The mechanism of Shenlong Jianji treatment of idiopathic pulmonary fibrosis inhibits fibroblast-to-myofibroblast transformation via the TGF-ß1/smads signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Shenlong Jianji (SLJJ) is a Chinese herbal compound composed of traditional medicines for supplementing Qi, nourishing Yin, promoting blood circulation, and removing obstruction in channels. It is widely used to treat idiopathic pulmonary fibrosis (IPF) in China. However, the underlying mechanism of SLJJ remains unclear. AIM OF THIS STUDY: To elucidate the efficacy and mechanisms of SLJJ in the treatment of IPF through in vivo and in vitro experiments. MATERIAL AND METHODS: 84 Wistar rats were randomly and equally divided into 7 groups: the control group (CTRL), the sham operation group (SHAM), the model group (IPF), the low dose of SLJJ group (L-SLJJ), the middle dose of SLJJ group (M-SLJJ), the high dose of SLJJ group (H-SLJJ), and the pirfenidone group (PFD). The rats in the CTRL, SHAM, and IPF groups were given normal saline each time for 28 days; the SLJJ groups were given Shenlong Jianji (9 g kg-1·d-1, 18 g kg-1·d-1, 36 g kg-1·d-1), and pirfenidone was administered as a sequential dose. After 28 days, the general condition of the rats was evaluated, and samples were collected. The lung coefficient was measured. The pathological changes of lung in each group were observed by H&E staining and Masson staining. α-SMA, collagen 1, and E-cadherin proteins were detected by immunohistochemistry. α-SMA, collagen 1, vimentin, E-cadherin, N-cadherin, TGF-ß1, smad2, and smad3 proteins were detected by WB in vivo.In vitro, A scratch test was used to assess the ratio of cell migration. α-SMA, vimentin, E-cadherin, and N-cadherin protein levels were evaluated by a cellular immunofluorescence assay. TGF-ß1/smads signaling pathway was detected by WB. HPLC-Q-TOF/MS analysis was used to identify the active compounds in the SLJJ. Molecular docking determined the free binding energy of the compound with the TGF-ß1 protein. RESULTS: SLJJ significantly improved the respiratory symptoms, heart rate, mental state, and food intake of IPF group rats and decreased the lung coefficient. In the IPF group, inflammatory cells were infiltrated, and the thickened alveoli wall and alveoli collapse were shown, while significantly alleviating pathological changes in the SLJJ and PFD groups. Masson staining showed that SLJJ and PFD decreased the collagen expression. Immunohistochemical results showed that the expressions of α-SMA, collagen 1, and N-cadherin decreased in the SLJJ and PFD groups, while E-cadherin increased significantly compared with the IPF group. SLJJ regulates TGF-ß1/smads signaling pathway proteins in vivo. SLJJ decreased the ratio of migration in HFL-1 cells; SLJJ reduced the fluorescence intensity of α-SMA, vimentin, and N-cadherin and increased the fluorescence intensity of E-cadherin in primary rat lung (PRL) fibroblast cells and HFL-1 cells. WB results showed that SLJJ significantly down-regulated α-SMA, Vimentin, N-cadherin, TGF-ß1, smad2, and p-smad2/3 proteins expression and up-regulated E-cadherin protein expression in vitro, whereas SRI-011381 (a TGF-ß1 agonist) antagonized the effects of SLJJ. CONCLUSION: SLJJ inhibits idiopathic pulmonary fibrosis. The TGF- ß1/Smads signaling pathway can be the target of SLJJ, which inhibits fibroblast-to-myofibroblast transformation and is expected to be a new drug for the treatment of IPF.

[Mechanism of Didang Decoction in prevention of anti-atherosclerosis and hyperlipidemia by HPLC-Q-TOF-MS/MS and network pharmacology based on theory of "nutrients return to heart and fat accumulates in channels"].

