Liraglutide ameliorates high glucose-induced oxidative stress injury in rat H9c2 cells through modulation of SIRT1 / 中华内分泌代谢杂志

Objective:To investigate the effect of liraglutide(LRG) on high glucose-induced oxidative stress injury in(H9c2) cardiomyocytes and its underlying mechanisms.Methods:A high glucose treatment was applied to H9c2 cells for 24 hours to establish an in vitro model of myocardial cell injury. Different concentrations of liraglutide(10, 100, 1000 nmol/L) were administered for intervention. Cell viability was evaluated using the CCK-8 assay, and changes in cell morphology were observed under an inverted microscope. After 24 hours of liraglutide(100 nmol/L) intervention following high glucose treatment, the levels of lactate dehydrogenase(LDH), superoxide dismutase(SOD), and malondialdehyde(MDA) in the cell supernatant were measured. RT-PCR and Western blotting were used to detect the mRNA and protein levels of silent information regulator factor 1(SIRT1) and forkhead box protein O1(FOXO1). Western blotting was also used to assess the acetylation level of FOXO1 protein. Small interfering RNA(siRNA) technology was employed to silence SIRT1 in H9c2 cells to confirm its role in the study. Results:Compared to the control group, the high glucose group showed decreased cell viability, cell structure damage, increased levels of LDH and MDA in the cell supernatant, decreased SOD levels, aggravated oxidative stress, decreased SIRT1 expression, and increased acetylation level of FOXO1(all P<0.05). Compared to the high glucose group, liraglutide intervention resulted in increased cell viability, improved cardiac cell morphology, reduced oxidative stress levels, increased SIRT1 expression, and decreased acetylation level of FOXO1(all P<0.05). When SIRT1 was downregulated, the protective effects of liraglutide were weakened(all P<0.05). Conclusions:Liraglutide has a protective effect against high glucose-induced oxidative stress injury in H9c2 cells, which may be associated with the upregulation of SIRT1 expression.

To investigate the effect of Astragaloside Ⅳ on high-glucose-induced cardiomyocytes pyroptosis / 中华老年医学杂志

Objective:To investigate the effect of Astragaloside Ⅳ on high glucose-induced cardiomyocyte pyroptosis.Methods:H9c2 cells were cultured in vitro and divided into control group(5.5 mmol/L glucose), high glucose group(33.3 mmol/L glucose), Astragaloside Ⅳ group(33.3 mmol/L glucose+ 100μmol/L Astragaloside Ⅳ), and NLRP3 inhibitor group(33.3 mmol/L glucose+ 1μmol/L MCC950). Cell counting kit 8(CCK-8)was used to detect the activity of H9c2 cells.Lactate dehydrogenase(LDH)kit was used to detect the content of LDH in cell supernatant.Superoxide anion fluorescent probe(DHE)was used to detect the level of intracellular reactive oxygen species(ROS). Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and Western blot were used to detect the mRNA and protein expression levels of pyroptosis-related genes.Immunofluorescence was used to detect the fluorescence intensity of NLRP3.Enzyme-linked immunosorbent assay(ELISA)was used to detect the level of inflammatory factors in cell supernatant.Results:When the concentration of Astragaloside Ⅳ was 100 μmol/L, it could significantly inhibit the decrease of cardiomyocyte viability induced by high glucose( P<0.01)and reduce LDH release( P<0.01). Compared with the control group, the level of ROS was increased( P<0.01), the mRNA and protein expressions of pyroptosis-related molecules were up-regulated( P<0.01 for all), the fluorescence intensity of NLRP3 was increased( P<0.01), and the levels of inflammatory factors in the cell supernatant were increased in the high glucose group( P<0.01). Compared with the high glucose group, the ROS level was decreased( P<0.01), the mRNA and protein expressions of pyroptosis-related molecules were down-regulated( P<0.05 or P<0.01), the fluorescence intensity of NLRP3 was decreased( P<0.01), and the levels of inflammatory factors in cell supernatant were decreased( P<0.05 or P<0.01)in Astragaloside Ⅳ group and inhibitor group. Conclusions:Astragaloside Ⅳ plays a protective role in high glucose-induced cardiomyocyte injury by inhibiting NLRP3/Caspase-1 signaling pathway and inhibiting pyroptosis.Moreover, it can improve the anti-inflammatory and antioxidant properties in cell models.

Effect of high glucose on intracellular cholesterol content in rat glomerular mesangial cells / 中国医师杂志

Objective:To investigate the effect of high glucose on intracellular cholesterol content in rat glomerular mesangial cells and the underlying mechanism.Methods:Rat glomerular mesangial cells were cultured in vitro and divided into high glucose culture group (high glucose group, medium glucose concentration of 30 mmol/L) and normal glucose culture group (normal group, medium glucose concentration of 5.5 mmol/L). Lipid content was determined by oil red O staining and spectrophotometer colorimetry at 24, 36 and 48 h of culture. Intracellular protein imprinting was used to detect the expression of low density lipoprotein cholesterol receptor (LDLR) and adenosine triphosphate binding cassette A1 (ABCA1). Results:Oil red O staining showed that the intracellular lipid drops in the high glucose group were less than those in the normal group at 36 h, and there was no significant difference between the two groups at 24 h and 48 h of culture. The total cholesterol (TC) and cholesterol ester (CE) in glomerular mesangial cells of rats in the high glucose group were significantly lower than those in the normal group ( P=0.028, 0.029), while there was no significant difference in the free cholesterol (FC) between the two groups ( P=0.306). There was no significant difference in TC, CE and FC between the two groups at 24 and 48 h of culture (all P>0.05). The expression of LDLR in mesangial cells of rats in high glucose group was significantly lower than that in normal group at 24, 36 and 48 h of culture ( P=0.043, 0.004, 0.028), and the expression of ABCA1 was significantly higher than that in normal group at 24, 36 and 48 h of culture, ( P=0.050, 0.009, 0.006). Conclusions:High glucose may reduce intracellular cholesterol content in rat glomerular mesangial cells by reducing LDLR protein expression and increasing ABCA1 protein expression.

