BACKGROUND:A short-peptide small molecule hydrogel (SMH) developed in the previous study has more obvious advantages than other hydrogels to improve local microenvironment, carry bioactive substances and interfere with stem cell signal transduction pathways. OBJECTIVE:To explore the effect of SMHs on bone marrow mesenchymal stem cells (BMSCs) proliferation, apoptosis and differentiation into myocardial cells. METHODS: (1) Passage 9 rat BMSCs in vitro were divided into control group and experimental group, followed by routine culture and culture in SMHs, respectively. At 7 days of culture, cell proliferation and apoptosis were detected. Cells in the two groups were exposed to anaerobic environment for 12 hours, and expression levels of Bcl-2, Bax and Caspase-3 in BMSCs were detected. (2) Passage 9 BMSCs were divided into four groups and then cultured in 5-azacytidine, SMHs, SMHs+5-azacytidine, and L-DMEM (normal control), respectively. After 4 weeks of induction, expression of CTnT, desmin and Cx-43 proteins was detected and expression levels of early cardiac transcription factors, NKX2.5 and GATA-4, were also measured. RESULTS AND CONCLUSION: (1) Compared with the control group, better proliferation and lower apoptosis of BMSCs were found in the experimental group. Under anaerobic conditions, the number of survival cells was reduced in both groups, but less apoptosis or necrosis was found in the experimental group than the control group (P < 0.05). Moreover, the level of Bcl-2 was higher in the experimental group than the control group (P < 0.01), while the levels of Bax and Caspases-3 protiens were lower in the experimental group than the control group (P < 0.01). (2) NKx2.5 and GATA-4 mRNA expression was found in both 5-azacytidine and SMHs+5-azacytidine groups, and moreover, the mRNA levels of early cardiac transcription factors were significantly higher in the SMHs+5-azacytidine group than in the 5-azacytidine group (P < 0.05). In the normal control group, cTnT expressed negatively, and desmin and Cx-43 expressed weakly. The expression of cTnT, desmin and Cx-43 proteins was higher in the SMHs+5-azacytidine group than in the 5-azacytidine and SMHs groups, while there was no significant difference between the latter two groups. To conclude, SMHs as a culture medium is conducive to the proliferation of BMSCs, reduces cell apoptosis, and promotes myocardial differentiation of BMSCs.

AIM:Tostudywhe ther theangiotens in-(1-7)[Ang-(1-7)]/Mas receptor axis protects cardio-myocytes against high glucose (HG)-induced injury by inhibiting nuclear factor-κB (NF-κB) pathway.METHODS:The cell viability was measured by CCK-8 assay.The intracellular levels of reactive oxygen species ( ROS) were detected by DCFH-DA staining .The number of apoptotic cells was tested by Hoechst 33258 nuclear staining .Mitochondrial membrane potential ( MMP) was examined by JC-1 staining.The levels of NF-κB p65 subunit and cleaved caspase-3 protein were de-termined by Western blotting.RESULTS: Treatment of H9c2 cardiac cells with 35 mmol/L glucose (HG) for 30, 60, 90, 120 and 150 min significantly enhanced the levels of phosphorated ( p) NF-κB p65, peaking at 60 min.Co-treatment of the cells with 1 μmol/L Ang-(1-7) and HG for 60 min attenuated the up-regulation of p-NF-κB p65 induced by HG. Co-treatment of the cells with Ang-(1-7) at concentrations of 0.1~30μmol/L and HG for 24 h inhibited HG-induced cy-totoxicity, evidenced by an increase in cell viability .On the other hand, 1 μmol/L Ang-(1-7) ameliorated HG-induced apoptosis, oxidative stress and mitochondrial damage , indicated by decreases in the number of apoptotic cells , cleaved caspase-3 level, ROS generation and MMP loss .However, the above cardioprotective effects of Ang-(1-7) were markedly blocked by A-779, an antagonist of Ang-(1-7) receptor (Mas receptor).Similarly, co-treatment of H9c2 cardiac cells with 100 μmol/L PDTC ( an inhibitor of NF-κB) and HG for 24 h also obviously reduced the above injuries induced by HG.CONCLUSION:Ang-(1-7)/Mas receptor axis prevents the cardiomyocytes from the HG-induced injury by inhibiting NF-κB pathway .

