Tetracycline combined with Cinnamon Twig Decoction Plus Kudzuvine Root impacted on nucleus pulposus cells proliferation and correlated factor expressions

English Keywords:Tetracycline  Cinnamon Twig Decoction Plus Kudzuvine Root  Nucleus pulposus cells  Cell proliferation  Rat

English Abstract:

  【Abstract】 Objectives: To investigate the effects of tetracycline(TET) combined with Cinnamon Twig Decoction Plus Kudzuvine Root(CK) on nucleus pulposus cell proliferation and the expressions of aggrecan, type Ⅱ collagen(Col2a), matrix metalloproteinase-13(MMP-13) and inducible nitric oxide synthase(iNOS). Methods: The nucleus pulposus cells of 8-week-old healthy male SD rats were isolated and identified in vitro. The successfully isolated nucleus pulposus cells were then treated with drug. TET group was treated with different concentrations (5, 10, 15, 20, 25μg/ml) of TET. CK group was treated with low, middle and high doses of CK. TET+CK group was treated with 20μg/ml of TET and middle dose of CK. Control group was treated without drug. Cell viabilities in different drug-treated groups were measured by CCK8 method. The relative mRNA expressions of aggrecan, Col2a, iNOS and MMP-13 in TET+CK group were measured by quantitative real-time polymerase chain reaction(qRT-PCR). The relative protein expressions of aggrecan, Col2a, iNOS, and MMP-13 were measured by Western bolt. The recombinant plasmid pcDNA3.1-iNOS was constructed by pcDNA3.1-CMV(+). The nucleus pulposus cells were transfected with pcDNA3.1-iNOS and treated with 20μg/ml of TET and middle dose of CK in transfer group. Nucleus pulposus cells in blank load transfection group were transfected with pcDNA3.1 and treated with 20μg/ml of TET and middle dose of CK. The control group was treated without drug or transfection. qRT-PCR was used to detect the mRNA expressions of iNOS and MMP-13 in each group. Western blot was used to detect the protein expressions of iNOS and MMP-13 in each group. Results: Immunohistochemical staining of type Ⅱ collagen and aggrecan was obviously positive staining in isolated cultured nucleus pulposus cells, and showed 96% and 98% positive cells respectively. The viability of nucleus pulposus cells with each concentration of TET and CK was not significantly different with that of control group(P＞0.05). The mRNA and protein expression levels of aggrecan and Col2a were significantly up-regulated(P＜0.05), while iNOS and MMP-13 were markedly down-regulated in the cells treated with 20μg/ml of TET and middle dose of CK when compared with those in control group(P＜0.05). The mRNA and protein expression levels of iNOS and MMP-13 were significantly up-regulated after transfection with recombinant plasmid pcDNA3.1-iNOS when compared with those in control group and blank load transfection group. Conclusions: TET combined with CK significantly promotes nucleus pulposus cell proliferation, the mRNA and protein expressions of aggrecan and Col2a, while inhibits the mRNA and protein expression of MMP-13 through down-regulating iNOS expression. It provides theoretical basis for drug prevention and treatment of intervertebral disc degeneration.