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ObjectiveTo investigate the mechanism of ethyl acetate extract of Tibetan medicine dampness bud Gentianopsis paludosa in the prevention and treatment of recurrent ulcerative colitis （UC） in rats with dampness-heat in large intestine syndrome based on the apoptotic pathway mediated by the B-cell lymphoma-2 （Bcl-2） and Bcl-2-associated X protein （Bax）. MethodUsing the disease-syndrome combination method， a recurrent UC model of dampness-heat in large intestine syndrome was constructed in rats. Seventy SPF-grade male SD rats were randomly divided into control group， model group， high-， medium-， and low-dose ethyl acetate of G.paludosa groups （150， 75， 37.5 mg·kg-1）， and mesalazine group （135 mg·kg-1）. The rats were orally administered with respective drugs for 14 days. The general conditions of the rats were recorded， and colon length and mucosal damage were observed. The colon wet weight index and organ coefficients of the liver， spleen， and thymus were calculated. Enzyme-linked immunosorbent assay （ELISA） was used to measure the levels of interleukin-6 （IL-6） and interleukin-1β （IL-1β） in the serum of each group. Hematoxylin-eosin （HE） staining was performed to observe pathological changes in the colon. Terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） was used to detect apoptosis in colonic epithelial cells. Western blot was used to measure the expression levels of Bcl-2， Bax， Caspase-3， Caspase-9， Zona Occludens-1 （ZO-1）， Claudin3， and Occludin in colonic tissue. Immunohistochemistry （IHC） was used to observe the expression of Bax and Caspase-3 in colonic epithelial cells. ResultCompared with the control group， the model group showed significant increases in the disease activity index （DAI） score， colonic mucosal damage index （CMDI）， intestinal epithelial apoptosis， liver and spleen indexes， and levels of inflammatory factors IL-1β and IL-6 in the serum （P<0.01）， decreased expression of intestinal mucosal protective proteins ZO-1， Claudin3， and Occluding （P<0.01）， increased expression of pro-apoptotic proteins Bax， Caspase-3， and Caspase-9 （P<0.01）， and decreased expression of anti-apoptotic protein Bcl-2 （P<0.01）. Compared with the model group， the high-， medium-， and low-dose ethyl acetate of G.paludosa groups all significantly improved the general condition of the rats， reduced colonic lesions， decreased intestinal epithelial cell apoptosis， reduced liver and spleen indexes， upregulated the expression of ZO-1， Claudin3， Occludin， and Bcl-2 proteins， and downregulated the expression of Bax， Caspase-3， and Caspase-9 proteins， with the high- and medium-dose ethyl acetate of G.paludosa groups showing the superior effects （P<0.05， P<0.01）. ConclusionEthyl acetate of G.paludosa can alleviate colonic mucosal damage and exert a therapeutic effect on UC by regulating the Bcl-2/Bax signaling pathway.
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ObjectiveTo predict the mechanism of Sinitang in treating myocardial ischemia-reperfusion injury （MI/RI） based on network pharmacology and verify the prediction results by cellular experiments. MethodThe traditional Chinese medicine system pharmacology database and analysis platform （TCMSP） was employed for retrieval of the main components and potential targets of Sinitang. Online Mendelian Inheritance in Man （OMIM） and GeneCards were employed to obtain the targets of Sinitang in treating MI/RI. STRING was employed to construct the protein-protein interaction （PPI） network， and DAVID to perform gene ontology （GO） annotation and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment. Finally， cellular experiments were carried out to verify the predicted anti-MI/RI mechanism of Sinitang. ResultA total of 105 active ingredients and 234 targets of Sinitang were screened out， among which 116 targets were predicted to be involved in the treatment of MI/RI. The GO annotation gave 587 entries， including 417 biological process entries， 101 cell component entries， and 69 molecular function entries. The KEGG analysis enriched 125 signaling pathways， involving vascular endothelial growth factor （VEGF）， phosphoinositide 3-kinase/protein kinase B （PI3K/Akt）， forkhead box transcription factor O （FoxO）， hypoxia-inducible factor-1 （HIF-1） apoptosis and other signaling pathways. The results of cell viability assay showed that Sinitang increased the survival rate of H9C2 cells damaged by hypoxia/reoxygenation （H/R）. Sinitang decreased the levels of tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， and creatine kinase-MB （CK-MB） in H9C2 cells damaged by H/R. The results of flow cytometry demonstrated that Sinitang decreased the apoptosis rate of H9C2 cells damaged by H/R. Western blot showed that Sinitang down-regulated the expression of Bcl-2 related X protein （Bax） and up-regulated that of B-cell lymphoma-2 （Bcl-2） in H/R-injured H9C2 cells. ConclusionSinitang treats MI/RI in a multi-target and multi-pathway manner， which involves the signaling pathways associated with apoptosis.