Antagonistic effect of SC79 on high glucose-induced apoptosis of RPE cells and its regulatory mechanism on AKT-XIAP signaling pathway / 中华实验眼科杂志

Objective:To investigate the antagonistic effect and potential mechanism of specific AKT activator SC79 on the apoptosis of human retinal pigment epithelial (ARPE)-19 cells induced by high glucose in vitro. Methods:The ARPE-19 cells were cultured in high glucose medium (containing 30 mmol/L glucose) plus 5, 10 or 20 μg/ml SC79, respectively.After 6-, 12- and 24-hour culture, the optimal experimental concentration and timing were determined according to cell proliferation rate.Then ARPE-19 cells were divided into four groups, normal control group cultured in normal medium containing 5.6 mmol/L glucose for 48 hours, mannitol group cultured in medium containing 5.6 mmol/L glucose and 24.4 mmol/L mannitol for 48 hours, high glucose group cultured in high glucose medium for 48 hours, and high glucose+ SC79 group cultured in normal medium containing 10 μg/ml SC79 for 12 hours plus in high glucose medium for 36 hours.The proliferation rate of APRE-19 cells was detected by MTS assay.The apoptosis rate was measured by flow cytometry.The relative expression levels of phosphorylated protein kinase B (p-Akt), X-linked inhibitor of apoptosis protein (XIAP), caspase-9, caspase-3 and its active fragments (active-caspase-3) were assayed by Western blot.The ARPE-19 cells were divided into Neg-shRNA group, AKT shRNA group and blank control group and were treated with the corresponding transfection complex and serum-free medium.The AKT mRNA expression was detected by real-time PCR.The transfected ARPE-19 cells were divided into Neg-shRNA+ SC79 group and AKT shRNA+ SC79 group and were cultured according to the culturing method of high-glucose+ SC79 group.The apoptosis rate of the two groups was tested by flow cytometry.Results:Among different concentrations of SC79 and treatment times, the proliferation rate of cells treated with 10 μg/ml SC79 for 12 hours was the highest.The proliferation rate of ARPE-19 cells in high-glucose group was significantly lower than that in normal control group, mannitol group and high-glucose+ SC79 group, and the differences were statistically significant (all at P<0.01). The apoptosis rate of cells in the high-glucose group was (52.27±3.21)%, which was significantly higher than (3.90±0.71)% in normal control group and (20.70±3.62)% in high-glucose+ SC79 group (both at P<0.01). The relative expression levels of p-Akt, XIAP, caspase-9 and caspase-3 were significantly lower and the relative expression level of active-caspase-3 was significantly higher in high glucose group than those in normal control group and high-glucose+ SC79 group (all at P<0.05). The relative expression level of AKT mRNA in normal control group, Neg-shRNA group and AKT shRNA group was 0.60±0.07, 0.59±0.03 and 0.11±0.10, respectively, showing a statistically significant difference among the groups ( F=30.44, P<0.01). The apoptosis rate of cells in the AKT shRNA+ SC79 group was significantly higher than that in high-glucose+ SC79 group and Neg-shRNA+ SC79 group (both at P<0.001). Conclusions:SC79 can partially antagonize the apoptosis of ARPE-19 cells induced by high glucose, which is related to the activation of AKT/XIAP pathway and the inhibition of the caspase family.

Role of ROS/TXNIP/NLRP3 signaling pathway in high glucose-induced pyroptosis of human embryonic trophoblast cells / 中华内分泌外科杂志

Objective:To investigate the changes of ROS/TXNIP/NLRP3 signaling pathway in pyroptosis of human embryonic trophoblast cells induced by high glucose.Methods:Human embryonic trophoblast cells were cultured in vitro to establish high glucose injury model, and they were randomly divided into control group, high glucose (HG) group and HG + ROS inhibitor N-acetyl-L-cysteine (HG + NAC) group. MTT assay was used to detect the cell survival rate. The level of ROS in each group was detected by dihydroethidine ROS fluorescence probe. Expression of TXNIP and NLRP3 mRNA was detected by real-time quantitative PCR (RT-qPCR). Western blot analysis was used to detect the expression levels of TXNIP, NLRP3, Caspase-1, interleukin (IL) -1β, tumor necrosis factor-α (TNF-α) and GSDMD proteins. In addition, pyroptosis was detected by flow cytometry.Results:The optimal glucose concentration for high glucose-induced injury of human embryonic trophoblast cells was 30 mmol/L. Compared with the control group (96.27±3.10) %, the survival rate of human embryonic trophoblast cells in HG group (55.44±2.15) % was significantly lower ( P<0.05), while the fluorescence intensity (ROS level) of 7 'dichlorofluorescein (DCF), the expression levels of TXNIP and NLRP3 proteins, the number of pyroptosis, expression levels of Caspase-1, GSDMD, IL-1β and TNF-α proteins were significantly higher ( P<0.05) ; Compared with HG group, the survival rate of human embryonic trophoblast cells in HG+NAC group (84.75±2.33) % was significantly higher ( P<0.05), the fluorescence intensity (ROS level) of DCF, the expression levels of TXNIP and NLRP3 proteins, the number of pyroptosis, and expression levels of Caspase-1, GSDMD, IL-1β and TNF-α proteins were significantly lower ( P<0.05) . Conclusion:Inhibition of ROS level in human embryonic trophoblast cells induced by high glucose may promote cell proliferation and reduce the occurrence of pyroptosis by inhibiting TXNIP/NLRP3 signaling pathway.

