| Objective1.By establishing a model of intervertebral disc degeneration,observe the situation of intervertebral disc degeneration,nucleus pulposus cell apoptosis and changes in the expression level of miR-155-5p;2.The target genes of miR-155-5p were predicted by bioinformatics,the target genes of miR-155-5p were identified by lentivirus transfection and dual luciferase detection,and the key factors of mitochondrial signal pathway were detected,so as to clarify the relationship between miR-155-5p and mitochondrial signal pathway;3.Bushen zhuangdu decoction containing serum intervened nucleus pulposus cells induced by H2O2,observed the apoptosis of nucleus pulposus cells,explored the relationship between miR-155-5p and its target gene and mitochondrial signal pathway,and verified whether Bushen zhuangdu decoction could inhibit nucleus pulposus cell apoptosis through up regulating miR-155-5p and mitochondrial signal pathway at the cellular level.4.Based on the above experiments,to explore whether Bushen zhuangdu decoction can improve the intervertebral disc degeneration by up regulating the expression of miR-155-5p and inhibiting the apoptosis of nucleus pulposus cells through inhibiting the key factors of mitochondrial signal pathway.Methods1.According to the random number table method,28 12-week-old SPF male rats were divided into 2 groups:sham operation group and model group,and 14 rats in each group.The sham operation group only exposed the intervertebral disc without puncture,and the model group received acupuncture on the intervertebral disc.After 8 weeks of continuous gastric administration,samples were taken for relevant tests,including HE staining to observe the degeneration of intervertebral disc tissue,in situ terminal transferase labeling technique to detect nucleus pulposus cell apoptosis,and qPCR to detect the expression changes of miR-155-5p.2.Predict the target genes regulated by miR-155-5p by using bioinformatics software,and predict whether the 3’UTR of Apaf1 gene has binding sites with miR-155-5p;test various indicators of the experiment,including:flow cytometry Detection of human nucleus pulposus cell apoptosis,dual luciferase reporter gene detection,qPCR detection of gene expression changes,Western blotting detection of apoptotic protein expression.3.According to the random number table method,37 12-week-old SPF male rats were divided into 4 groups:normal group,low-dose Chinese medicine group,middle-dose Chinese medicine group,and high-dose Chinese medicine group.The rats in the normal group were given intragastric administration with Bushen Zhuangdu Decoction,and the rats in the normal group were given the same amount of normal saline.This process lasted for 2 weeks;then blood was drawn from the abdominal aorta,and the serum was separated for subsequent experiments.Carry out the intervention of Bushen Zhuangdu prescription-containing serum to detect related indicators,including transmission electron microscopy,flow cytometry to detect apoptosis,western blotting to detect apoptotic protein expression,and qPCR to detect gene expression changes.4.Divide 70 12-week-old SPF male rats into 5 groups according to the random number table method:sham operation group,model group,low-dose Chinese medicine group,medium-dose Chinese medicine group,and high-dose Chinese medicine Group,assigned to each group of 14 animals.The sham operation group only exposed the intervertebral disc without puncture,and the other four groups received acupuncture on the intervertebral disc.The sham operation group and the model group were given 3ml of normal saline every day,and the remaining low,medium and high dose groups were given traditional Chinese medicine solution by gavage.After 8 weeks of continuous intragastric administration,samples were taken for related tests,including HE staining to observe the degeneration of intervertebral disc tissue,in situ terminal transferase labeling technique to detect nucleus pulposus cell apoptosis,qPCR to detect changes in gene expression,and Western blotting to detect nucleus pulposus Changes in protein expression in tissues.Results 1.The expression of miR-155-5p in the nucleus pulposus tissue of rat intervertebral disc degeneration(1)The histopathological score of intervertebral disc tissue of rats in each group was significantly higher than that of the sham operation group(P<0.05)(2)Compared with the sham operation group,the apoptosis index of the nucleus pulposus cells of the rat intervertebral discs in each group was significantly higher in the model group(P<0.05).(3)The expression of miR-155-5p in the nucleus pulposus cells of the intervertebral discs of rats in each group was significantly lower in the model group compared with the sham operation group(P<0.05).2.The mechanism of miR-155-5p on apoptosis of human nucleus pulposus cells(1)Predicted by bioinformatics methods,it is found that the 3’-UTR of human Apaf1 has a binding site for miR-155-5p,indicating that miR-155-5p is likely to target and regulate Apaf1.(2)Compared with the wild-type NC group,the wild-type mimic group significantly reduced the expression of Apaf1(P<0.05),which indicates that miR-155-5p can bind to Apaf1 3’-UTR,that is,miR-155-5p It can target and regulate the expression of Apaf1.