Inhibitory Effect of shRNA TRPV4 Gene on Intervertebral Disc Degeneration in Scoliosis Rat Model

doi:10.3969/j.issn.1674-5817.2019.05.004

Abstract

Abstract: Objective To explore the mechanism of short hairpin (shRNA) transient receptor potential vanilloid 4 (TRPV4) gene on intervertebral disc degeneration intervention in SD rat scoliosis model. Methods The classical scoliosis model of bipedal rats was used to construct the animal model. Based on the principle of siRNA interference, shRNA-TRPV4 interference vector was constructed. According to the purpose of the experiment, animals were divided into three groups, control group, model group and shRNA group. The degree of scoliosis and cobb angle were observed by radiography, degenerative intervertebral disc tissue was stained by pathological HE, expression of collagen II and Aggrecan in nucleus pulposus tissue was detected by immunohistochemical staining, inducible nitric oxide synthase (iNOS) was detected by RT-PCR and Western Blotting at gene and protein levels as well as inflammatory related factors cyclooxygenase 2 (COX-2) and matrix metalloproteinase-9 (MMP-9) and the expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2 antagonist of cell death(Bad), Bcl-2 associated X protein(Bax), cysteinyl aspartate specific proteinase-3(Caspase-3), Caspase-6 and Caspase-9. Results The transfection efficiency of 0.011 μL shRNA-TRPV4 lentivirus was 90.3%. The control group had no scoliosis, the model group had left scoliosis, and the cobb angle was 35.22°±2.18°, the shRNA group had left scoliosis, and the Cobb angle was 24.07°±3.09°. RT-PCR results showed that iNOS, COX-2, MMP-9, Bad, Bax and apoptosis-related factors such as Caspase-3, Caspase-6, Caspase-9 in the model group was higher than that of control group (P<0.05), while the expression of anti-apoptotic factor Bcl-2 was lower than that of control group (P<0.05). Western blotting showed that the expression of iNOS and MMP-9 in nucleus pulposus of model group was higher than that of control group (P<0.05), but lower than that of shRNA group (P<0.05). Immunohistochemical staining showed that the expression level of Type II collagen and Agrecan protein was lower than that of control group (P<0.05), but higher than that of model group (P<0.05). Conclusion ShRNA-TRPV4 can inhibit the apoptosis of nucleus pulposus cells in scoliosis rat model, reduce the expression of iNOS and inflammatory factors COX-2 and MMP-9 in nucleus pulposus tissue, and protect the degeneration of nucleus pulposus tissue.

Key words: Nucleus pulposus cells, Scoliosis, Apoptosis, Mechanically sensitive ion channels, Transient receptor potential vanilloid 4 (TRPV4)

CLC Number:

Q95-33

LI Yan, KONG Jian-jun, CHEN Zi-qi, ZHU Cong-cong. Inhibitory Effect of shRNA TRPV4 Gene on Intervertebral Disc Degeneration in Scoliosis Rat Model[J]. Laboratory Animal and Comparative Medicine, 2019, 39(5): 356-363.

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