Effects of Qi-Fang-Xi-Bi-Granules on Cartilage Morphology and C/ebpα Promoter Methylation in Rats with Knee Osteoarthritis.

OBJECTIVE: To investigate the effects of Qi-Fang-Xi-Bi-Granules (QFXBGs) on cartilage morphology and methylation of C/ebpα (CCAAT/enhancer binding proteinα) at the promoter region. METHODS: Knee osteoarthritis (KOA) modeling was performed in rats in accordance with Hulth's method, and control group received sham operation. Eight weeks after KOA modeling, the rats in the KOA modeling group were further divided into 6 groups. Each group was given the appropriate drug. After 8 weeks, half of the rats were used for Micro-CT scan, HE staining, ABH/OG staining, immunohistochemistry, and TUNNEL staining of the knee joint tissue, and the other half were used to examine C/ebpα promoter methylation. RESULTS: The three dose groups of QFXBGs all showed lower degrees of surface fissures and flaking, thicker cartilage layer, and restored chondrocyte and subchondral bone morphology, compared with the KOA model group. C/ebpα-22 promoter methylation levels in the high- and low-dose groups were significantly higher than that in the KOA modeling group (p < 0.05), while C/ebpα-2 promoter methylation level in the medium-dose group was significantly higher than that in the KOA modeling group (p < 0.05). CONCLUSIONS: QFXBGs may alleviate articular cartilage degeneration through promoting C/ebpα-2 or C/ebpα-22 methylation at specific promoter sites.

Analysis of DNA methylation in chondrocytes in rats with knee osteoarthritis.

BACKGROUND: Knee osteoarthritis (KOA) is a degenerative knee disease commonly found in the ageing population. DNA methylation works with histone acetylation to participate in aging. Alterations of DNA methylation may involve the joint chondrocyte degeneration in KOA. The aim of this study is to detect DNA methylation changes in chondrocytes of rats with KOA. METHODS: The rat KOA model was established with the Hulth method (n = 10), while rats receiving sham operation served as the control (n = 10). At 16 weeks after modeling, the knee joint tissue was collected from half of the rats in each group for Micro-CT scanning, Haematoxylin& Eosin (HE) staining, ABH/OG staining, immunohistochemistry for Bax, Bcl-2 and Fas, and TUNNEL staining. Meanwhile, the articular cartilage was collected from the other half to detect promoter methylation in target genes with the MethylTarget approach. RESULTS: Micro-CT scanning, HE staining, ABH/OG staining, immunohistochemistry, and TUNNEL staining all showed more severe cartilage injury in the KOA group than in the control group, indicating successful establishment of KOA model. The methylation rate in the KOA group was significantly decreased for C/ebpα-2 (within a CpG island -452 bp to the initiation codon on chromosome 1 91,363,511), Cdk2 (within a CpG island -55 bp to the initiation codon on chromosome 7 3,132,362), Bak1 (within a CpG island 6452 bp to the initiation codon on chromosome 20 5,622,277), and Fas (within a CpG island on the entire chromosome 1 gene), compared with the sham group (P = 0.005, 0.008, 0.022 and 0.027, respectively). CONCLUSION: The chondrocyte apoptosis and significantly reduced methylation levels of C/ebpα-2, Cdk2, Bak1, and Fas may participate in the pathogenesis of KOA. However, the exact mechanisms remain to be determined.

The action and mechanism of myrislignan on A549 cells in vitro and in vivo.

Myrislignan is a natural compound with little pharmacological study. In our investigation, we investigated the effect of myrislignan in the induction of apoptosis in A549 cells in vitro and in vivo. Myrislignan inhibited the proliferation of A549 cells in a dose- and time-dependent manner assayed by MTT. In addition, Hoechst flow cytometry showed that myrislignan significantly induced apoptosis and cell cycle arrest in A549 cells. The apoptosis and anti-cell proliferation was mediated by the activation of mitogen-activated protein kinase and the inhibition of epidermal growth factor receptor signal pathway, change of mitochondrial membrane potential, the releasing of c-Myc, the downregulation of the level of the anti-apoptotic protein Bcl-2, and the upregulation of the level of the pro-apoptotic protein Bax. In conclusion, those results reveal a potential mechanism for the anti-cancer effect of myrislignan on human lung cancer, while suggesting that myrislignan may be a promising compound for the treatment of lung cancer.

Molecular Role of EGFR-MAPK Pathway in Patchouli Alcohol-Induced Apoptosis and Cell Cycle Arrest on A549 Cells In Vitro and In Vivo.