(3)Flow cytometric detection of the apoptosis index of nucleus pulposus cells,compared with the normal control group,the apoptosis index of nucleus pulposus cells in the model group was significantly increased(P<0.05);the model group+mimics was significantly lower than the model group+NC(P<0.05);The model group+anti-mimics was significantly higher than the model group+NC(P<0.05).(4)The expression level of miR-155-5p in nucleus pulposus cells in each group was compared with the normal control group,and the expression level in the model group was significantly reduced(P<0.05);model group+overexpression lentivirus and model group+control virus The group was significantly higher(P<0.05);the model group+interfering virus group was significantly lower than the model group+control virus group(P<0.05).Compared with the normal control group,the expression of Bcl-2 in the nucleus pulposus cells of each group was significantly reduced in the model group(P<0.05);the overexpression lentivirus group and the model group+ control virus group were significantly higher(P<0.05).The interference virus group was significantly lower than the model group+control virus group(P<0.05).The expression levels of Cytc and Apaf1 in nucleus pulposus cells in each group were significantly higher in the model group compared with the normal control group(P<0.05);compared with the overexpression lentivirus group and the model group+control virus group,they were significantly reduced(P<0.05),The interference virus group and the model group+control virus group were significantly higher(P<0.05).(5)Compared with the normal control group,the expression of Bcl-2 protein in nucleus pulposus cells of each group was significantly reduced in the model group(P<0.05);the overexpression lentivirus group and the model group+control virus group were significantly increased(P<0.05),the interference virus group was significantly lower than the model group+control virus group(P<0.05).The expression levels of Cytc and Apaf1 proteins in nucleus pulposus cells in each group were significantly higher in the model group compared with the normal control group(P<0.05);compared with the overexpression lentivirus group and the model group+control virus group,they were significantly reduced(P<0.05)),the interference virus group and the model group+control virus group were significantly higher(P<0.05).3.Effect of serum containing Bushen Zhuangdu Recipe on mitochondrial apoptosis pathway of nucleus pulposus cells and miR-155-5p(1)Transmission electron microscope observation results:the normal group has normal cell volume,uniform cytoplasm,and the presence of microvilli on the surface;the model group has reduced cell volume,condensed nuclear chromatin,concentrated intracellular organelles,and is in the late stage of apoptosis.Apoptotic bodies can be observed in nuclear cells.In the low-dose drug-containing serum group,nuclear chromatin was marginalized and there were non-quantitative apoptotic bodies;in the medium-dose drug-containing serum group,the organelles were clear,and a small amount of apoptotic bodies were visible;the high-dose drug-containing serum was not seen in the cytoplasm Apoptotic bodies,cell volume is normal.(2)Flow cytometric detection of nucleus pulposus cell apoptosis index,compared with the normal control group,the nucleus pulposus cell apoptosis index in the model group was significantly increased(P<0.05);low,medium,and high-dose drug-containing serum groups and models The comparison between the two groups was significantly lower(P<0.05);among the low,medium,and high dose groups,the difference between the low and medium dose groups was statistically significant(P<0.05).(3)Bcl-2 protein expression of nucleus pulposus cells in each group,compared with the normal control group,the Bcl-2 protein expression of the model group was significantly reduced(P<0.05);low,medium,and high-dose drug-containing serum groups and models The group was significantly higher(P<0.05);there were statistical differences between the low and high dose groups(P<0.05)and the low and medium dose groups(P<0.05).The expression of Cytc and Apaf1 protein in nucleus pulposus cells in each group was significantly higher in the model group compared with the normal control group(P<0.05);the low,medium,and high-dose drug-containing serum group was significantly reduced compared with the model group(P<0.05);Comparison among the low,medium and high dose groups,there are differences among the three groups(P<0.05).