High-glucose Induced Mitochondrial Dynamics Disorder of Spinal Cord Neurons in Diabetic Rats and its Effect on Mitochondrial Spatial Distribution.

STUDY DESIGN: A randomized, double-blind, controlled trial. OBJECTIVE: Few studies have investigated the changes in mitochondrial dynamics in spinal cord neurons. Meanwhile, the distribution of mitochondria in axons remains unclear. In the present study, the investigators attempted to clarify these questions and focused in observing the changes in mitochondrial spatial distribution under a high-glucose environment. SUMMARY OF BACKGROUND DATA: Mitochondrial dynamics disorder is one of the main mechanisms that lead to nervous system diseases due to its adverse effects on mitochondrial morphology, function, and axon distribution. High-glucose stress can promote the increase in mitochondrial fission of various types of cells. METHODS: The lumbar spinal cord of type 1 diabetic Sprague-Dawley rats at 4 weeks was observed. VSC4.1 cells were cultured and divided into three groups: normal control group, high-glucose intervention group, and high-glucose intervention combined with mitochondrial fission inhibitor Mdivi-1 intervention group. Immunohistochemistry and immunofluorescence methods were used to detect the expression of mitochondrial marker VDAC-1 in the spinal cord. An electron microscope was used to observe the number, structure, and distribution of mitochondria. Western blot was used to detect VDAC-1, fusion protein MFN1, MFN2, and OPA1, and fission protein FIS1 and DRP1. Living cell mitochondrial staining was performed using MitoTracker. Laser confocal microscopy and an Olympus live cell workstation were used to observe the mitochondrial changes. RESULTS: The mitochondrial dynamics of spinal cord related neurons under an acute high-glucose environment were significantly unbalanced, including a reduction of fusion and increase of fission. Hence, mitochondrial fission has the absolute advantage. The total number of mitochondria in neuronal axons significantly decreased. CONCLUSION: Increased mitochondrial fission and abnormal distribution occurred in spinal cord related neurons in a high-glucose environment. Mdivi-1 could significantly improve these disorders of mitochondria in VSC4.1 cells. Mitochondrial division inhibitors had a positive significance on diabetic neuropathy. LEVEL OF EVIDENCE: N/A.

Lycium barbarum polysaccharide protects diabetic peripheral neuropathy by enhancing autophagy via mTOR/p70S6K inhibition in Streptozotocin-induced diabetic rats.

Lycium barbarum polysaccharide (LBP), the major active component of Lycium barbarum, has been found to be effective in the management of some diabetic complications. We evaluated the protective effect of LBP in diabetic peripheral neuropathy (DPN) and explored the possible mechanisms. We found that LBP mildly decreased blood glucose levels and partially rescued allodynia and hyperalgesia in the diabetes mellitus (DM) rats. For the electrophysiological function of the sciatic nerve, the decrease in sensory nerve conduction velocity (SNCV) and sensory nerve action potential (SNAP) amplitudes in DM rats were partially rescued. Moreover, DM-induced structural damage to the nerve fiber myelination showed great improvement by 12 weeks of LBP treatment. The decreased expression of the myelin-related proteins, myelin protein zero (P0) and myelin basic protein (MBP), in the DM sciatic nerve was also markedly rescued after 12 weeks of LBP treatment. Furthermore, the possible role of mammalian target of rapamycin (mTOR)-mediated autophagy during these protective processes was examined. The expression of microtubule-associated protein light chain 3-II(LC3-II) and Beclin1 in the sciatic nerve was significantly decreased while the expression of P62 increased in DM rats, demonstrating an decreased activation of autophagy. As expected, the LC3-II and Beclin1 protein levels were markedly increased, and P62 was markedly decreased after LBP treatment. The expression of mTOR, p-mTOR, p70 ribosomal protein S6 kinase (p70S6K) and p-p70S6K in the DM group were markedly increased, while all of these proteins decreased in LBP group. These results demonstrate that LBP exerts protective effects on DPN, which is likely to be mediated through the induction of autophagy by inhibiting the activation of the mTOR/p70S6K pathways.

HSPB8 Promotes the Fusion of Autophagosome and Lysosome during Autophagy in Diabetic Neurons.

Although autophagy has been proposed to play an emerging role in diabetic neuropathy, autophagy and its possible role remains unclear. Moreover, only few studies about diabetes have explored the autophagy mediated by heat shock protein beta-8 (HSPB8) and Bcl-2 associated athanogene 3 (BAG3). In the present study, we examined the autophagy induced by high glucose levels in an in vivo rat model of diabetes induced by streptozotocin (STZ) and an in vitro model of retinal ganglion cell-5 (RGC5) cells under high glucose conditions. In the spinal cord tissues of the STZ-induced diabetic rats, the levels of light chain 3 (LC3) and Beclin-1-marked autophagy rose with increasing HSPB8 and BAG3 levels. By confocal immunofluorescence, HSPB8 and LC3 were observed to be co-localized in the spinal cord tissues. In the RGC5 cells, high-glucose stimulation upregulated the expression of LC3-Ⅱ, Beclin-1, and HSPB8 in a dose-dependent manner. When the RGC5 cells were subjected to high-glucose conditions, HSPB8 overexpression, along with upregulated LC3-Ⅱ and Beclin-1 expression, increased the autophagic rate, whereas siRNA-silenced HSPB8 decreased the autophagic rate. Furthermore, in GFP-mRFP-LC3 probe experiments, HSPB8 overexpression promoted autophagosome-lysosome fusion, whereas HSPB8 silencing disrupted this process. In the cells treated with HSPB8 and siRNA, the fusion was impaired, as indicated by the elevated p62 expression. HSPB8 overexpression can partly rescue the blocking of the autophagy flux with chloroquine through the reduction of p62 expression level. Our study demonstrated that HSPB8 is involved in the high glucose-induced autophagy under the in vivo and in vitro conditions and critically participated in the autophagosome-lysosome fusion during the autophagy flux.