Effects of low-level laser on the expression of interleukin-6, tumor necrosis factor‑α, osteoprotegerin, and receptor activator of nuclear factor-κB ligand in human periodontal ligament cells / 华西口腔医学杂志

OBJECTIVES@#This study aims to determine the effects of low-level laser (LLL) on the expression of interleukin-6 (IL-6), tumor necrosis factor (TNF)-α, osteoprotegerin (OPG), and receptor activator of nuclear factor-κB ligand (RANKL) in human periodontal ligament cells (HPDLCs) stimulated by high glucose; and identify the molecular mechanism of LLL therapy in the regulation of periodontal inflammation and bone remodeling during orthodontic treatment in diabetic patients.@*METHODS@#HPDLCs were cultured in vitro to simulate orthodontic after loading and irradiated with LLL therapy. The cultured cells were randomly divided into four groups: low glucose Dulbecco's modification of Eagle's medium (DMEM)+stress stimulation (group A), high glucose DMEM+stress stimulation (group B), hypoglycemic DMEM+LLL therapy+stress stimulation (group C), and hyperglycemic DMEM+LLL therapy+stress stimulation (group D). Groups C and D were further divided into C1 and D1 (energy density: 3.75 J/cm2) and C2 and D2 (energy density: 5.625 J/cm2). Cells in groups A, B, C, and D were irradiated by LLL before irradiation. At 0, 12, 24, 48, and 72 h, the supernatants of the cell cultures were extracted at regular intervals, and the protein expression levels of IL-6, TNF-α, OPG, and RANKL were detected by enzyme-linked immunosorbent assay.@*RESULTS@#1) The levels of IL-6 and TNF-α secreted by HPDLCs increased gradually with time under static pressure stimulation. After 12 h, the levels of IL-6 and TNF-α secreted by HPDLCs in group A were significantly higher than those in groups B, C1, and C2 (P<0.05), which in group B were significantly higher than those in groups D1, and D2 (P<0.01). 2) The OPG protein concentration showed an upward trend before 24 h and a downward trend thereafter. The RANKL protein concentration increased, whereas the OPG/RANKL ratio decreased with time. Significant differen-ces in OPG, RANKL, and OPG/RANKL ratio were found among group A and groups B, C1, C2 as well as group B and groups D1, D2 (P<0.05).@*CONCLUSIONS@#1) In the high glucose+stress stimulation environment, the concentrations of IL-6 and TNF-α secreted by HPDLCs increased with time, the expression of OPG decreased, the expression of RANKL increased, and the ratio of OPG/RANKL decreased. As such, high glucose environment can promote bone resorption. After LLL therapy, the levels of IL-6 and TNF-α decreased, indicating that LLL therapy could antagonize the increase in the levels of inflammatory factors induced by high glucose environment and upregulate the expression of OPG in human HPDLCs, downregulation of RANKL expression in HPDLCs resulted in the upregulation of the ratio of OPG/RANKL and reversed the imbalance of bone metabolism induced by high glucose levels. 2) The decrease in inflammatory factors and the regulation of bone metabolism in HPDLCs were enhanced with increasing laser energy density within 3.75-5.625 J/cm2. Hence, the ability of LLL therapy to modulate bone remodeling increases with increasing dose.

miR-148a-3p improves mitochondrial injury and apoptosis induced by high-glucose through inhibiting CRT expression / 西安交通大学学报(医学版)

【Objective】 To evaluate the effects of miR-148a-3p on calreticulin (CRT) expression and mitochondrial function in cardiomyocytes incubated with high glucose. 【Methods】 miR-148a-3p minic and inhibitor were used to intervene the H9c2 cardiomyocytes of rats. The expression of CRT protein was detected. Then the cells were divided into control group, high-glucose group (HG), HG +miR-148a-3p minic group, HG + miR-148a-3p minic + TG (CRT agonist) group, HG + miR-148a-3p inhibitor group, and HG + miR-148a-3p inhibitor + CRT- (CRT-siRNA) group. The content of adenosine triphosphate (ATP) and the level of reactive oxygen species (ROS), the activity of mitochondrial respiratory chain complex enzyme and apoptotic rate were detected. 【Results】 miR-148a-3p minic significantly inhibited the expression of CRT protein in cardiomyocytes, while miR-148a inhibitor increased the expression of CRT. miR-148a-3p minic inhibited the decrease of ATP production, the increase of ROS production and cell apoptosis, and the inactivity of mitochondrial respiratory chain complex enzyme in cardiomyocytes induced by high glucose, while TG weakened the above effects of miR-148a-3p minic. miR-148a inhibitor aggravated the mitochondrial injury and apoptosis of cardiomyocytes induced by high glucose, but the effects of miR-148a-3p inhibitor were partially blocked by CRT-siRNA. 【Conclusion】 miR-148a-3p negatively regulates the expression of CRT in cardiomyocytes and protects the mitochondrial injury and apoptosis induced by high-glucose through inhibiting CRT.

