Pyroptosis mediates high glucose-induced inflammation and injury in MC3T3-E1 osteoblasts

AIM To investigate whether pyroptosis contributes to the inflammation and injury in mouse embryonic osteoblastic cell line MC3T3-E1 induced by high glucose (HG; 45 mmol/L glucose). METHODS The cell viability was measured by CCK-8 assay. The protein expression levels of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) and caspase-1 (CASP1) were determined by Western blot. The secretion levels of interleukin-18 (IL-18) and IL-1β were measured by ELISA. The intracellular level of reactive oxygen species (ROS) was detected by 2',7'-dichlorodihydrofluorescein diacetate staining followed by photofluorography. Mitochondrial membrane potential (MMP) was examined by rhodamine 123 staining followed by photofluorography. The alkaline phosphatase (ALP) activity was determined using the ALP kit, and the number of mineralized nodules was detected by alizarin red S staining. RESULTS After the MC3T3-E1 osteoblasts were treated with HG for 24 h, the protein expression levels of NLRP3 and CASP1, and the secretion levels of IL-18 and IL-1β were significantly increased. The decrease in cell viability, and the increases in ROS generation and MMP loss were also observed. Moreover, the differentiation and mineralization of MC3T3-E1 osteoblasts were inhibited, evidenced by decreases in both ALP activity and mineralized nodule number. Knockdown of CASP1 by siRNA attenuated the HG-induced osteoblast inflammation and injury mentioned above. CONCLUSION Pyroptosis mediates HG-induced inflammation and injury in MC3T3-E1 osteoblasts.     

Importance of mitochondrial calcium uniporter in high glucose-induced cardiac myocyte apoptosis

AIM: To investigate the role of mitochondrial calcium uniporter (MCU) in high glucose(HG)-induced apoptosis of cardiac myocytes. METHODS: Cardiac myocytes were exposed to normal glucose (5.5 mmol/L glucose+ 19.5 mmol/L mannitol), HG (25 mmol/L glucose), or HG combined with 5 μmol/L spermine for 72 h. Mitochondrial free Ca²⁺ concentration ([Ca²⁺]_m), MCU at mRNA and protein levels, pyruvate dehydrogenase (PDH) activity, mitochondrial membrane potential (Δψ_m), the levels of ATP and reactive oxygen species (ROS), and apoptosis were determined. RESULTS: The [Ca²⁺]_m, the mRNA and protein levels of MCU, PDH activity, ATP levels, and Δψ_m were reduced (P<0.05), while ROS content and the protein levels of caspase-9 and caspase-3 were increased in HG group (P<0.05). Adding 5 μmol/L spermine returned these parameters toward control levels (P<0.05). Moreover, apoptosis was reduced by adding spermine and HG treatment (P<0.05). CONCLUSION: HG-induced cardiac myocyte apoptosis may be associated with the decreased MCU expression and activity, abnormal mitochondrial Ca²⁺ handling, deviant mitochon-drial respiratory chain, and mitochondrial dysfunction.     

Role of chlorogenic acid in endothelial dysfunction in db/db mice

AIM: To explore the effects of chlorogenic acid (CGA) on endothelial dysfunction in db/db mice and the possible mechanism. METHODS: Male db/db mice (n=12) were divided into control group and CGA group, with 6 mice in each group. The mice in CGA group were treated with diet containing 0.02% CGA, while the mice in control group were given normal diet only. The observation period was 12 weeks. Fasting blood glucose level, tail blood pressure and the body weight were analyzed each week. At the end of the 12th week, the mice were anesthetized and blood was taken from carotid artery. The plasma levels of heme oxygenase-1 (HO-1), catalase (CAT), NAD(P)H dehydrogenase quinone 1 (NQO1) and glutathione peroxidase-1 (GPx-1) were measured by ELISA. The mouse aortas were isolated, and the superoxide anion and nitric oxide (NO) levels were measured by DHE and DAF-2 DA staining, respectively. Wire Myograph System was used to detect the vasorelaxation of db/db mouse aorta. The protein levels of peroxisome proliferator-activated receptor α (PPARα), nuclear factor E2-related factor 2 (Nrf2), phosphorylated AMP-activated protein kinase (p-AMPK), phosphorylated endothelial NO synthase (p-eNOS), P22^phox and P47^phox were determined by Western blot. RESULTS: Dietary CGA decreased fasting blood glucose and body weight in db/db mice as compared with control group (P<0.01 or P<0.05). The plasma levels of HO-1, CAT, NQO1 and GPx-1 in CGA group were higher than those in control group (P<0.01 or P<0.05). Administration of CGA for 12 weeks attenuated superoxide anion level, increased NO level in the mouse endothelium and improved endothelium-dependent relaxation of the db/db mouse aorta. CGA also increased the protein levels of PPARα, Nrf2, p-AMPK and p-eNOS, and decreased P22^phox and P47^phox levels (P<0.01). CONCLUSION: Dietary CGA improves db/db mouse endothelium-dependent relaxation. This effect may be related to the increases in the levels of antioxidant molecules PPARα, Nrf2 and p-AMPK, and the up-regulation of antioxidant capacity, thus decreasing the oxidative stress, promoting eNOS phosphorylation, and increasing NO level.     

