Effect of Wulao Qisun Prescription on Proliferation and Osteogenic Differentiation of AS Fibroblasts by Regulating Wnt/β-catenin Signaling Pathway
Objective:To investigate the effect and underlying mechanism of the Wulao Qisun prescription on pathological new bone formation in ankylosing spondylitis(AS).Methods:Synovial fibroblasts were isolated from the hip joints of AS patients and observed under a microscope to assess cell morphology.The cells were identified using immunofluorescence staining.The isolated AS fibroblasts were divided into blank group,low drug-containing serum group,medium drug-containing serum group,high drug-containing serum group,and positive drug group.After drug intervention,cell proliferation was measured using the cell counting kit-8(CCK-8)assay to observe fibroblast growth and determine the optimal intervention time.Alkaline phosphatase(ALP)activity was measured using the alkaline phosphatase assay.Protein expression of osteocalcin(OCN),osteopontin(OPN),and runt-related transcription factor 2(Runx2)was detected by Western blot.The mRNA expression levels of Wnt5a,β-catenin,and Dickkopf-1(DKK-1)were measured by real-time quantitative polymerase chain reaction(Real-time PCR).Results:Compared with the blank group,each drug-containing serum group of Wulao Qisun prescription and the positive drug group inhibited the proliferation of AS fibroblasts and reduced ALP expression(P＜0.01).Compared with the blank group,the low drug-containing serum group of Wulao Qisun prescription downregulated β-catenin mRNA expression(P＜0.05).The medium and high drug-containing serum groups and the positive drug group significantly downregulated Wnt5a andβ-catenin mRNA expression(P＜0.05,P＜0.01),with the positive drug group showing the most pronounced effect(P＜0.01).The high drug-containing serum group and the positive drug group significantly upregulated DKK-1 mRNA expression(P＜0.01).Compared with the blank group,the low drug-containing serum group of Wulao Qisun prescription inhibited the expression of OPN and Runx2 proteins(P＜0.05,P＜0.01),while the medium and high drug-containing serum groups and the positive drug group inhibited the expression of OCN,OPN,and Runx2 proteins(P＜0.05,P＜0.01).Conclusion:The Wulao Qisun prescription can inhibit the proliferation and osteogenic differentiation of AS fibroblasts,thereby delaying the formation of pathological new bone in AS.The possible mechanism involves the regulation of Wnt/β-catenin-related gene expression,further inhibiting the transcription of downstream target genes.
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