Effects of DNMT3A on mouse cardiac fibroblast proliferation and migration under high glucose environment / 中国药理学通报

Aim To investigate the effect of DNA methyltransferase 3A (DNMT3A) on the proliferation and migration of cardiac fibroblasts (CFs) in C57 mice under high glucose environment. Methods The hearts of C57 mice were taken from 1 to 3 days. After cutting and digesting, CFs were extracted by differential adherance centrifugattion and observed under microscope. After cell attachment, the cells were cultured under low glucose (5.5 mmol • L

The protective effect of terpinen-4-ol on inflammatory injury of VSMCs induced by high glucose through improving autophagic flux disorder / 中国药理学通报

Aim To investigate the effect of terpinen-4-ol (T40) on inflammatory injury of vascular smooth muscle cells (VSMCs) induced by high glucose based on the improvement of autophagic flow disorder and involved molecular signals. Methods The scratch test was used to analyze the migration ability of VSMCs, the levels of IL-1β and IL-6 in cell culture supernatant were measured by ELISA, the expression levels of inflammation-related proteins NF-κb p65, p-NF-κb p65, IL-1β, IL-18 and autophagy-related proteins p62, LC3-HYLC3-I, Beclinl, p-Beclinl were de-tected by Western blot. Results T40 inhibited migration of VSMCs induced by high glucose, reduced the secretion and release of pro-inflammatory factors IL-1β and IL-6, inhibited the expression of p-NF-κb p65/ NF-κb p65, IL-1β, IL-18, downregulated the expression of p62, LC3-TJ/LC3- I and p-Beclinl at same time. After interfering the autophagic flux of VSMCs with autophagy inhibitor chloroquine (CQ) , T40 pre-treatment significantly inhibited the protein expression levels of the above inflammatory factors and autophagy-related signals which mediated by CQ. Conclusion T40 inhibits the inflammatory injury of VSMCs induced by high glucose through improving the autophagic flow disorder.

Terpinen-4-ol inhibits proliferation of VSMCs exposed to high glucose via regulating KLF4/NF-κB signaling pathway / 中国中药杂志

This study aimed to observe the effect of terpinen-4-ol(T4O) on the proliferation of vascular smooth muscle cells(VSMCs) exposed to high glucose(HG) and reveal the mechanism via the Krüppel-like factor 4(KLF4)/nuclear factor kappaB(NF-κB) signaling pathway. The VSMCs were first incubated with T4O for 2 h and then cultured with HG for 48 h to establish the model of inflammatory injury. The proliferation, cell cycle, and migration rate of VSMCs were examined by MTT method, flow cytometry, and wound healing assay, respectively. The content of inflammatory cytokines including interleukin(IL)-6 and tumor necrosis factor-alpha(TNF-α) in the supernatant of VSMCs was measured by enzyme-linked immunosorbent assay(ELISA). Western blot was employed to determine the protein levels of proliferating cell nuclear antigen(PCNA), Cyclin D1, KLF4, NF-κB p-p65/NF-κB p65, IL-1β, and IL-18. The KLF4 expression in VSMCs was silenced by the siRNA technology, and then the effects of T4O on the cell cycle and protein expression of the HG-induced VSMCs were observed. The results showed that different doses of T4O inhibited the HG-induced proliferation and migration of VSMCs, increased the percentage of cells in G_1 phase, and decreased the percentage of cells in S phase, and down-regulated the protein levels of PCNA and Cyclin D1. In addition, T4O reduced the HG-induced secretion and release of the inflammatory cytokines IL-6 and TNF-α and down-regulated the expression of KLF4, NF-κB p-p65/NF-κB p65, IL-1β, and IL-18. Compared with si-NC+HG, siKLF4+HG increased the percentage of cells in G_1 phase, decreased the percentage of cells in S phase, down-regulated the expression of PCNA, Cyclin D1, and KLF4, and inhibited the activation of NF-κB signaling pathway. Notably, the combination of silencing KLF4 with T4O treatment further promoted the changes in the above indicators. The results indicate that T4O may inhibit the HG-induced proliferation and migration of VSMCs by down-regulating the level of KLF4 and inhibiting the activation of NF-κB signaling pathway.

miR-519d-3p alleviates high glucose-induced human retinal microvascular endothelial cells dysfunction and inhibits angiogenesis by targeting hypoxia inducible factor 1 subunit alpha / 国际眼科杂志(Guoji Yanke Zazhi)

AIM:To clarify the effect of miR-519d-3p on high glucose-induced human retinal microvascular endothelial cells(HRMEC)dysfunction and angiogenesis, and to elucidate the regulatory mechanism of miR-519d-3p on hypoxia inducible factor 1 subunit alpha(HIF-1α).METHODS: The normal glucose(NG)and high glucose(HG)cell models were established by inducing HRMEC with 5 and 30 mmol/L glucose, respectively. Control group: HG cell model was transfected with negative control mimics; mannitol group: the control group was added with 25 mmol/L mannitol; miR-519d-3p overexpression group: HG cell model was transfected with miR-519d-3p mimics; miR-519d-3p combined with HIF-1α overexpression group: HG cell model was co-transfected with miR-519d-3p mimics and HIF-1α overexpression vector. The expression of miR-519d-3p in each group was tested by real-time fluorescence quantitative PCR. The expression of HIF-1α protein in each group was tested by Western blotting. The binding sites between miR-519d-3p and HIF-1α were detected by luciferase reporter gene assay. The cell proliferation of each group was detected by CCK-8. The cell apoptosis of each group was tested by Hoechst 33342 staining. The protein expression of extracellular fluid inflammatory factors tumor necrosis factor-α(TNF-α), interleukin(IL)-1β and IL-6 in each group was tested by ELISA. The formation of new capillary lumen-like structures was detected by tubule formation assay.RESULTS: Compared with the NG, miR-519d-3p expression was significantly reduced in the HG cell model, while HIF-1α protein expression was significantly increased in the HG(all P&#x003C;0.01). Compared with the control group, HIF-1α protein expression was significantly reduced in the miR-519d-3p overexpression group(P&#x003C;0.01). The “CGUGAAA” sequence of miR-519d-3p could specifically bind to the “GCACUUU” sequence of HIF-1α 3'-untranslated region(3'-UTR). Compared with the control group, the miR-519d-3p overexpression group showed a significant increase in 24, 48 and 72h absorbance values, a significant decrease in cell apoptotic rate, a significant decrease in the concentrations of TNF-α, IL-1β and IL-6, and a significant decrease in the number of new capillary lumen-like structures(all P&#x003C;0.01). Compared with the miR-519d-3p overexpression group, the miR-519d-3p combined with HIF-1α overexpression group showed a significant decrease in 24, 48 and 72h absorbance values, a significant increase in cell apoptotic rate, a significant increase in the concentrations of TNF-α, IL-1β and IL-6, and a significant increase in the number of new capillary lumen-like structures(all P&#x003C;0.01). There was no difference between the control group and mannitol group in the comparison of the above indicators(all P&#x003E;0.05).CONCLUSION: miR-519d-3p expression is down-regulated while HIF-1α protein expression is up-regulated in high glucose induced HRMEC model. HIF-1α is a target gene of miR-519d-3p. The miR-519d-3p targets HIF-1α to increase cell proliferation and reduce cell apoptosis and inflammation, thereby alleviating high glucose-induced HRMEC dysfunction and inhibiting angiogenesis.