Atherosclerosis(AS) is caused by impaired lipid metabolism, which deposits lipids in the intima, causes vascular fibrosis and calcification, and then leads to stiffening of the vascular wall. Hyperlipidemia(HLP) is one of the key risk factors for AS. Based on the theory of "nutrients return to the heart and fat accumulates in the channels", it is believed that the excess fat returning to the heart in the vessels is the key pathogenic factor of AS. The accumulation of fat in the vessels over time and the blood stasis are the pathological mechanisms leading to the development of HLP and AS, and "turbid phlegm and fat" and "blood stasis" are the pathological products of the progression of HLP into AS. Didang Decoction(DDD) is a potent prescription effective in activating blood circulation, removing blood stasis, resolving turbidity, lowering lipids, and dredging blood vessels, with the functions of dispelling stasis to promote regeneration, which has certain effects in the treatment of atherosclerotic diseases. This study employed high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry(HPLC-Q-TOF-MS/MS) to screen the main blood components of DDD, explored the targets and mechanisms of DDD against AS and HLP with network pharmacology, and verified the network pharmacological results by in vitro experiments. A total of 231 blood components of DDD were obtained, including 157 compounds with a composite score >60. There were 903 predicted targets obtained from SwissTargetPrediction and 279 disease targets from GeneCards, OMIM, and DisGeNET, and 79 potential target genes of DDD against AS and HLP were obtained by intersection. Gene Ontology(GO) analysis suggested that DDD presumably exerted regulation through biological processes such as cholesterol metabolism and inflammatory response, and Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis suggested that signaling pathways included lipid and atherosclerosis, insulin resistance, chemo-carcinogenesis-receptor activation, and AGE-RAGE signaling pathways in diabetic complications. In vitro experiments showed that DDD could reduce free fatty acid-induced lipid accumulation and cholesterol ester content in L02 cells and improve cellular activity, which might be related to the up-regulation of the expression of PPARα, LPL, PPARG, VEGFA, CETP, CYP1A1, and CYP3A4, and the down-regulation of the expression of TNF-α and IL-6. DDD may play a role in preventing and treating AS and HLP by improving lipid metabolism and inflammatory response, and inhibiting apoptosis with multi-component, multi-target, and multi-pathway characteristics.

DiDang decoction improves mitochondrial function and lipid metabolism via the HIF-1 signaling pathway to treat atherosclerosis and hyperlipidemia.

ETHNOPHARMACOLOGICAL RELEVANCE: DiDang Decoction (DDD) is a traditional classical prescription that has been used to treat atherosclerosis (AS) and hyperlipidemia (HLP) in China. Nevertheless, the underlying mechanism of DDD remains unclear. AIM OF THE STUDY: To validate the mechanism of DDD in AS and HLP based on network pharmacology and in vitro experiments. MATERIALS AND METHODS: The chemical components of DDD were obtained from the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP) database and literature mining, and the disease targets of AS and HLP were obtained from the Gencards, OMIM, and DisGeNET databases. The intersection genes were imported into the STRING database to construct protein-protein interaction (PPI) network, and the DAVID database was used for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Combined with the results of KEGG pathway analysis, the HIF-1 signaling pathway was selected for further in vitro experiments. RESULTS: The results showed that network pharmacology predicted 112 targets related to DDD treatment of AS and HLP, and the top 10 related pathways are: Lipid and atherosclerosis, AGE-RAGE signaling pathway in diabetic complications, Chemical carcinogenesis - receptor activation, Pathways in cancer, Proteoglycans in cancer, Fluid shear stress and atherosclerosis, HIF-1 signaling pathway, Alcoholic liver disease, PPAR signaling pathway, and Coronavirus disease-COVID-19. In vitro experiments showed that DDD effectively reduced lipid accumulation in FFA-treated L02 cells; DDD attenuated mitochondrial damage and reduced ROS content; DDD inhibited ferroptosis and apoptosis; DDD up-regulated the expression of HIF-1α, Glutathione Peroxidase 4(GPX4), and Bcl2 proteins, and down-regulated expression of Bax protein. CONCLUSION: DDD exerts therapeutic effects on AS and HLP through multiple targets and pathways, and improves mitochondrial function, reduces ROS content, inhibits ferroptosis and apoptosis by activating the HIF-1 signaling pathway, which provides reliable theoretical and experimental support for DDD treatment of AS and HLP.