Berberine Protects Against Hypoxia And Serum Deprivation-induced Mesenchymal Stem Cells Apoptosis And Protects Against Carbon Tetrachloride-induced Mouse Acute Liver Injury

ObjectiveBy using an in vitro model of hypoxia consisting of hypoxia and serum deprivation to mimic the in vivo ischemic microenvironment, we sought to investigate whether the Chinese traditional medicine berberine could protect against hypoxia and serum deprivation-induced mesenchymal stem cells injury.MethodsWe isolated mesenchymal stem cells from bone marrow and created in vitro model of hypoxia consisting of hypoxia and serum deprivation for 6 hours to induce mesenchymal stem cell injury. Different concentrations of berberine were given one hour before hypoxia and were still present during the process of hypoxia. Hoechst33342 staining and flow cytometric analysis were determined for cell apoptosis. Cell viability assay was used to test the berberine-induced protective effects against mesenchymal stem cells apoptosis.ResultsAfter 6 hours of hypoxia and serum deprivation, MSCs underwent severe injury. Hoechst 33342 staing showed that there was a lot of early-stage aopotosis in the cellsexposed hypoxia, compared to the control group. Pretreatment of berberine showed that it protected against cell injury in a concentration dependent manner, with the highest effects at a dose of 10μM, which almost completely reversed the cell death. Hoechst 33342 staining and flow cytometry conformed that berberine could protect anainst hypoxia and serum deprivation-induced MSCs apoptosis.ConclusionsOur data demonstrated that after hypoxia and serum deprivation, MSCs underwent early-stage apoptosis. Different concentrations of berberine could protect against MSCs deaths. Hoechst 33342 staining and flow cytometry confirmed that berberine could attenuate MSCs apoptosis induced by hypoxia and serum deprivation. ObjectiveWe created a model of hypoxia and serum deprivation-induced MSCs apoptosis,we investigated whether berberine could alleviate reactive oxygen species (ROS)production and inhibit mitochondrial dependent apoptotic pathway, thus protected mesenchymal stem cells apoptosis against hypoxia and serum deprivation. We also explored other mechanisms that could mediate berberine-induced protection.MethodsMesenchymal stem cells were exposed to hypoxia and serum deprivation for 6 hours; 10μM berberine was treated 1 hour before hypoxia and was present during the process of hypoxia. Production of cellular ROS was evaluated by analyzing changes in fluorescence intensity resulting from the oxidation of the intracellular fluoroprobe DCFH-DA. Loss of the mitochondrial membrane potential was assessed using the fluorescent probe JC-1. Western blot was used to test the JNK phosphrylation and realease of cytochorome c from the mitochondria to the cytosol. Apoptotic protein caspase-3 was also tested for its activation. At the same time we also used the PI3K/Akt inhibitors wortmannin and LY294002 see if they were also involved the protective effects of berberine.ResultsBerberine could reduce the ROS production of mesenchymal stem cells exposed to hypoxia and serum deprivation, the antioxidant NAC could also inhibit the release of ROS. The JC-1 probe assay showed that berberine could inhibit the loss of mitochondrial membrane potential compared to hypoxia and serum deprivation group. Western blot analysis showed that berberine could inhibit the JNK phosphrylation and realease of cytochorome c from the mitochondria to the cytosol and also berberine reduced the apoptosis related protein cleaved (activated) caspase-3. Our results also showed that berberine could increase the phosphrylation of Akt in the early stage of hypoxia and serum deprivation, and both PI3K/Akt inhibitors wortmannin and LY294002 could block the protective effects of berberine. Further study showed that wortmannin and LY294002 could block berberine induced ROS production JNK activation and subsequent mitochondrial apoptotic pathway, thus blocked the protective effects of berberine.ConclusionsROS plays a very important role in hypoxia and serum deprivation-induced MSCs apoptosis. Berberine could inhibit the production of ROS and phosphrylation of JNK, alleviate the release of cytochorome c and activation of caspase-3, and thus confer its ObjectiveBy creating the model of carbon tetrachloride-induced acute liver injury, we sought to investigate whether an antioxidant berberine could protect against liver cell deaths and the mechanisms within this process.MethodsBerberine was give orally once everyday for a successive of 7 days, and then mice were inject intraperitoneally with carbon tetrachloride to induce acute liver injury. After 24 hours, mice blood and liver tissue were collected. Serum ALT, AST, SOD and MDA were measured to test the liver function and oxidation. Liver tissue was also fixed with polyformaldehyde and was stained for TUNEL assay.ResultsAfter 24 hours'treatment, there were huge increase in the serum ALT and AST, as compared to control group. When pretreated with berberine, the ALT and AST level was reduced, with the best effects at the dose of 200mg/kg. The serum SOD was upregulated and MDA was downregulated when pretreated with berberine, compared with the acute liver injury group, with the highest protective effects at the dose of 200mg/kg. The TUNEL assay showed that berberine could significantly reduce the aopotosis.ConclusionsBy creating the carbon tetrachloride-induced acute lung injury model, we found that the antioxidant berberine could alleviate acute liver injury-induced cell apoptosis. We also found that berberine could increase the serum antioxidant SOD level, reduce the ROS production (indicated by the decrease of MDA, which is the end product of ROS) and thus confer its anti-apoptotic effects.