Red light-emitting diode light mediated by the KEAP1-NRF2/HO-1 pathway induces osteogenic differentiation and attenuates the oxidative stress damage of human periodontal ligament stem cells induced by high glucose / 口腔疾病防治

Objective@# To explore the effects of red LED light mediated by the Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (KEAP1-NRF2/HO-1) pathway on osteogenic differentiation and oxidative stress damage of human periodontal ligament stem cells (hPDLSCs) induced by high glucose, which provides a basis for the application of red light-emitting diode (LED) light in cell antioxidative damage.@*Methods@#hPDLSCs were identified by flow cytometric analysis, alkaline phosphatase (ALP) staining and Alizarin red-S staining; hPDLSCs were pretreated in a high glucose environment for 48 hours and irradiated with 1, 3, or 5 J/cm2 red LED light. A CCK-8 assay was performed to choose the radiant exposure that had the strongest effect on promoting the cell proliferation rate for subsequent experiments. hPDLSCs were divided into a control group, a high glucose group and a high glucose+light exposure group. ALP staining, ALP activity, Alizarin red-S staining and quantitative calcified nodules were used to detect the osteogenic differentiation of hPDLSCs; qRT-PCR and Western blot were used to detect the gene and protein expression levels of ALP, runt-related transcription factor 2 (RUNX2) and osterix (OSX); the relative mRNA expression levels of antioxidant enzyme-related genes superoxide dismutase 2 (SOD2) and catalase (CAT) in hPDLSCs were detected by qRT-PCR; reactive oxygen species (ROS) levels were detected by fluorescence microscopy and flow cytometry; the tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) levels in cell supernatants were detected by ELISA; the NRF2-specific inhibitor ML385 was used to inhibit the NRF2 pathway; ALP staining and ALP activity were used to detect the markers of early osteogenic differentiation; qRT-PCR was used to detect the gene expression of ALP, RUNX2 and OSX; and the protein expression levels of KEAP1, NRF2 and HO-1 were detected by Western blot.@*Results @# Identified, and irradiant exposure of 5 J/cm2 was chosen for subsequent experiments. Red LED light irradiation (5 J/cm2) improved the osteogenic differentiation of hPDLSCs induced by high glucose (P<0.05), increased the mRNA and protein levels of ALP, RUNX2 and OSX (P<0.05), upregulated the mRNA expression levels of SOD2 and CAT (P<0.05), reduced the levels of ROS (P<0.05), and reduced TNF-α and IL-1β levels in the cell supernatants (P<0.05). When ML385 was added to inhibit the NRF2 pathway, the ALP activity of cells was decreased (P<0.05); the gene expression levels of ALP, RUNX2 and OSX were downregulated (P<0.05); the protein level of KEAP1 was upregulated (P<0.05); and the protein levels of NRF2 and HO-1 were downregulated (P<0.05)@*Conclusion@#Red LED light may promote the proliferation and osteoblastic differentiation of hPDLSCs induced by high glucose through the KEAP1-NRF2/HO-1 pathway and reduce the oxidative stress damage to hPDLSCs induced by high glucose.

Teriparatide regulates osteoblast differentiation in high-glucose microenvironment through the cAMP/PKA/CREB signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effect of teriparatide on the differentiation of MC3T3-E1 cells in high-glucose microenvironment and explore the possible mechanism.@*METHODS@#MC3T3-E1 cells cultured in normal glucose or high-glucose (25 mmol/L) medium were treated with 10 nmol/L teriparatide with or without co-treatment with H-89 (a PKA inhibitor). CCK-8 assay was used to detect the changes in cell proliferation, and cAMP content in the cells was determined with ELISA. Alkaline phosphatase (ALP) activity and mineralized nodules in the cells were detected using ALP kit and Alizarin red staining, respectively. The changes in cell morphology were detected by cytoskeleton staining. Real-time PCR was used to detect the mRNA expressions of PKA, CREB, RUNX2 and Osx in the treated cells.@*RESULTS@#The treatments did not result in significant changes in proliferation of MC3T3-E1 cells (P > 0.05). Compared with the cells in routine culture, the cells treated with teriparatide showed significantly increased cAMP levels (P < 0.05) with enhanced ALP activity and increased area of mineralized nodules (P < 0.05). Teriparatide treatment also resulted in more distinct visualization of the cytoskeleton in the cells and obviously up-regulated the mRNA expressions of PKA, CREB, RUNX2 and Osx (P < 0.05). The opposite changes were observed in cells cultured in high glucose. In cells exposed to high glucose, treatment with teriparatide significantly increased cAMP levels (P < 0.05), ALP activity and the area of mineralized nodules (P < 0.05) and enhanced the clarity of the cytoskeleton and mRNA expressions of PKA, CREB, RUNX2 and Osx; the effects of teriparatide was strongly antagonized by co-treatment with H-89 (P < 0.05).@*CONCLUSION@#Teriparatide can promote osteoblast differentiation of MC3T3-E1 cells in high-glucose microenvironment possibly by activating the cAMP/PKA/CREB signaling pathway.