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BACKGROUND: Bisphosphonates are a novel inhibitor of bone resorption that can inhibit the activity and function of osteoclasts. OBJECTIVE: To observe the effects of sodium ibandronate on the expression of dentin matrix protein 1, Caspace3, Bcl-2 and Bax in condylar cartilage in osteoporosis rats. METHODS: Thirty-six female rats were randomly divided into sham group, osteoporosis group and sodium ibandronate group, twelve in each group. The sham group did not excise ovaries during surgery. Bilateral ovaries of rats were removed in the osteoporosis and sodium ibandronate groups. On the 7th day after operation, rats in the sodium ibandronate group were intraperitoneally given sodium ibandronate 10 µg/kg, once every 7 days. After 90 days, the rat ovaries were taken. Bone mineral density was measured in each group. The changes of condylar cartilage were observed by toluidine blue staining and TUNEL staining. The expression of dentin matrix protein 1 protein was detected by immunohistochemistry. The levels of Caspase3, Bcl-2 and Bax were detected by western blot assay. The study protocol was approved by the Animal Ethics Committee of Nanhua Hospital in China with the approval No. SLXD_201804010. RESULTS AND CONCLUSION: Compared with the sham group or sodium ibandronate group, the bone mineral density in the osteoporosis group was significantly decreased (P < 0.05). The results of toluidine blue staining showed that the hypertrophic layer of condylar cartilage in the sodium ibandronate group was significantly thicker than that in the osteoporosis group. Compared with the sham group or sodium ibandronate group, the number of apoptotic cells in condylar cartilage and subchondral bone increased significantly in the osteoporosis group (P < 0.05). The expression of dentin matrix protein 1 protein was significantly lower in the osteoporosis group than the sham group, but it increased after treatment with sodium ibandronate (P < 0.05). Compared with the sham group, the expression of Caspase 3 and Bax in the osteoporosis group increased significantly, and the expression of Bcl-2 decreased. However, treatment with sodium ibandronate decreased the expression of Caspase 3 and Bax and increased the expression of Bcl-2 significantly. Overall, our findings reveal that sodium ibandronate can inhibit the apoptosis of condylar chondrocytes and the number of osteoclasts in osteoporotic state, which may be related to the regulation of Caspase 3, Bcl-2, Bax and dentin matrix protein 1 expression.
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OBJECTIVE: To investigate the effect of ultraviolet B( UVB) on autophagy and apoptosis in human epidermal melanoma A375 cells. METHODS: i) A375 cells at logarithmic growth phase were exposed to UVB at doses of 10. 0 and15. 0 m J/cm~2. Then cells were collected at time point of 3,6,9 and 12 hours after irradiation. The effect of UVB on cell autophagy was observed by monodansylcadaverine staining and the effect of UVB on cell apoptosis was observed by acridine orange/ethidium bromide staining. ii) A375 cells of 10. 0 m J/cm~2 group and 15. 0 m J/cm~2 group were exposed to corresponding dose of UVB irradiation. Then cells were collected at time point of 18,24,36 and 48 hours after irradiation,and cell survival rate was examined using CCK-8 assay. iii) A375 cells were irradiated with UVB at doses of 10. 0 and15. 0 m J/cm~2 and then cells were collected at time point of 3,6,9 and 12 hours after irradiation. After that,A375 cells were irradiated at doses of 2. 5,5. 0,7. 5,10. 0 and 15. 0 m J/cm~2 of UVB,then cells were collected at time point of 9 hours after irradiation. The expressions of B-lymphoblastoma-2( Bcl-2),Bcl-2 related X protein( Bax),Bcl-2 interacting protein( Beclin-1) and microtubule-associated protein 1 light chain 3( LC3) Ⅱ were detected by Western blotting. A375 cells with no UVB irradiation were set as the control( pseudo-irradiation) in each experiment. RESULTS: i) Both autophagy and apoptosis of A375 cells induced by UVB irradiation at doses of 10. 0 and 15. 0 m J/cm~2 increased with time after irradiation. The effect on autophagy decreased at 12 hours time point with 15. 0 m J/cm~2 UVB irradiation. ii) The cell viability increased with time after irradiation in the 10. 0 and 15. 0 m J/cm~2 groups( P < 0. 05). From 18-48 hours after irradiation,the cell viability of the 10. 0 and 15. 0 m J/cm~2 groups was lower than that of the control group( P < 0. 05).From 24-48 hours after irradiation,the cell viability of the 15. 0 m J/cm~2 group was lower than that of the 10. 0 m J/cm~2 group( P < 0. 05). iii) The relative expression of Beclin-1 and LC3 Ⅱ protein at the 10. 0 m J/cm~2 group increased with time after 0-12 hours irradiation( P < 0. 05). The above changes of the 15. 0 m J/cm~2 group were observed within 0 to 9 hours,and the above two autophagy-related proteins were significantly decreased at the 12 hours time point( P < 0. 05).The relative expression of Bcl-2 protein at the 10. 0 and 15. 0 m J/cm~2 groups decreased with increasing time from 3 to 12 hours after irradiation( P < 0. 05). The relative expression of Bax protein increased with time from 0 to 12 hours after irradiation( P < 0. 05). The relative expression of Beclin-1 and LC3 protein in cells at 0. 0-10. 0 m J/cm~2,and the relative expression of Bax protein in cells at 0. 0-15. 0 m J/cm~2 increased with increase of irradiation dose( P < 0. 05). The relative expression of Bcl-2 protein decreased with increase of irradiation dose at 5. 0-15. 0 m J/cm~2( P < 0. 05). CONCLUSION: Autophagy and apoptosis of A375 cells can be induced by UVB irradiation at doses of 10. 0 and 15. 0 m J/cm~2. Autophagy induced by UVB irradiation at 10. 0 m J/cm~2 partially resisted the induction of apoptosis by UVB and enhanced cell viability. 15. 0 m J/cm~2 UVB-induced autophagy was insufficient to exert the above-mentioned effects,and the induction of apoptosis was the dominant effect.