The aim of this research is to evaluate the effect of tetramethylpyrazine (TMP) and connective tissue growth factor (CTGF) miRNA plasmids on the expressive levels of CTGF, transforming growth factor-beta (TGF-beta) and type Ⅰ collagen of rat hepatic stellate cells (HSC) which are stimulated by high glucose. The rat HSCs which were successfully transfected rat CTGF miRNA plasmids and the rat HSCs which were successfully transfected negative plasmids were cultured in vitro. After stimulus of the TMP and the high glucose, the protein levels and gene expressive levels of CTGF, TGF-beta and type Ⅰ collagen were tested. The results indicated that high glucose increased the expression of CTGF mRNA, CTGF protein, TGF-beta mRNA,TGF-beta protein and type Ⅰ collagen (P<0.05). The expressive levels of CTGF mRNA, CTGF protein, TGF-beta mRNA, TGF-beta and type Ⅰ collagen in TMP group were lower than those in high glucose group and showed statistically significant differences (P0.05). Compared with high glucose group, the expressive levels of CTGF mRNA, CTGF protein, TGF-beta mRNA, TGF-beta and type Ⅰ collagen in rat CTGF miRNA plasmid interference group were significantly lower (P<0.05). However, no statistically significant difference was found in CTGF mRNA and CTGF protein levels between TMP group and CTGF miRNA group (P>0.05), while type Ⅰ collagen levels showed statistically significant differences (P<0.05). It is concluded that high glucose could promote the expressions of CTGF, TGF-beta and type Ⅰ collagen, and TMP and rat CTGF miRNA plasmids could reduce the expressions of CTGF, TGF-beta, type Ⅰ collagen.
Objective To investigate the mechanism of p38 mitogen activated protein kinase (MAPK) signaling pathway in regulating the hyperplasia and hypertrophy of human lumbar ligamentum flavum via transforming growth factor β1 (TGF-β1)/connective tissue growth factor (CTGF). Methods The lumbar ligamentum flavum tissue taken from patient with lumbar intervertebral disc herniation was isolated by collagenase-predigested explant cultures. The ligamentum flavum cells were treated with the extracellular regulated protein kinase pathway blocker PD98059, c-Jun N-terminal kinase pathway blocker SP600125, and p38 pathway blocker SB203580, and then the mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ were detected by real-time fluorescence quantitative PCR (qRT-PCR). The ligamentum flavum cells were divided into 4 groups, and transfected with small interfering RNA (siRNA), p38 siRNA, siRNA+3 ng/mL TGF-β1, and p38 siRNA+3 ng/mL TGF-β1 in groups A, B, C, and D, respectively. After 24 hours of transfection, immunofluorescence staining was performed to observe the expressions of p38 and phosphorylation p38 (p-p38); the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ in each group were detected by qRT-PCR; the protein expression of CTGF in each group was detected by Western blot. Results p38 pathway blocker SB203580 could significantly reduce the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ (P<0.05). After 24 hours of transfection, immunofluorescence staining showed positive staining with p38 and p-p38 expressions in groups A, C, and D and negative staining in group B. Compared with group A, the relative mRNA expressions of CTGF, collagen type Ⅰ, and collagen type Ⅲ and relative protein expression of CTGF in group B decreased significantly (P<0.05), while those in groups C and D increased significantly (P<0.05); and those indicators significantly increased in group C than in group D (P<0.05). Conclusion TGF-β1/CTGF based on the p38 MAPK signaling pathway play an important role in the occurance and development of hypertrophy of human lumbar ligamentum flavum.