Effect of ROS/NLRP3/Caspase-1 signaling pathway on the apoptosis of retinal pigment epithelial cells induced by high glucose / 中华实验眼科杂志

Objective:To investigate the effect of the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3 (NLRP3) inflammasome on the proliferation and apoptosis of human retinal pigment epithelial cell line ARPE-19 exposed to high glucose and its mechanism.Methods:ARPE-19 cells cultured in vitro were divided into normal control group and high-glucose group, and were cultured in conventional medium and medium containing 30 mmol/L glucose for 48 hours, respectively.The content of reactive oxygen species (ROS) were detected by fluorescent probe, and the activity of superoxide dismutase (SOD) and the concentration of malondialdehyde (MDA) were tested by biochemical assay.The cells of the two groups were cultured with 0, 2, 5, 10, 15 and 20 μmol/L NLRP3 inhibitor CY-09 for 48 hours, respectively.The proliferation rate of ARPE-19 cells under various concentrations of CY-09 treatment was detected by cell counting kit-8, and the appropriate concentration of CY-09 was determined.ARPE-19 cells were divided into normal control group, normal+ CY-09 group, high-glucose group and high glucose+ CY-09 group.The culture medium in the normal+ CY-09 group and high glucose+ CY-09 group was supplemented with 15 μmol/L CY-09.Flow cytometry was used to detect the apoptosis rate of each group, and Western blot was used to detect the relative expression levels of NLRP3, apoptosis-associated point protein (ASC), Caspase-1 precursor (pro-Caspase-1) and active fragments (cleaved-Caspase-1), B lymphocytoma-2 protein (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3 precursor (pro-Caspase-3) and active fragments (cleaved-Caspase-3). Results:The intensity of ROS fluorescence and MDA concentration were 120 020±3 245, (4.92±0.09) nmol/mg in the high-glucose group, which were both significantly higher than 35 426±811 and (1.78±0.03) nmol/mg in the normal control group, and the SOD activity was (35.65±1.22) μmol/(min·mg) in the high-glucose group, which was significantly lower than (74.96±1.41) μmol/(min·mg) in the normal control group, showing statistically significant differences between the two groups ( t=35.760, 46.960, 29.830; all at P<0.05). The proliferation rate of RPE cells in high-glucose group was significantly lower than that in normal control group, and the difference was statistically significant ( t=18.820, P<0.05). With the increase of CY-09 concentration, the proliferation rate of cells in the high-glucose group was gradually increased.The proliferation rates of cells treated with 10, 15 and 20 μmol/L CY-09 were all significantly higher than those treated with 0 μmol/L CY-09, showing statistically significant differences between them (all at P<0.05). The proliferation rates of cells treated with 15 μmol/L and 0 μmol/L CY-09 were not significantly different in the normal control group ( P>0.05). The apoptosis rate of cells in the high-glucose group was (21.68±0.41)%, which was significantly higher than (6.67±1.05)% in the normal control group and (13.96±0.07)% in the high-glucose+ CY-09 group, and the differences were statistically significant (both at P<0.05). The relative expression levels of NLRP3, ASC, cleaved-Caspase-1, cleaved-Caspase-3 and Bax proteins were significantly higher and the relative expression levels of Bcl-2 protein was significantly lower in the high-glucose group compared with the normal control group, and the differences were statistically significant (all at P<0.05). The relative expression levels of NLRP3, ASC, the active fragment of cleaved-Caspase-1, Bax and cleaved-Caspase-3 proteins were decreased and the relative expression levels of Bcl-2 protein were increased in the normal+ CY-09 group and high glucose+ CY-09 group compared with the normal control group and high glucose group, and the differences were statistically significant (all at P<0.05). Conclusions:NLRP3 inflammasome mediates the high glucose induced RPE cells apoptosis through ROS/NLRP3/Caspase-1 signaling pathway.

Liraglutide Inhibits High Glucose⁃induced Injury of Mouse Islet Cells / 中国生物化学与分子生物学报

Liraglutide is an analog of glucagon⁃like peptide⁃1 and plays an important role in the treatment of diabetes. But the specific mechanism of liraglutide in improving the function of pancreatic β cells is still unclear. Therefore, high glucose (33 mmol/ L) was used to induce islet MIN6 cells for 48 hours to establish a high glucose injury model in this study. The CCK⁃8 assay was used to detect cell viability, and the results showed that the viability of MIN6 cells in the high glucose group decreased (P<0. 05) compared with the control group, and the cell viability increased after liraglutide treatment (P<0. 05). Using the mouse insulin detection kit and ATP content detection kit, we found that both the insulin re⁃ lease (P<0. 01) and ATP content decreased (P<0. 001) in the high glucose group compared with the control group, and after liraglutide treatment both insulin release (P < 0. 05) and ATP content (P < 0. 001) increased. We used the mitochondrial membrane channel pore (MPTP) fluorescence detection kit in MIN6 cells and found that the green fluorescence intensity of the high glucose group was significant⁃ ly decreased (P<0. 001) compared with the control group, and after liraglutide treatment the green fluo⁃ rescence intensity was significantly increased (P<0. 001). The DCFH⁃DA probe combined with flow cy⁃ tometry was used to detect the level of reactive oxygen species (ROS). We found that compared with the control group, the ROS level in the high glucose group was significantly increased (P<0. 001), and de⁃ creased by liraglutide treatment (P<0. 01). Intracellular malondialdehyde (MDA) contents, superoxide dismutase (SOD) and catalase (CAT), and lactate dehydrogenase (LDH) activities in the cell superna⁃ tant were measured, and the levels of MDA and LDH were significantly increased (P<0. 05), and the levels of SOD and CAT were significantly decreased (P<0. 01) in the high glucose group compared with the control group. After liraglutide treatment, the levels of MDA and LDH were decreased (P<0. 05), and the levels of SOD and CAT were increased (P<0. 05). The results of Western blotting showed that the expression of UCP2 was upregulated in the high glucose group (P<0. 01) compared with the control group, and after liraglutide treatment the expression of UCP2 decreased (P<0. 05). The results indica⁃ ted that liraglutide has significant effects in high⁃glucose induced mitochondrial damage, oxidative stress and insulin secretion in MIN6 cells, which will provide a theoretical basis for clinical utilization of liraglu⁃ tide.

