Study On The Expression Of Phosphodiesterase Isoforms In Ligamentum Flavum Fibroblasts And The Anti-fibrotic Effect Of Rolipram On Fibroblasts

Ligamentum flavum hypertrophy is a common spinal degenerative disease that can cause spinal stenosis and nerve compression.It has been reported that inflammation and fibrosis are two important factors leading to the hypertrophy of the ligamentum flavum.Therefore,substances with anti-inflammatory and anti-fibrotic effects may be beneficial to the treatment of ligamentum flavum hypertrophy.The phosphodiesterase family(PDEs)has11 subfamilies and multiple genotypes.Changes in its expression and activity are related to inflammation and fibrosis.We studied the expression of PDE subtypes in the hypertrophic ligamentum flavum and analyze the relationship between the expression of PDE and ligamentum flavum hypertrophy.Then we studied the anti-fibrotic effect and mechanism of Rolipram,a PDE4 inhibitor.We collected clinical hypertrophic ligamentum flavum specimens as the experimental group and the ligamentum flavum specimens of patients with simple intervertebral disc herniation as the control group.Isolate and culture ligamentum flavum fibroblasts.Quantitative PCR and tissue staining were used to detect the expression of multiple PDE subtypes,type Ⅰ/Ⅲ collagen,fibronectin(FN1)and transforming growth factor-β1(TGF-β1).TGF-β1recombinant protein was used to stimulate fibroblasts to make a cell model of ligamentum flavum fibrosis.Rolipram,β-catenin agonists,PI3 K inhibitors,and ERK1/2 inhibitors were given to treat the fibroblasts.Sirius Red staining was used to observe the cell status.Cell scratch test observes the proliferation and migration ability of cells.The expression of fibrosis markers such as ColⅠ,Col Ⅲ and FN1 in fibroblasts after administration was detected.Detect the expression of β-catenin and ERK1/2 signaling pathway proteins.A total of 11 PDE subtypes were expressed in ligamentum flavum fibroblasts.Among them,the expression of PDE4 A and 4B was significantly up-regulated in the hypertrophic group(P<0.01).Their expression is linearly related to the thickness of the ligamentum flavum.The differences in other PDE subtypes are not obvious.After stimulation by the recombinant protein TGF-β1,the cell morphology changed from a spindle to a polygonal shape.The ability of cell proliferation and migration is strengthened.The expression of Col Ⅰ,FN1,endogenous TGF-β1 and p-ERK1/2 increased.The change of p-β-catenin was not obvious.After treatment with Rolipram,the above-mentioned fibrosis-related indicators decreased significantly.the normal expression of p-ERK1/2 was restored.The normal expression of p-ERK1/2 was restored.PDE4A and PDE4 B were up-regulated in the hypertrophic group.This suggests that it is related to the hypertrophy of ligamentum flavum.Recombinant protein TGF-β1 stimulates fibroblast fibrosis.Rolipram exerts a good anti-fibrosis effect by blocking the activation of p-ERK1/2 stimulated by TGF-β1.