Aim To investigate the role of ATP-sensi-tive potassium channels-Akt pathway in exogenous hy-drogen sulfide( H2 S) inhibiting the high glucose( HG)-induced injury in H9c2 cardiac cells. Methods The expression level of Akt protein was tested by Western blot assay. The cell viability was measured by cell counter kit-8(CCK-8 assay). The number of apoptotic cells was tested by Hoechst 33258 nuclear staining fol-lowed by photofluorography. The intracellular levels of reactive oxygen species ( ROS ) were detected by DCFH-DA staining followed by photofluorography. Mi-tochondrial membrane potential ( MMP ) was examined by JC-1 staining followed by photofluorography. Results H9c2 cells were treated with 35 mmol·L-1 glucose (high glucose, HG) for 0 ~24 h respectively. After treating for 3 h, the expression level of phosphorated ( p )-Akt protein began to be obviously reduced, the maximum reduced expression level was observed after the cells were exposed to HG for 24 h. Pretreatment of the cells with 50 μmol · L-1 pinacidil ( Pin, a KATP channel opener) or 400 μmol·L-1 NaHS( a donor of H2 S) prior to exposure to HG considerably blocked the down regulation of p-Akt expression level induced by HG. However, pretreatment with 1 mmol · L-1 KATP channel blocker glibenclamide( Gli) obviously attenua-ted the inhibitory effect of NaHS on HG-induced down-regulation of p-Akt expression level. On the other hand, the protective effects of NaHS against the HG-induced cardiomyocyte injury were markedly blocked by 30 μmol·L-1 Ly294002(an inhibitor of Akt), as indicated by the decrease in cell viability and MMP dissipation as well as the increases in the number of apoptotic cells and ROS generation. Conclution KATP channels-Akt pathway mediates the protective effect of H2 S against the HG-induced cardiac injury.

AIM:To investigate the roles of ATP-sensitive potassium ( KATP ) channels in high glucose-induced cardiac injury and the inhibitory effect of hydrogen sulfide ( H2 S) on the cardiomyocyte injury.METHODS:The expres-sion level of KATP channel protein was tested by Western blot.The cell viability was measured by CCK-8 assay.The number of apoptotic cells was observed by Hoechst 33258 nuclear staining.Mitochondrial membrane potential ( MMP) was exam-ined by JC-1 staining.RESULTS:After the H9c2 cells were treated with 35 mmol/L glucose ( high glucose, HG) for 1~24 h, the protein level of KATP channel was significantly reduced at 6 h, 9 h, 12 h and 24 h, reaching the minimum level at 12 h and 24 h.Pretreatment of the cells with 400μmol/L NaHS ( a donor of H2 S) prior to exposure to HG for 12 h con-siderably blocked the down-regulation of KATP channels induced by HG.Pretreatment of the cells with 100 μmol/L mito-chondrial KATP channel opener diazoxide, 50μmol/L non-selective KATP channel opener pinacidil or NaHS obviously inhibi-ted HG-induced injuries, leading to an increase in the cell viability, and decreases in the number of apoptotic cells and the MMP loss.Pretreatment with 100μmol/L mitochondrial KATP channel antagonist 5-hydroxydecanoic acid or 1 mmol/L non-selective KATP channel antagonist glibenclamide attenuated the above cardioprotective effects of NaHS.CONCLUSION:KATP channels mediate the inhibitory effect of H2 S on HG-induced cardiac injury.

Aim To investigate the role of the interac-tion between necroptosis ( Nec ) and p38 mitogen-acti-vated protein kinase ( MAPK) pathway in the high glu-cose (HG)-induced H9c2 cardiac cells injury.Meth-ods The cell viability was measured by cell counter kit-8 assay .The intracellular level of reactive oxygen species ( ROS ) was tested by DCFH-DA stating fol-lowed by photofluorography .Mitochondrial membrane potential ( MMP) was detected by Rhodamine 123 stai-ning followed by photofluorography . The expression levels of receptor interaction protein 3 ( RIP3, an indi-cator of Nec ) and p38 MAPK protein were tested by Western blot assay .Results The treatment of H9c2 cardiac cells with 35 mmol? L-1 glucose ( high glu-cose, HG) for 24 h induced considerable injuries , in-cluding a decrease in cell viability , increases in ROS generation as well as MMP loss .The co-treatment of the cells with 100 μmol? L-1 necrostatin-1(Nec-1,a specific inhibitor of Nec ) and HG for 24 h or the pre-treatment of the cells with 3 μmol? L-1 SB 2 0 3 5 8 0 ( an inhibitor of p38MAPK) for 60 min before HG exposure attenuated the above injuries induced by HG .Moreo-ver, the treatment of the cells with HG for 1,3,6,9, 12 ,24 ,36 and 48 h significantly increased the expres-sion levels of RIP3, peaking at 24 h.The co-treatment of the cells with 100 μmol? L-1 Nec-1 or the pre-treatment of the cells with 3 μmol? L-1 SB203580 considerably blocked the up-regulation of RIP3 expres-sion induced by HG .On the other hand , the co-treat-ment of the cells with 100 μmol? L-1 Nec-1 alleviated the HG-induced up-regulation of the expression of p-p38MAPK.Conclusion The interaction between Nec and p38 MAPK pathway mediates the HG-induced inju-ry in H9c2 cardiac cells.