(4)The expression of miR-155-5p in nucleus pulposus cells in each group and the expression of miR-155-5p in nucleus pulposus cells in each group were significantly lower in the model group compared with the normal control group(P<0.05);Compared with the low,medium,and high-dose drug-containing serum groups,the low,medium,and high three groups were significantly higher(P<0.05).Compared with the low,medium,and high groups,the low,medium,and high three groups also had statistics.Academic difference(P<0.05).The expression of Bcl-2 in nucleus pulposus cells in each group was significantly reduced in the model group compared with the normal control group(P<0.05);compared with the model group,Bcl-2 in the middle and high-dose drug-containing serum group The expression level increased significantly(P<0.05);there were statistically significant differences among the low,medium,and high-dose drug-containing serum groups(P<0.05);The expression levels of Cytc and Apafl in nucleus pulposus cells in each group were significantly increased in the model group compared with the normal control group(P<0.05);compared with the model group,the Cytc expression levels in the low,medium,and high-dose drug-containing serum groups were significant Decrease(P<0.05),and there are statistical differences between the low,medium and high dose drug-containing serum groups(P<0.05);4.Effect of Bushen Zhuangdu Recipe on mitochondrial apoptosis pathway and miR-155-5p in degenerated nucleus pulposus tissue in rats(1)The histopathological scores of the intervertebral discs of rats in each group were significantly higher in the model group compared with the sham operation group(P<0.05);compared with the low,medium,and high-dose Chinese medicine group,the model group was low There was a significant decrease in the three groups of,medium and high(P<0.05),and there were statistical differences between the high,medium,low,and high dose groups(P<0.05).(2)Compared with the sham operation group,the apoptosis index of the nucleus pulposus cells of the rat intervertebral discs in each group was significantly higher(P<0.05);the model group was compared with the low,medium and high dose Chinese medicine group,low,There was a significant decrease in the middle and high three groups(P<0.05),and there were also statistical differences in the low,medium,and high three groups(P<0.05).(3)The expression of miR-155-5p in the nucleus pulposus cells of rat intervertebral discs in each group was significantly lower in the model group compared with the sham operation group(P<0.05);the model group was compared with the low,medium and high dose Chinese medicine group There was a significant increase in the middle and high groups(P<0.05),and there was a statistical difference between the low and high groups(P<0.05).Compared with the sham operation group,the expression of Bcl-2 in the nucleus pulposus cells of the rats in each group was significantly lower in the model group(P<0.05);compared with the medium and high-dose Chinese medicine group,the medium and high-dose groups were significantly higher in the model group(P<0.05);The comparison between the low-dose and high-dose groups was also statistically different(P<0.05).The expression of Cytc in the nucleus pulposus cells of rat intervertebral discs in each group was significantly higher in the model group compared with the sham operation group(P<0.05);compared with the low,medium and high dose Chinese medicine group,the model group was significantly higher in the low,medium and high dose groups Decrease(P<0.05),and there are also statistical differences among the three groups of low,medium and high(P<0.05).Compared with the sham operation group,the expression of Apaf1 in the nucleus pulposus cells of the rats in each group was significantly higher in the model group(P<0.05);compared with the low,medium and high dose Chinese medicine group,the model group was significantly higher in the low,medium and high dose groups Decrease(P<0.05),there is a statistical difference between the low and high groups(P<0.05).(4)Compared with the sham operation group,the expression of Bcl-2 protein in the nucleus pulposus cells of rats in each group was significantly lower in the model group(P<0.05);compared with the low,medium,and high dose Chinese medicine group,the model group showed low,medium and high doses of traditional Chinese medicine.There was a significant increase in the middle and high-dose groups(P<0.05),and there was also a statistical difference between the low,medium,and high-dose groups(P<0.05).The expression levels of Cytc and Apaf1 protein in the nucleus pulposus cells of rats in each group were significantly higher in the model group compared with the sham operation group(P<0.05);compared with the low,medium,and high dose Chinese medicine group,the model group was divided into three groups:low,medium and high All were significantly reduced(P<0.05),and there were also statistical differences among the three groups of low,medium and high(P<0.05).Conclusion1.The target gene of miR-155-5p is Apaf1.Both overexpression and knockdown of miR-155-5p can affect the apoptosis of nucleus pulposus cells.The mechanism of affecting the apoptosis of nucleus pulposus cells is through the targeted regulation of Apaf1 to participate in mitochondria Apoptosis pathway.2.The serum containing Bushen Zhuangdu Recipe can inhibit the apoptosis of nucleus pulposus induced by hydrogen peroxide.The mechanism is to up-regulate miR-155-5p,down-regulate the expression of the target gene Apaf1,and inhibit the mitochondrial apoptosis pathway,thereby inhibiting the nucleus pulposus cell apoptosis.3.Bushen Zhuangdu Decoction can up-regulate miR-155-5p,down-regulate the target gene Apaf1,inhibit mitochondrial apoptosis pathway,reduce nucleus pulposus cell apoptosis,and improve intervertebral disc degeneration. |
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