Genipin Protects High Glucose-injured MIN6 Cells / 中国生物化学与分子生物学报

Genipin (Gen) is an important antioxidant that plays a crucial role in the process of intracellular resistance to oxidative stress. In order to study the effect of genipin on MIN6 cells injured by high glucose, the CCK-8 method was used to detect the cell survival rate. The cell viability of the high-glucose injury group decreased (P <0. 05). But after genipin acted on the cells injured by high glucose, the cell viability increased (P <0. 05). The mouse insulin detection kit and ATP content detection kit found that the insulin release in the high glucose injury group decreased (P < 0. 001) and the ATP content decreased (P <0. 001). After genipin was given, the insulin release increased (P <0. 01), ATP content increased (P <0. 01). The fluorescent probe DCFH-DA detected the intracellular reactive oxygen species (ROS) level and found that the ROS content in the high glucose-injury group was significantly increased (P <0. 01). After genipin was administered, ROS content decreased (P < 0. 05). Glutathione(GSH) / oxidized glutathione (GSSG), intracellular malondialdehyde (MDA), superoxide dismutase(SOD) and catalase (CAT) and lactate dehydrogenase (LDH) activity in the cell supernatant revealed that the GSH / GSSH ratio, SOD and CAT levels in the high glucose injury group decreased (P <0. 05), and the intracellular MDA and LDH levels were significant increased (P<0. 001) .After administration of genipin, the GSH / GSSH ratio, SOD and CAT levels increased (P <0. 01), MDA and LDH levels were significantly reduced (P <0. 01). Mitochondrial membrane potential (MMP) levels decreased in the highglucose injury group (P <0. 01). After genipin acted on the cells injured by high glucose, the MMP level increased (P < 0. 05). Western blot detected uncoupling protein 2 (UCP2), antioxidative proteinsglutathione reductase (GR) and glutaredoxin 1 (Grx1) contents. The results showed that UCP2 contents in the high glucose injury group increased (P <0. 01) and the content of oxidized protein decreased (P < 0. 01). After genipin was administered, the expression of UCP2 decreased (P < 0. 05), and the expression of antioxidative protein increased (P < 0. 05). Experiments suggest that genipin has anantioxidant protective effect on MIN6 cells damaged by high glucose and maintains the function of MIN6cells to promote insulin secretion. This experiment provides experimental data for the antioxidation of genipin on MIN6 cells injured by high glucose, and also provides new ideas for the follow-up study of genipin in the treatment and prevention of diabetes.

PPARfJ involved in endothelial cell injury induced by high glucose via nitrative stress / 中国药理学通报

Aim To investigate the role of PPARβ and nitrative stress in human umbilical vein endothelial cells(HUVECs)injury induced by high glucose.Methods The cell viability was detected by CCK-8.The cell proliferation was detected by EdU proliferation detection kit.The protein expression level of PPARβ,eNOS,iNOS,and 3-nitrotyrosine was detected by Western blot.The content of peroxynitrite and nitric oxide(NO)was determined by peroxynitrite kit and Griess Reagent,respectively.Results Glucose(30,40,50 mmol·L-1)significantly reduced the cell viability of HUVECs in a dose-dependent manner.Glucose at 30 mmol·L-1(high glucose,HG)significantly reduced the proliferation of HUVECs,down-regulated the expression of PPARβ,eNOS at protein level and NO content,and increased iNOS,3-nitrotyrosine protein expression and peroxynitrite level.The above effects of HG were reversed by PPARβ agonist GW0742(1 μmol·L-1).Both PPARβ antagonist GSK0660(1 μmol·L-1)and NOS inhibitor L-NAME(10 μmol·L-1)blocked the protective effects of GW0742.Conclusion The down-regulation of PPARβ is involved in the injury of HUVECs induced by high glucose,which may be mediated,at least partly,by the stimulation of nitrative stress.