Ultrastructural impairment of islet microvascular endothelial cells in STZ-induced type 1 diabetic mice

AIM: To investigate the ultrastructural changes of islet microvascular endothelial cells in STZ-induced type 1 diabetic mice. METHODS: BALB/c mice were randomly divided into diabetic group and control group. The expression of insulin and platelet-endothelial cell adhesion molecule-1 (CD31) in islet microvessels was detected by immunohistochemical staining. The ultrastructural changes of islet β cells and islet microvessels were observed under transmission electron microscope. RESULTS: Compared with control group, the number of islet β cells, ratio of β cells/α cells, average number of secretory granules in β cells and insulin expression area per islet in diabetic group were significantly decreased (P<0.01). Besides, diabetic group had fewer microvessels with lower expression of CD31 (P<0.01). Mitochondria in islet microvascular endothelial cells and pericytes in diabetic group were swelling. The basement membrane of islet microvessels became thicker in diabetic group (P<0.01). CONCLUSION: Islet microvascular endothelial cells were impaired in type 1 diabetic mice.     

Protective effect of curcumin analogue L6H4 on diaphragm of type 2 diabetic rats

AIM: To investigate the protective effect of curcumin analogue L6H4 on diaphragm of type 2 diabetic rats.METHODS: SPF male Sprague-Dawley rats (n=40) were randomly divided into 5 groups: normal control (NC) group, high fat (HF) group, high fat+L6H4 treatment (FT) group, diabetes mellitus (DM) group and DM+L6H4 treatment (DT) group. The rats in the later 4 groups were fed with high-fat diet. After 4 weeks of high-fat diet fee-ding, the rats in DM and DT groups were intraperitoneally injected with streptozotocin to induce type 2 diabetes melliutus. The rats in FT and DT groups were given L6H4 by gavage for 8 weeks. Blood glucose and blood lipid levels were detected biochemically. Fasting serum insulin (FINS) level was measured by radioimmunoassay and insulin resistance index (HOMA-IR) was calculated. Serum adiponectin (APN) level was measured by ELISA. The morphological changes of the diaphragm were observed under light and transmission electron microscopes. Lipid deposition and the activity of succinate dehydrogenase (SDH) and NADH-tetrazolium reductase (NADH-TR) were observed by enzyme histochemical staining. The content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) in the diaphragm were measured by thiobarbituric acid method and hydroxylamine method, respectively. The protein expression of adiponectin receptor 1 (AdipoR1) in the diaphragm was determined by immunohistochemistry and Western blot. RESULTS: The levels of blood lipids, blood glucose, FINS and HOMA-IR in HF and DM groups were higher than those in NC group, but decreased after L6H4 treatment. The serum APN level in HF and DM groups was lower than that in NC group, but increased after treatment with L6H4. The muscle fibers of the diaphragm were shrunk, fat particles accumulated in the muscle fibers, and the mitochondria were slightly swollen in HF and DM groups. The diaphragmatic fibrosis was obvious in DM group. These lesions were relieved after L6H4 treatment. Compared with NC group, the level of MDA and the activity of SDH and NADH-TR in the diaphragm were increased in HF and DM groups, but decreased after treatment with L6H4. The activity of SOD and the expression of AdipoR1 in the diaphragm were lower than those in NC group, but increased after L6H4 treatment.CONCLUSION: The curcumin analogue L6H4 exerts a protective effect on diaphragm in type 2 diabetic rats. The strengthened protein expression of AdipoR1, the increased serum level of APN, and anti-lipid peroxidation may be involved in the process.     

MicroRNA-9-5p mediates sympathetic overactivity by targeting KCNN3 in paraventricular nucleus of rats with T2D

AIM To investigate whether microRNA-9-5p （miR-9-5p） mediates sympathetic overactivity by targeting KCNN3 （potassium intermediate/small conductance calcium-activated channel, subfamily N, member 3） gene,which encoded small-conductance calcium-activated potassium channel 3 （SK3） protein, in paraventricular nucleus （PVN） of rats with type 2 diabetes mellitus （T2D）. METHODS A rat model of T2D was established by high-fat diet combined with intraperitoneal injection of 30 mg/kg streptozotocin. The levels of miR-9-5p and KCNN3 mRNA in PVN were detected by real-time PCR. The relationship between KCNN3 and miR-9-5p was predicted by TargetScan. Recombinant adeno-associated virus （rAAV）-miR-9-5p or KCNN3 were bilaterally microinjected into the PVN to observe the changes in plasma glucose levels and sympathetic drive indicators. The number of FosB and SK3 positive cells was measured by immunofluorescence staining. The protein expression of SK3 was determined by Western blot. The relationship between KCNN3 and miR-9-5p were confirmed by cell transfection and dual-luciferase reporter assay. RESULTS Compared with the rats in diabetes control （DC） group, the blood glucose, sympathetic drive indexes and the level of miR-9-5p in PVN were significantly increased, while the SK3 expression in PVN was obviously reduced in the diabetes mellitus （DM） rats. After microinjecion of rAAV-miR-9-5p in PVN, the sympathetic drive indexes, blood glucose, and the number of FosB-positive cells were increased significantly, but the SK3 protein expression was significantly reduced （P<0.05）. However, up-regulation of KCNN3 in PVN had the opposite effect. These responses were obviously enhanced in DM rats compared with DC rats. The results of cell transfection and dual-luciferase reporter assay demonstrated that miR-9-5p bound to the 3’-UTR of KCNN3 and inhibit its expression. CONCLUSION miR-9-5p was up-regulated in PVN of the rats with T2D, and it may mediate sympathoexcitation by targeting KCNN3.