Nowadays, chemotherapy is still the main effective treatment for cancer. Herb prescriptions containing Pogostemon cablin Benth (also known as "Guang-Huo-Xiang") have been widely used in Chinese medicine today. In our research, we found that patchouli alcohol, a compound isolated from the oil of Pogostemon cablin Benth, exerted antitumor ability against human lung cancer A549 cells ability both in vitro and in vivo. MTT assay was used to assess cell viability. Hoechst 33342 staining and TUNEL cover glass staining provided the visual evidence of apoptosis. Caspase activity measurement showed that patchouli alcohol activated caspase 9 and caspase 3 of mitochondria-mediated apoptosis. Consistently, patchouli alcohol inhibited the xenograft tumor in vivo. Further investigation of the underlying molecular mechanism showed that MAPK and EGFR pathway might contribute to the antitumor effect of patchouli alcohol. Our study proved that patchouli alcohol might be able to serve as a novel antitumor compound in the clinical treatment of lung cancer.

Anti-proliferation effects, efficacy of cyasterone in vitro and in vivo and its mechanism.

Cyasterone was demonstrated potential inhibition effect in mouse skin carcinoma cells in published report. However, the molecular mechanisms of the cyasterone on cells remain unknown. Herein, we investigated the effects of cyasterone-induced apoptosis in A549 and MGC823 cells in vitro. MTT assay showed that cyasterone caused a significantly decreasing of the proliferation of A549 and MGC823 cells in a time-and dose-dependent manner with IC50 values of 38.50±3.73µg/mL on A549 cells and 32.96±1.24µg/mL on MGC823 cells at 48h, respectively. Hoechst staining and TUNEL staining results indicated the quintessential apoptosis features in immunofluorescence image. Apoptosis and cell cycle were determined by flow cytometry. Cyasterone treatment triggered inhibition of epidermal growth factor receptor- phosphatidylinositol 3 kinase/protein kinase B (EGFR-AKT) signaling pathways and activation of P38 pathways. Furthermore, cyasterone inhibited MGC823 cells xenografted tumor growth in vivo with few changes in body weights. In conclusion, our findings provide the evidence that cyasterone inhibits growth of A549 and MGC823 cells, via regulating EGFR signaling pathway. Our results indicated that cyasterone, a natural EGFR inhibitor, maybe a promising anti-cancer agent.

Anti-proliferation effects of trifolirhizin on MKN45 cells and possible mechanism.

Trifolirhizin is a compound isolated from Sophora flavescens. It has been shown to exert cytotoxicity on several cancer cell lines. However, the underlying mechanism remains unknown. MKN45 cells were used as a research model. We assessed the cytotoxicity of trifolirhizin to MKN45 by MTT. Hoechst staining and TUNEL method were used to demonstrate apoptosis. Flow cytometry was used to determine cell cycle and ratio of apoptosis. Caspase activity assay was used to examine the activation of caspase cascade pathways. Western blotting was used to explore the protein levels. Consistently, trifolirhizin inhibited MKN45 xenograft tumor growth in vivo. Trifolirhizin caused a significantly decreased proliferation of MKN45 cells in a time- and dose-dependent manner, with IC50 values of 33.27±2.06 µg/ml at 48 h. Western blot assay manifested that trifolirhizin activated the EGFR-MAPK signaling pathways. This study indicated that trifolirhizin may be a therapeutic application in human gastric cancer therapy.

Lobetyol activate MAPK pathways associated with G1/S cell cycle arrest and apoptosis in MKN45 cells in vitro and in vivo.

AIM: The agent lobetyol, which is isolated from Lobelia chinensis, was previously shown to be cytotoxicity againts several cancer cell lines in published report. Today, we perform a study in vitro and in vivo to analyze its anti-carcinoma effect in MKN45 cells and to explore the molecular mechanism. MAIN METHODS: The growth inhibition of lobetyol on MKN45 cells was analyzed with MTT and flow cytometry. Hoechst 33342 staining and TUNEL cover glass staining were used to provide the visual evidence of apoptosis. Western blotting assay was performed to study the activation or blocking of related signaling pathways. KEY FINDINGS: Lobetyol induce apoptosis and cell cycle arrest in a time- and dose-dependent manner in MKN45 cells in our study. This process is mediated by the MAPK signaling pathways. This study confirmed the cytotoxicity of lobetyol in MKN45 cells and provided an insight into the molecular mechanism, which demonstrates the potential of lobetyol as an anti-tumor agent.