Role of FOXM1 in collagen synthesis in MRC-5 cells induced by high glucose / 中国药理学通报

Aim To explore the role of transcription factor F0XM1 in collagen synthesis in MRC-5 cells in-duced by high glucose.Methods I ▪ The optimal time and concentration of the hyperglycemia model in MRC-5 cells were explored by CCK8: the time gradi¬ents: 6,12,24,48,72 h; the concentration gradients: 5.5,15 ,30,45 mmol • L 1 , and 30 mmol • L 1 man- nitol was used to be the hypertonic control group.(2) Hie effect of collagen synthesis in MRC-5 cells induced by high glucose was detected: the cells were divided into normal control group, hypertonic control group (30 mmol • L 1 mannitol) and high glucose (30 mmol • L 1 ) group.WB and qPCR assays were used to de¬tect the expression of conllagen synthesis factors ( Fn, COL 1, COL m, a-SMA, MMP9, TIMP1 ) and TGF-p signaling pathway factors (TGF-pi , p-Smad2/ Smad2).(3 The role of FOXMl in promoting collagen synthesis by high glucose was investigated: the cells were divided into normal control group, hypertonic control group (30 mmol • L 1 mannitol) , high glucose ( 30 mmol • L 1 ) group and high glucose (30 mmol • L 1 ) + thiostrepton ( 1 (xmol) group, and the expres¬sions of FOXM1 , collagen synthesis factors were detec¬ted by WB and qPCR assays.Results Mannitol had no significant effect on proliferation of MRC-5 cells, hut their proliferation activity was significantly lower than that of control group when MRC-5 cells were trea¬ted with 30 mmol • L 1 high glucose for 24 h; the ex¬pressions of COL 1 , COL IH , F0XM1 and other fac¬tors were promoted when MRC5 cells were treated with high glucose; the expression of F0XM1 was signifi¬cantly inhibited after the addition of thiostrepton, and the expressions of collagen synthesis factors also de-creased compared with high glucose group, and the a- bove differences were all statistically significant (P < 0.05).Conclusion FOXM1 is a factor related to the increase of collagen synthesis in MRC-5 cells induced by high glucose.

Effect of Dapagliflozin on high glucose-induced apoptosis and oxidative stress in human retinal vascular endothelial cells / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To explore the effect of dapagliflozin on the apoptosis and oxidative stress of high glucose-induced human retinal vascular endothelial cells and its regulatory effect on forkhead FOXO4. <p>METHODS: High glucose-induced human retinal vascular endothelial cells(HRVECs)were used to establish a cell injury model(high glucose group). Experimental groups include high glucose+dapagliflozin low-dose group(1ng/L dapagliflozin), high glucose+dapagliflozin medium-dose group(5ng/L dapagliflozin), high glucose+dapagliflozin high-dose group(10ng/L dapagliflozin), high glucose+dapagliflozin high-dose+pcDNA group, high glucose+dapagliflozin high-dose+pcDNA-FOXO4 group, and normal sugar group(5.5mmol/L D-glucose). Flow cytometry was used to detect the apoptosis rate. The levels of superoxide dismutase(SOD)and malondialdehyde(MDA)were tested with corresponding kits. Western blot assay was used to detect the protein level of FOXO4. <p>RESULTS: Compared with the normal sugar group, the apoptosis rate(<i>P</i><0.05), the level of MDA(<i>P</i><0.05)and FOXO4(<i>P</i><0.05)were increased, but the level of SOD was decreased(<i>P</i><0.05)in high-glucose group. Compared with the high glucose group, cell apoptosis rate(<i>P</i><0.05), the level of MDA(<i>P</i><0.05)and the protein level of FOXO4 were decreased(<i>P</i><0.05), but the level of SOD was increased(<i>P</i><0.05)in high glucose+medium-dose dapagliflozin group and high glucose+high-dose dapagliflozin group. Compared with high glucose+dapagliflozin high-dose+pcDNA group, the apoptosis rate(<i>P</i><0.05)and the level of MDA(<i>P</i><0.05)were increased, but the level of SOD was decreased(<i>P</i><0.05)in high glucose+dapagliflozin high-dose+pcDNA-FOXO4 group(<i>P</i><0.05). <p>CONCLUSION: Dapagliflozin could inhibit oxidative stress and cell apoptosis in high glucose-induced HRVECs by down-regulating FOXO4, thereby reducing cell damage.

Mechanism of Didangtang Against Inflammatory Cascade Triggered by Foam Cell Pyroptosis in High-glucose Environment / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Didangtang （DDT） against the inflammatory cascade triggered by foam cell pyroptosis in high-glucose environment. MethodOxidized low density lipoprotein （ox-LDL， 100 mg·L-1） was used to induce pyroptosis of foam cells. The control group （5.5 mmol·L-1 glucose）， foam cell group （100 mg·L-1 ox-LDL）， high-glucose group （33.3 mmol·L-1 glucose）， DDT group （10% DDT-containing serum）， and NOD-like receptor family pyrin domain-containing 3 （NLRP3） inhibitor group （MCC950， 10 nmmol·L-1） were designed. The cell membrane damage was observed by lactate dehydrogenase （LDH） release assay. The expression of cysteinyl aspartate-specific proteinase-1 （Caspase-1） was detected by immunofluorescence method， and expression of key proteins NLRP3， Caspase-1， gastermin D （GSDMD）， interleukin-1β （IL-1β）， and interleukin-18 （IL-18） in the pyroptosis pathway was determined by Western blot. The release of IL-18 and IL-1β， monocyte chemoattractant protein-1 （MCP-1）， and tumor necrosis factor α （TNF-α） in cell supernatant was measured by enzyme-linked immunosorbent assay （ELISA）. ResultThe expression of NLRP3， Caspase-1， and GSDMD was up-regulated （P<0.01） and the release of IL-1β， IL-18， MCP-1， IL-1α， and TNF-α was increased （P<0.01） in foam cell group compared with those in the control group. The expression of NLRP3， Caspase-1， and GSDMD was higher （P<0.01） and the release of inflammatory factors was more （P<0.01） in the high-glucose group than in the foam cell group. DDT and MCC950 can inhibit expression of NLRP3， Caspase-1， GSDMD and reduce the release of IL-1β， IL-18， MCP-1， IL-1α， and TNF-α. ConclusionDDT can suppress the pyroptosis of foam cells induced by NLRP3/Caspase-1 pathway in high-glucose environment and thereby alleviate the inflammatory cascade.