AIM:To investigate whether the opening of ATP-sensitive K+(KATP) channels protects H9c2 car-diac cells against high glucose ( HG)-induced injury and inflammation by inhibiting the Toll-like receptor 4 ( TLR4 )/nu-clear factor-κB ( NF-κB) pathway.METHODS:The protein levels of TLR4 and NF-κB p65 were determined by Western blot.The levels of interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) were detected by ELISA.The cell viabil-ity was measured by CCK-8 assay.Mitochondrial membrane potential (MMP) was examined by rhodamine 123 (Rh 123) staining followed by photofluorography.The intracellular levels of reactive oxygen species ( ROS) were detected by 2′, 7′-
dichlorfluorescein-diacetate (DCFH-DA) staining followed by photofluorography.The number of apoptotic cells was ob-served by Hoechst 33258 nuclear staining followed by photofluorography.RESULTS: After the H9c2 cardiac cells were treated with HG (35 mmol/L glucose) for 24 h, the protein levels of TLR4 and phosphorylated NF-κB p65 ( p-NF-κB p65) were significantly increased.Pretreatment of the cells with 100 μmol/L diazoxide ( DZ, a KATP channel opener) for 30 min before exposure to HG considerably blocked the up-regulation of the TLR4 and p-NF-κB protein levels induced by HG.Moreover, co-treatment of the cells with 30 μmol/L TAK-242 (an inhibitor of TLR4) obviously inhibited the HG-in-duced up-regulation of the p-NF-κB p65 protein level.On the other hand, pretreatment of the cells with 100 μmol/L DZ had a clear myocardial protection effect, which attenuated the HG-induced cytotoxicity, inflammatory response, mitochon-drial damage, oxidative stress and apoptosis, evidenced by an increase in the cell viability, and decreases in the levels of IL-1βand TNF-α, MMP loss, ROS generation and the number of apoptotic cells.Similarly, co-treatment of H9c2 cardiac cells with 30μmol/L TAK-242 or 100μmol/L PDTC ( an inhibitor of NF-κB) and HG for 24 h also obviously reduced the above injuries and inflammation induced by HG.CONCLUSION: The opening of KATP channels protects H9c2 cardiac cells against HG-induced injury and inflammation by inhibiting the TLR4/NF-κB pathway.

AIM:To study whether hydrogen sulfide (H2S) protects H9c2 cardiomyocytes against high glucose ( HG)-induced injury by inhibiting necroptosis .METHODS:The protein levels of RIP3 ( an indicator of necroptosis ) and cleaved caspase-3 were determined by Western blot .The cell viability was measured by CCK-8 assay.The intracellular le-vels of reactive oxygen species (ROS) were detected by 2’, 7’-dichlorfluorescein diacetate staining followed by photofluo-rography.Mitochondrial membrane potential (MMP) was examined by rhodamine 123 staining followed by photofluorogra-phy.The number of apoptotic cells was observed by Hoechst 33258 nuclear staining followed by photofluorography .RE-SULTS:After the H9c2 cells were treated with HG (35 mmol/L glucose) for 0~24 h, the protein expression of RIP3 in the H9c2 cells was significantly increased at 3 h, 6 h, 9 h, 12 h and 24 h, reaching the maximum level at 24 h.Pretreat-ment of the cells with 400μmol/L NaHS (a donor of H2S) or co-treatment of the cells with necrostatin-1 (Nec-1;a speci-fic inhibitor of necroptosis) considerably blocked the up-regulation of RIP3 protein induced by HG.Moreover, pretreatment with NaHS or co-treatment with Nec-1 obviously inhibited HG-induced injuries , leading to an increase in the cell viability , and decreases in the generation of ROS and MMP loss .On the other hand , pretreatment with NaHS also reduced the num-ber of apoptotic cells and the protein level of cleaved caspase-3 in the HG-treated H9c2 cardiomyocytes .CONCLUSION:H2 S protects H9c2 cardiomyocytes against HG-induced injury by inhibiting necroptosis .