[Aqueous extract of Epimedium sagittatum mitigates pulmonary fibrosis in mice].

This study aims to investigate the intervention effect of the aqueous extract of Epimedium sagittatum Maxim on the mouse model of bleomycin(BLM)-induced pulmonary fibrosis, so as to provide data support for the clinical treatment of pulmonary fibrosis. Ninety male C57BL/6N mice were randomized into normal(n=10), model(BLM, n=20), pirfenidone(PFD, 270 mg·kg~(-1), n=15), and low-, medium-, and high-dose E. sagittatum extract(1.67 g·kg~(-1), n=15; 3.33 g·kg~(-1), n=15; 6.67 g·kg~(-1), n=15) groups. The model of pulmonary fibrosis was established by intratracheal instillation of BLM(5 mg·kg~(-1)) in the other five groups except the normal group, which was treated with an equal amount of normal saline. On the day following the modeling, each group was treated with the corresponding drug by gavage for 21 days. During this period, the survival rate of the mice was counted. After gavage, the lung index was calculated, and the morphology and collagen deposition of the lung tissue were observed by hematoxylin-eosin(HE) and Masson staining, respectively. The levels of reactive oxygen species(ROS) in lung cell suspensions were measured by flow cytometry. The levels of glutathione peroxidase(GSH-Px), total superoxide dismutase(T-SOD), and malondialdehyde(MDA) the in lung tissue were measured. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling(TUNEL) was employed to examine the apoptosis of lung tissue cells. The content of interleukin-6(IL-6), chemokine C-C motif ligand 2(CCL-2), matrix metalloproteinase-8(MMP-8), transforming growth factor-beta 1(TGF-ß1), alpha-smooth muscle actin(α-SMA), E-cadherin, collagen Ⅰ, and fibronectin in the lung tissue was measured by enzyme-linked immunosorbent assay(ELISA). The expression levels of F4/80, Ly-6G, TGF-ß1, and collagen Ⅰ in the lung tissue were determined by immunohistochemistry. The mRNA levels of CCL-2, IL-6, and MMP-7 in the lung tissue were determined by qRT-PCR. The content of hydroxyproline(HYP) in the lung tissue was determined by alkaline hydrolysation. The expression of α-SMA and E-cadherin was detected by immunofluorescence, and the protein levels of α-SMA, vimentin, E-cadherin in the lung tissue were determined by Western blot. The results showed the aqueous extract of E. sagittatum increased the survival rate, decreased the lung index, alleviated the pathological injury, collagen deposition, and oxidative stress in the lung tissue, and reduced the apoptotic cells. Furthermore, the aqueous extract of E. sagittatum down-regulated the protein levels of F4/80 and Ly-6G and the mRNA levels of CCL-2, IL-6, and MMP-7 in the lung tissue, reduced the content of IL-6, CCL-2, and MMP-8 in the alveolar lavage fluid. In addition, it lowered the levels of HYP, TGF-ß1, α-SMA, collagen Ⅰ, fibronectin, and vimentin, and elevated the levels of E-cadherin in the lung tissue. The aqueous extract of E. sagittatum can inhibit collagen deposition, alleviate oxidative stress, and reduce inflammatory response by regulating the expression of the molecules associated with epithelial-mesenchymal transition, thus alleviating the symptoms of bleomycin-induced pulmonary fibrosis in mice.

The effects of Jiawei Duhuo Jisheng mixture on Wnt/ß-catenin signaling pathway in the synovium inflamed by knee osteoarthritis: An in vitro and in vivo experiment.

ETHNOPHARMACOLOGICAL RELEVANCE: Knee osteoarthritis (KOA) is one of the common age-degenerative diseases. Recent studies have demonstrated that the pathogenesis of KOA is closely related to synovial lesions. Jiawei Duhuo Jisheng mixture (JDJM) has shown great potential in the treatment of KOA. However, the effect and mechanism of JDJM on synovial lesions of KOA remain unclear. AIM OF THE STUDY: The regulatory effect of JDJM on the Wnt/ß-catenin signaling pathway in KOA inflamed synovium was studied via in vitro and in vivo experiments, respectively. MATERIALS AND METHODS: For the in vitro experiment, fibroblasts were isolated from the rabbit synovium with KOA. The fibroblasts were grouped as follows: the vehicle group was given 0.5% FBS; the inhibitor group was treated with 0.5% volume fraction of XAV939; the normal serum groups and JDJM serum groups were treated with 5%, 10%, and 20% volume fractions of normal serum and JDJM-containing serum. The expression levels of Wnt3a, ß-catenin, Cyclin D1, metalloproteinase-7(MMP-7) and cyclooxygenase-2(COX-2) were detected by different assays 48 and 72 h after the intervention. For the in vivo experiment, the rabbit KOA model was prepared using the improved Hulth modeling method, whereby all rabbits were randomly divided into normal control, model control, positive control, and traditional Chinese medicine (TCM) groups. The expression levels of Wnt3a, ß-catenin, Cyclin D1, MMP-7 and COX-2 were detected by different assays in the 2, 4, and 8 weeks of treatment. RESULTS: In the two test results of in vitro experiments, the normal serum group was compared with the JDJM-containing serum group with the same volume fraction, demonstrating that mRNA transcription and protein expression levels of Wnt3a, ß-catenin, Cyclin D1, MMP-7, and COX-2 in the latter decreased (P < 0.05), with more pronounced effects observed in the group treated with 20% volume fraction of JDJM serum. Compared with the inhibitor group, there was no significant difference (P > 0.05) in the mRNA transcription and protein expression levels, i.e., Wnt3a, ß-catenin, Cyclin D1, and MMP-7 were observed in the JDJM serum groups, except for a significant decrease (P < 0.05) in the level of mRNA transcription and protein expression of COX-2. Based on the in vivo experiment, compared to the model control group, articular cartilage, synovial hyperplasia, and the inflammatory reaction of the TCM group at different treatment times were significantly improved. The mRNA transcription level of Wnt3a, ß-catenin, Cyclin D1, MMP-7 and COX-2 detected by RT-qPCR and the protein expression level of Wnt3a, ß-catenin, Cyclin D1, MMP-7 and COX-2 detected by Western blot were significantly reduced (P < 0.05), and the effect was more evident at the eighth week. CONCLUSION: JDJM can regulate the synovial Wnt/ß-catenin signaling pathway in KOA models, reduce the mRNA transcription and protein expression levels of Wnt3a, ß-catenin, Cyclin D1, MMP-7, and COX-2 in the synovium, thus inhibiting synovial inflammation and protecting articular cartilage, which could be the key mechanism of action in treating this disease.