Abstract:

Objective To investigate the effects of Buyang Huanwu Decoction （BYHWD） on the abnormal proliferation and fibrosis of human ligamentum flavum cells （LFCs） induced by transforming growth factor-β1 （TGF-β1）， and to elucidate the underlying molecular mechanisms mediated by the PHLPP1/AKT/mTORC1 signaling pathway. Methods Primary human LFCs were cultured in vitro and stimulated with TGF-β1 to establish a fibrotic cell model， followed by intervention with BYHWD-medicated serum. Cell proliferation was assessed using CCK-8 and EdU assays. Western blotting was performed to detect the protein expression levels of proliferation-related markers （PCNA， Ki-67）， fibrosis-related markers （collagen Ⅰ， collagen Ⅲ， α-SMA， and fibronectin）， and key signaling proteins （PHLPP1， p-AKT， total AKT， p-S6K， total S6K， p-S6， and total S6）. Furthermore， a lentiviral vector carrying shPHLPP1 was constructed to perform PHLPP1 knockdown rescue experiments， thereby verifying the pathway dependence of BYHWD efficacy. Results BYHWD-medicated serum markedly suppressed TGF-β1-induced proliferation of LFCs and downregulated the expression of PCNA and Ki-67. Concurrently， BYHWD reduced the protein levels of collagen I， collagen III， α-SMA， and fibronectin， thereby alleviating cellular fibrosis. Mechanistically， BYHWD upregulated PHLPP1 expression and decreased the ratios of phosphorylated to total proteins （p-AKT/AKT， p-S6K/S6K， and p-S6/S6）， thus blocking the hyperactivation of the AKT/mTORC1 cascade. Notably， PHLPP1 knockdown significantly reversed the inhibitory effects of BYHWD on cell proliferation and fibrosis， as well as its suppressive effect on the AKT/mTORC1 pathway. Conclusions BYHWD attenuates the aberrant proliferation and fibrosis of LFCs by upregulating PHLPP1 and suppressing the hyperactivation of the AKT/mTORC1 signaling pathway. This study provides experimental evidence and identifies potential molecular targets for the clinical application of BYHWD in treating ligamentum flavum hypertrophy and lumbar spinal stenosis associated with Qi deficiency and blood stasis syndrome.

Key words: Buyang Huanwu Decoction, ligamentum flavum, PHLPP1, fibrosis