Regulation of ERK/P27Kip1 signaling pathway by isorhynchophylline improves bleomycin induced pulmonary fibrosis in mice
Pulmonary fibrosis (PF) is a chronic interstitial lung disease characterized by abnormal proliferation of lung fibroblasts, excessive accumulation of extracellular matrix (ECM), inflammatory damage, and tissue structure destruction, ultimately leading to the formation of lung scars, lung dysfunction, and respiratory failure. Various factors such as oxidative stress, inflammatory response, fibroblast proliferation and activation, and abnormal ECM deposition are involved in the pathogenesis of pulmonary fibrosis (PF). More importantly, the process of proliferation and differentiation of lung fibroblasts into myofibroblasts (MFb) may be the key to the formation of pulmonary fibrosis. Numerous studies have shown that under the stimulation of cytokines such as transforming growth factor – β 1 (TGF – β 1), lung fibroblasts can proliferate and differentiate into alpha smooth muscle actin (α – SMA) positive MFb. MFb, through its synthesis of a large amount of ECM and pro fibrotic factors, is considered a key pathogenic mechanism for the progression of pulmonary fibrosis. Therefore, exploring the mechanisms of proliferation and differentiation of lung fibroblasts and identifying their inhibitory targets may be a potential strategy to alleviate pulmonary fibrosis.

Isorhynchophylline (IRN) is a tetracyclic indole alkaloid isolated from the traditional Chinese medicine Hook Vine. Previous studies have found that IRN has various effects such as antioxidant, anti-inflammatory, anti proliferative, and neuroprotective. Guo et al. found that IRN alleviates monocrotaline induced pulmonary arterial hypertension by inhibiting the proliferation of pulmonary artery smooth muscle cells and reducing pulmonary vascular remodeling. Further research has found that IRN has a certain alleviating effect on bronchial asthma by inhibiting the proliferation of bronchial smooth muscle cells. The latest research has found that isorhynchophylline has a certain protective effect on silica induced lung injury in mice. Our previous research found that mimosa alkaloids can significantly inhibit the proliferation and differentiation of pulmonary fibroblasts induced by TGF – β 1 by inhibiting the phosphorylation of extracellular regulated protein kinases 1/2 (ERK1/2) and upregulating the expression of p27Kip1. IRN significantly inhibits the proliferation of neonatal rat ventricular myocytes induced by angiotensin II (AngII) by suppressing the phosphorylation of ERK1/2. Based on the above research background, this study aims to explore the anti PF effect and mechanism of IRN by targeting the ERK1/2 and p27Kip1 signaling pathways, providing new ideas for the research and development of traditional Chinese medicine with anti PF effects.

Pulmonary fibrosis is a progressive interstitial disease characterized by excessive proliferation of fibroblasts and deposition of extracellular matrix, ultimately leading to the destruction of lung structure and formation of fibrotic lesions, with a poor prognosis. At present, the main drugs used to treat pulmonary fibrosis are glucocorticoids and immunosuppressants, but they have serious side effects. In recent years, with the continuous deepening of research on pulmonary fibrosis, the exploration and application of using traditional Chinese medicine to treat pulmonary fibrosis have gradually increased. Traditional Chinese medicine and its active ingredients have shown good anti pulmonary fibrosis activity by reducing the expression of inflammatory factors, inhibiting the proliferation of lung fibroblasts, inducing apoptosis of lung fibroblasts, and blocking the fibrosis process. They can effectively improve fibrosis symptoms and delay the progression of the disease. Previous studies have found that monomers of alkaloids in traditional Chinese medicine, such as methylprednisolone and oxymatrine, have certain effects in alleviating pulmonary fibrosis. Our research group also found in the early stage that mimosa alkaloids and rutaecarpine have a certain alleviating effect on bleomycin induced pulmonary fibrosis. Other studies have shown that traditional Chinese medicines such as Danshensu and Danshensu B improve bleomycin induced pulmonary fibrosis by inhibiting the proliferation and differentiation of lung fibroblasts. Previous studies have found that IRN alleviates MCT induced PAH by inhibiting the proliferation of PASMCs and reducing pulmonary vascular remodeling, and has a certain alleviating effect on bronchial asthma by inhibiting the proliferation of bronchial smooth muscle cells. However, this study found that after 21 days of treatment with IRN, collagen deposition and expression of collagen I in lung tissue of PF mice were significantly reduced, the expression of α SMA was significantly reduced, and the degree of lung injury was significantly reduced. Further in vitro experiments revealed that IRN can significantly inhibit TGF – β 1-induced proliferation of lung fibroblasts, significantly reduce TGF – β 1-induced cell migration, and to varying degrees inhibit the expression of TGF – β 1-induced alpha SMA and collagen I in lung fibroblasts. These results suggest that isorhynchophylline may improve BLM induced pulmonary fibrosis by inhibiting the proliferation and differentiation of pulmonary fibroblasts.

Research has shown that the proliferation, migration, and differentiation of pulmonary fibroblasts into MFb play an important role in the development of pulmonary fibrosis. Among them, TGF – β 1 plays a key role in the proliferation and differentiation of fibroblasts towards MFb. Research has found that TGF – β 1 can promote the proliferation and differentiation of lung fibroblasts by activating the Smad signaling pathway, and can also induce the proliferation and differentiation of lung fibroblasts by activating the MAPK signaling pathway. Cyclin dependent kinase inhibitor protein p27Kip1 is a member of the Cip/Kip family of CDK inhibitors and a negative regulator of protein kinase CDK2/cyclin E. It can block the cell cycle in the G0/G1 phase. The level of p27Kip1 is relatively high in the G0/G1 phase of the cell cycle. Under mitotic stimulation, p27Kip1 rapidly degrades, allowing CDK2/cyclin E to promote cell proliferation. Our research group found that TGF – β 1 induces the proliferation and differentiation of pulmonary fibroblasts by upregulating the phosphorylation level of ERK1/2 and inhibiting the expression of p27Kip1, while the ERK1/2 inhibitor PD98059 inhibits the proliferation and activation of pulmonary fibroblasts induced by TGF – β 1 by inhibiting ERK1/2 phosphorylation and upregulating the expression of p27Kip1. Meanwhile, we also found that mimosa alkaloids inhibited TGF – β 1-induced proliferation and activation of pulmonary fibroblasts by upregulating the expression of p27Kip1. Previous studies have found that IRN alleviates MCT induced PAH by inhibiting phosphorylation of ERK1/2 and upregulating expression of p27Kip1, thereby suppressing platelet activating factor induced PASMCs. Further research has also found that IRN inhibits the development of myocardial hypertrophy by suppressing the MAPK signaling pathway. This study found that IRN administration for 3 weeks significantly reduced the expression of TGF – β 1 in lung tissue of mice with pulmonary fibrosis, inhibited the phosphorylation of ERK1/2, upregulated the expression of p27Kip1, and downregulated the expression of CDK2/cyclin E1. Further in vitro experiments revealed that IRN can significantly inhibit TGF – β 1-induced ERK1/2 phosphorylation, significantly upregulate the expression of p27Kip1, and significantly downregulate the expression of CDK2/cyclin E1. These results suggest that IRN may inhibit the proliferation and differentiation of pulmonary fibroblasts by regulating ERK/p27Kip1.

In summary, our study suggests that IRN may inhibit the transformation of fibroblasts into myofibroblasts by suppressing the ERK signaling pathway and upregulating the expression of p27Kip1, thereby alleviating BLM induced PF. This study provides new theoretical basis for the clinical application of IRN and new ideas for the treatment of PF.