and then the LIDD model was established by the method of the whole layer puncture of the anulus fibrosus.Second day after modeling,low-dose group of Chinese medicine,medium-dose group of Chinese medicine and high-dose group of Chinese medicine were given low dose,middle dose and high dose of Bushen Zhuangdu recipe decoction by gavage.At the same time,sham operation group and model group were given equal volume of normal saline by gavage.After 8 weeks,the rat plasma and L4 to the L6 intervertebral discs were removed.The degeneration of the intervertebral discs was observed by HE staining,and the pathological score was performed according to the degree of degeneration.The expression of specific miRNAs in the plasma and lumbar intervertebral discs were detected by real-time quantitative RT-PCR.The specific miRNA target gene and its signal transduction pathway expression in the lumbar intervertebral discs were analyzed by western blot and real-time quantitative RT-PCR.Results1.The clinical research? 23 differentially expressed microRNAs were screened by using microRNA microarray,including 16 up-regulated microRNAs and 7 down-regulated microRNAs.? According to the microRNA microarray' s probe detectable flag(?1 undetected case),17 differentially expressed microRNAs were chosen for an independent real-time quantitative RT-PCR in the situ validation.Only miRNA-4763-3p and miRNA-4767 down-regulated expression were statistically significant(P<0.05).? According to the results of the situ validation and the literature study,miRNA-4763-3p,miRNA-4767 and miRNA-125a-5p were enrolled in the further validation test.The expression of miRNA-4767 and miRNA-125a-5p were significantly down-regulated(P<0.05),MiRNA-4763-3p expression was not significantly reduced(P>0.05).? Receiver operating characteristic(ROC)curve analyses indicated thatmiRNA-4767 and miRNA-125a-5p were the valuable biomarkers for LIDD(P<0.05).And the area under the receiver operating characteristic curve(AUC)was 0.735(95%CI:0.591-0.850)and 0.863(95%CI:0.759-0.967).2.The experimental study? Morphological changes of HE staining in lumbar intervertebral disc in each groupCompared with the sham operation group,the lumbar intervertebral disc of the model group was significantly degenerated and the tissue degeneration score was significantly increased(P<0.05).Compared with the model group,the lumbar disc degeneration in the low-dose group of Chinese medicine,medium-dose group of Chinese medicine and high-dose group of Chinese medicine showed a significant improvement.And the scores of histological degeneration of the three Chinese medicine group were significantly decreased(P<0.05).There were also significant differences in the histological degeneration scores of the three Chinese medicine groups(P<0.05).? Changes of plasma miRNA-125a-5p expression in each groupCompared with the sham operation group,the expression of miRNA-125a-5p in plasma of the model group was decreased,and the difference was statistically significant(P<0.05).Compared with the model group,the expression of miRNA-125a-5p in the plasma of the low-dose group of Chinese medicine,medium-dose group of Chinese medicine and high-dose group of Chinese medicine was significantly increased(P<0.05).There was a significant difference in the expression of miRNA-125a-5p in plasma of the three Chinese medicine groups(P<0.05).? Changes of miRNA-125a-5p expression in lumbar intervertebral disc tissue in each groupCompared with the sham operation group,the expression of miRNA-125a-5p in the intervertebral disc tissue of the model group was decreased,and the difference was statistically significant(P<0.05).Compared with the model group,the expression of miRNA-125a-5p in the intervertebral disc tissue of the low-dose group of Chinese medicine,medium-dose group of Chinese medicine and high-dose group of Chinese medicine was significantly increased(P<0.05).There was a significant difference in the expression of miRNA-125a-5p in the intervertebral disc tissue of the three Chinese medicine groups(P<0.05).? The gene expression of miRNA-125a-5p target gene TP53INP1,p21 and Caspase-3 in each groupCompared with the sham operation group,the gene expression of TP53INP1,p21 and Caspase-3 in the model group were significantly increased,and the difference was statistically significant(P<0.05).Compared with the model group,the gene levels of TP53INP1,P21 and caspase-3 in the low-dose group of Chinese medicine,medium-dose group of Chinese medicine and high-dose group of Chinese medicine were significantly lower(P<0.05).There were significant differences in the gene expression of TP53INP1,p21 and Caspase-3 of the three Chinese medicine groups(P<0.05).? The protein expression of miRNA-125a-5p target gene TP53INP1,p21 and Active Caspase-3 in each groupCompared with the sham operation group,the protein expression of TP53INP1,p21 and Active Caspase-3 in the model group were significantly increased,and the difference was statistically significant(P<0.05).Compared with the model group,the protein levels of TP53INP1,P21 and Active Caspase-3 in the low-dose group of Chinese medicine,medium-dose group of Chinese medicine and high-dose group of Chinese medicine were significantly lower(P<0.05).There were significant differences in the protein expression of TP53INP1,p21 and Active Caspase-3 of the three Chinese medicine groups(P<0.05).Conclusion1.There were significant differences in the expression of microRNAs in peripheral blood plasma of patients with lumbar intervertebral disc degeneration.Plasma miRNA-4767 and miRNA-125a-5p were significantly reduced in patients with LIDD.2.Plasma miRNA-4767 and miRNA-125a-5p had a good diagnostic performance for the diagnosis of lumbar intervertebral disc degeneration,and could act as novel biomarkers of LIDD.3.Bushen Zhuangdu recipe could effectively delay the pathological changes of lumbar intervertebral disc degeneration in rats,reduce the pathological scores of LIDD,and its therapeutic effect had a certain dose-dependent.4.The expression of miRNA-125a-5p in the plasma and degenerated intervertebral disc tissue of rats with LIDD was significantly decreased,while the expression of miRNA-125a-5p target gene TP53INP1,p21 and Caspase-3 were significantly increased.This showed that that miRNA-125a-5p and its target gene TP53INP1-mediated signaling pathway were activated during LIDD.The present study also indicated that miR-125a is a promising potential therapeutic target for LIDD.5.Bushen Zhuangdu recipe could promote the expression of miRNA-125a-5p in the degenerated intervertebral disc tissue and plasma of rats with LIDD.At the same time,Bushen Zhuangdu recipe could reduce the gene expression of TP53INP1,p21 and Caspase-3 and the protein expression of TP53INP1,p21 and Active Caspase-3.Its therapeutic mechanism to LIDD is related to the regulation of miRNA-125a-5p and its target gene TP53INP1-mediated pathway activity.And its intervention effect had a certain dose-dependent. |
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