In vivo direct reprogramming of liver cells to insulin producing cells by virus-free overexpression of defined factors.

Direct reprogramming of autologous cells from diabetes patients to insulin producing cells is a new method for pancreatic cell replacement therapy. At present, transdifferentiation among mature cells is achieved mainly by introducing foreign genes into the starting tissue with viral vector, but there are potentical safety problems. In the present study, we delivered plasmids carrying Pdx1, Neurog3 and MafA genes (PNM) into mouse hepatocytes by hydrodynamics tail vein injection, investigated islet ß cells markers in transfected cells from protein and mRNA level, and then observed the long-term control of blood glucose in diabetic mice. We found that hepatocytes could be directly reprogrammed into insulin-producing cells after PNM gene transfection by non-viral hydrodynamics injection, and fasting blood glucose was reduced to normal, and lasted until 100 days after transfection. Intraperitoneal glucose tolerance test (IPGTT) showed that glucose regulation ability was improved gradually and the serum insulin level approached to the level of normal mice with time. Insulin-positive cells were found in the liver tissue, and the expression of various islet ß-cell-specific genes were detected at the mRNA level, including islet mature marker gene Ucn3. In conclusion, we provide a new approach for the treatment of diabetes by in vivo direct reprogramming of liver cells to insulin producing cells through non-viral methods.

Metformin exerts glucose-lowering action in high-fat fed mice via attenuating endotoxemia and enhancing insulin signaling.

AIM: Accumulating evidence shows that lipopolysaccharides (LPS) derived from gut gram-negative bacteria can be absorbed, leading to endotoxemia that triggers systemic inflammation and insulin resistance. In this study we examined whether metformin attenuated endotoxemia, thus improving insulin signaling in high-fat diet fed mice. METHODS: Mice were fed a high-fat diet for 18 weeks to induce insulin resistance. One group of the mice was treated with oral metformin (100 mg·kg(-1)·d(-1)) for 4 weeks. Another group was treated with LPS (50 µg·kg(-1)·d(-1), sc) for 5 days followed by the oral metformin for 10 d. Other two groups received a combination of antibiotics for 7 d or a combination of antibiotics for 7 d followed by the oral metformin for 4 weeks, respectively. Glucose metabolism and insulin signaling in liver and muscle were evaluated, the abundance of gut bacteria, gut permeability and serum LPS levels were measured. RESULTS: In high-fat fed mice, metformin restored the tight junction protein occludin-1 levels in gut, reversed the elevated gut permeability and serum LPS levels, and increased the abundance of beneficial bacteria Lactobacillus and Akkermansia muciniphila. Metformin also increased PKB Ser473 and AMPK T172 phosphorylation, decreased MDA contents and redox-sensitive PTEN protein levels, activated the anti-oxidative Nrf2 system, and increased IκBα in liver and muscle of the mice. Treatment with exogenous LPS abolished the beneficial effects of metformin on glucose metabolism, insulin signaling and oxidative stress in liver and muscle of the mice. Treatment with antibiotics alone produced similar effects as metformin did. Furthermore, the beneficial effects of antibiotics were addictive to those of metformin. CONCLUSION: Metformin administration attenuates endotoxemia and enhances insulin signaling in high-fat fed mice, which contributes to its anti-diabetic effects.

Study on modified shengmai yin injection for prevention and treatment of brain impairment in endotoxin shock rats.

OBJECTIVE: To examine the effects of modified Shenmai Yin on invigorating vital energy, promoting blood flow, and protection against neural impairment in an endotoxin-induced shock rat model. METHODS: Ninety-six SD rats were randomly divided into four groups: sham operation (saline 20 ml/kg), shock model (lipopolysaccharide, LPS, 8 mg/kg), Reformed Shengmai Yin (Pulse-activating Decoction) (LPS 8 mg/kg + reformed Shengmai Yin Injection 10 ml/kg), and dexamethasone (LPS 8 mg/kg + dexamethasone 5 mg/kg) groups. Each group was subdivided into 1 h, 2 h, 3 h, and 6 h time points for observation. The carotid artery was separated and connected with a biological functional system to monitor mean arterial pressure (MAP). Brain water levels, malonaldehyde (MDA) content, and superoxide dismutase (SOD) activity were also determined. RESULTS: In the shock model group, MAP was progressively decreased after injection of LPS, brain water and MDA contents were increased, brain SOD activity was decreased, and capillary vessel edema in brain tissue was also observed. All these parameters were improved significantly in both treatment groups, although the effects were more marked with Shengmai Yin than with dexamethasone. CONCLUSION: Modified Shengmai Yin exhibits strong anti-shock and neuroprotective effects against Endotoxin-induced shock.

[Effect of salvianolic acid B on brain energy metabolism and hydrocephalus of cerebral ischemia in mice at different time].

Mice pathological model of acute cerebral ischemia was established. In order to observe the effect of salvianolic acid B (Sal B) on brain energy metabolism and hydrocephalus in the brain of mice at different ischemic times, the energy charge (EC), content of phosphocreatine (PCr), level of lactic acid (Lac), activity of Na+ -K+ -ATPase, brain index and water content of brain were measured at 6, 12, 18, 24, and 30 min, separately after ligating bilateral common carotid arteries in mice. NIH mice were randomly divided into sham-operated group (sham), cerebral ischemia group (ischemia), Sal B-treated group (Sal B) and nimodipine-collated group (Nim). At 6 min after cerebral ischemia, EC, content of PCr and activity of Na +-K -ATPase began to decrease, while level of Lac, brain index and water content of brain increased gradually. However, Sal B (22.5 mg x kg(-1) improved pathophysiological changes at different ischemic times. Especially at 30 min after cerebral ischemia in Sal B group, EC (P < 0.01), content of PCr (P < 0.01 and activity of Na+ -K+ -ATPase ( < 0.05) increased significantly. Meanwhile, level of Lac (P < 0.01, brain index (P < 0.01) and water content of brain (P < 0.05) were lower obviously than those of cerebral ischemia group. Sal B could alleviate hydrocephalus by the improvement of energy metabolism in mice with acute cerebral ischemia, that provides scientific evidence that Sal B can be used for the clinical application of ischemic diseases.

Immunogenicity of insulin-producing cells derived from human umbilical cord mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have been considered as hypo-immunogenic and immunosuppressive. However, a thorough understanding of the immunological properties after MSC differentiation in vitro and in vivo has not been reached. We asked whether it would be immunogenic after differentiation or influenced by the immune microenvironment after transplantation. In different disease models, the immunological changes of MSCs after differentiation greatly varied, with contradicting results. In order to clarify this, we used a modified four-step induction method to induce human umbilical cord MSCs (hUCMSCs) to differentiate into insulin-producing cells (IPCs), and investigate the immunological changes after differentiation and immune reactions after transplantation into diabetic mice. We found that the induced IPCs are hypo-immunogenic, lacking HLA-DR, CD40 and CD80 expression. Of note, we observed immune cell infiltration to peritoneal cavity and left kidney capsule after local transplantation of induced IPCs. This indicated that hUCMSC-derived IPCs maintained hypo-immunogenic in vitro, but became immunogenic after transplanting to the host, possibly due to the changes of immune microenvironment and thereafter immunological enhancement and immune cell infiltration.