ABSTRACT
ObjectiveTo investigate the protective effect and regulatory mechanism of berberine （BBR） against the senescence of ovarian granulosa cells. MethodA cell senescence model in the human ovarian granulosa-like tumor （KGN） cell line was induced by H2O2. A control group， a model group， and high-dose （1 μmol·L-1） and low-dose （0.5 μmol·L-1） BBR groups were set up. The cells in the model group and the BBR groups were incubated with 10 μmol·L-1 H2O2 for 40 min. The effect of BBR on KGN cell proliferation was detected by cell counting kit-8 （CCK-8） assay. The effect of BBR on the senescence of KGN cells was detected by β-galactosidase staining. The effects of BBR on the apoptosis and ROS content of KGN cells were detected by flow cytometry. The effects of BBR on the mRNA expression of B-cell lymphoma-2 （Bcl-2）/Bcl-2-associated X protein （Bax）， cysteinyl aspartate-specific protease-3 （Caspase-3）， forkhead transcription factor O1 （FoxO1）， and catalase （CAT） was detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. Western blot was used to detect the effects of BBR on protein expression of silent information regulator1 （SIRT1）， superoxide dismutase 2 （SOD2）， c-Jun N-terminal kinase （JNK）， FoxO1， autophagy-associated protein microtubule-associated protein light chain 3Ⅱ （LC3BⅡ）， mammalian ortholog of yeast Atg6 （Beclin-1）， and ubiquitin-binding protein p62. ResultAfter H2O2 induction for 40 min， the cell proliferation rate of the model group decreased compared with that of the control group （P<0.01）， and the cell proliferation rates of the BBR groups increased compared with that of the model group （P<0.05）. The results of β-galactosidase staining showed that the cells of the model group showed significant senescence compared with those of the control group （P<0.01）， and the cellular senescence in the BBR groups was reduced compared with that of the model group （P<0.01）. As revealed by flow cytometry， compared with the control group， the model group showed increased apoptosis rate （P<0.01）， and compared with the model group， BBR groups showed decreased apoptosis rates （P<0.05）. Meanwhile， the ROS content in the model group increased compared with that in the control group （P<0.01）， and compared with the model group， the BBR groups showed reduced cellular ROS content （P<0.01）. The Real-time PCR results showed that compared with the control group， the model group showed decreased mRNA expression of CAT and Bcl-2/Bax in KGN cells and increased mRNA expression of Caspase-3 and FoxO1 （P<0.05）， and compared with the model group， the BBR groups showed increased mRNA expression of CAT and Bcl-2/Bax （P<0.05） and reduced mRNA expression of Caspase-3 and FoxO1 in KGN cells （P<0.05）. As revealed by Western blot results， SIRT1， SOD2， and p62 protein levels decreased in the model group compared with those in the control group （P<0.01）， and JNK FoxO1， LC3BⅡ， and Beclin-1 protein levels increased （P<0.05）. After BBR intervention， SIRT1， SOD2， and p62 protein levels increased （P<0.01）， and JNK， FoxO1， LC3BⅡ， and Beclin-1 protein levels decreased compared with those in the model group （P<0.05）. ConclusionBBR has an inhibitory effect on ovarian granulosa cell senescence， and the mechanism is related to the inhibition of apoptosis and autophagy mediated by the SIRT1/FoxO1 pathway.
ABSTRACT
Objective To explore the role of resolvin D1 in reducing brain injury after porcine cardiopulmonary resuscitation and its potential mechanisms.Methods Twenty-eight male domestic pigs weighing (36 ±3)kg were utilized.The animals were randomly divided into 4 groups (n =7 each):sham operation group (group S),cardiopulmonary resuscitation group (group CPR),low-dose resolvin D1 gToup (group LRD),and high-dose resolvin D1 group (group HRD).The animals in group S only got the general preparation without the procedure of cardiac arrest and resuscitation.The pig model was established by 8 mins of untreated ventricular fibrillation and then 5 mins of cardiopulmonary resuscitation.At 5 min post-resuscitation,the doses of resolvin D10.3 μg/kg,and 0.6 μg/kg were correspondingly injected via the femoral vein in LRD and HRD groups,and meanwhile the same amount of vehicle was given into the animals inthe other two groups.At 3 h,6 h and 24 h post-resuscitation,the concentrations of neuron specific enolase (NSE) and S100B protein (S100B) in serum was measured.At 24 h post-resuscitation,neurological deficit score (NDS) was evaluated;thereafter the pigs were sacrificed,and cerebral cortex was obtained for the determination of tumor necrosis factor-alpha (TNF-α),interleukin-6 (IL-6),and malondialdehyde (MDA) contents,and superoxide dismutase (SOD) activity.Results Compared to group S,post-resuscitation brain injury was observed in the other three groups,which was indicated by significantly increased NDS score,and markedly elevated concentrations of serum NSE and S100B.Compared to group CPR,the NDS was significantly decreased at 24 h post-resuscitation,and the concentrations of serum NSE and S100B were significantly reduced at 6 h and 24 h post-resuscitation in LRD and HRD groups.Compared to group LRD,the NDS score and its serum markers were further significantly decreased in group HRD.The inflammatory response and oxidative stress in brain tissue were observed in all the animals experiencing cardiac arrest and resuscitation,which were indicated by increased contents of TNF-α,IL-6 and MDA and decreased SOD activity.Compared to group CPR,the contents of TNF-α,IL-6 and MDA were significantly decreasedwhile SOD activity was significantly increased in LRD and HRD groups.The indicators of inflammatory response and oxidative stress in brain tissue were further significantly improved in group HRD when compared to group LRD.Conclusions Resolvin D1 can reduce post-resuscitation brain injury in a dose-dependent manner in swine,and the mechanism is related to the inhibition of inflammatory response and oxidative stress.