MiR-126 Suppresses the Glucose-Stimulated Proliferation via IRS-2 in INS-1 ß Cells.

BACKGROUND: Increasing evidence suggests that miR-126 participates in the glucose homeostasis through its target molecules. Although bioinformatics analysis predicts that miR-126 can bind with the insulin receptor substrate-2(IRS-2) mRNA at the "seed sequence", but there are still no definitely reports to support it. In this study, we provided evidences that IRS-2 was one of the target genes of miR-126. And miR-126 has a proliferation inhibiting effects in INS-1 ß cells, mainly through the suppression of IRS-2. METHODS: The 3'-UTR of IRS-2 regulated by miR-126 was analyzed by the luciferase assay and western blot. Furthermore, proliferation of INS-1 ß cells stimulated by glucose was tested, and the association between IRS-2 and miR-126 were analyzed. RESULTS: We found that mutation of only three of the 6 "seed sequences" can eliminate the inhibition effect of miR-126. In INS-1 ß cells, administration of miR-126 suppresses the proliferation, together with the unbalanced down-regulation of IRS-2 and IRS-1. Over-expression of IRS-2 can reverse the proliferation effect of miR-126, while not of IRS-1. These results suggested that miR-126 inhibited the ß-cell proliferation via the inhibition of IRS-2 instead of IRS-1.Additionally, we also found that high glucose and insulin could stimulate the rapid production of endogenous miR-126 within 6 hours, together with the short term suppression of IRS-1 and IRS-2 expression, and intensify the unbalanced expression of IRS-1 and IRS-2. CONCLUSIONS: IRS-2 was one of the targets of miR-126. MiR-126 inhibited the ß-cell proliferation through IRS-2 instead of IRS-1. MiR-126 may take part in the glucose homeostasis both through its target IRS-2 and IRS-1. The unbalance between IRS-1 and IRS-2 caused by miR-126 may play an important role in type 2 diabetes.

Dendritic and Langerhans cells respond to Aß peptides differently: implication for AD immunotherapy.

Both wild-type and mutated beta-amyloid (Aß) peptides can elicit an immune response when delivered subcutaneously. However, only mutated forms of Aß can sensitize dendritic cells when administered intravenously or intraperitoneally. To understand the role of mutation and delivery routes in creating immune responses, and the function of dendritic cells as therapeutic agents, we used fluorescent-conjugated WT Aß1-40 (WT40) and artificially mutated Aß1-40 (22W40) peptides to treat dendritic and Langerhans cells from young and/or old mice at different time points. The cell types were analyzed by flow cytometry and confocal microscopy to identify differences in function and antigen presentation, and Luminex and Western blots for cell activation and associated mechanisms. Our results demonstrated that the artificial mutant, 22W40, enhanced dendritic cell's phagocytosis and antigen presentation better than the WT40. Interestingly, Langerhans cells were more effective at early presentation. The artificial mutant 22W40 increased CD8α+ dendritic cells, CD8+ T-cells, and IFN-γ production when co-cultured with self-lymphocytes and dendritic cells from aged mice (30-month-old). Here, the 22W40 mutant peptide has been found to be potent enough to activate DCs, and that dendritic cell-based therapy may be a more effective treatment for age-related diseases, such as Alzheimer's disease (AD).

An evidence-based update on the pharmacological activities and possible molecular targets of Lycium barbarum polysaccharides.

Lycium barbarum berries, also named wolfberry, Fructus lycii, and Goji berries, have been used in the People's Republic of China and other Asian countries for more than 2,000 years as a traditional medicinal herb and food supplement. L. barbarum polysaccharides (LBPs) are the primary active components of L. barbarum berries and have been reported to possess a wide array of pharmacological activities. Herein, we update our knowledge on the main pharmacological activities and possible molecular targets of LBPs. Several clinical studies in healthy subjects show that consumption of wolfberry juice improves general wellbeing and immune functions. LBPs are reported to have antioxidative and antiaging properties in different models. LBPs show antitumor activities against various types of cancer cells and inhibit tumor growth in nude mice through induction of apoptosis and cell cycle arrest. LBPs may potentiate the efficacy of lymphokine activated killer/interleukin-2 combination therapy in cancer patients. LBPs exhibit significant hypoglycemic effects and insulin-sensitizing activity by increasing glucose metabolism and insulin secretion and promoting pancreatic ß-cell proliferation. They protect retinal ganglion cells in experimental models of glaucoma. LBPs protect the liver from injuries due to exposure to toxic chemicals or other insults. They also show potent immunoenhancing activities in vitro and in vivo. Furthermore, LBPs protect against neuronal injury and loss induced by ß-amyloid peptide, glutamate excitotoxicity, ischemic/reperfusion, and other neurotoxic insults. LBPs ameliorate the symptoms of mice with Alzheimer's disease and enhance neurogenesis in the hippocampus and subventricular zone, improving learning and memory abilities. They reduce irradiation- or chemotherapy-induced organ toxicities. LBPs are beneficial to male reproduction by increasing the quality, quantity, and motility of sperm, improving sexual performance, and protecting the testis against toxic insults. Moreover, LBPs exhibit hypolipidemic, cardioprotective, antiviral, and antiinflammatory activities. There is increasing evidence from preclinical and clinical studies supporting the therapeutic and health-promoting effects of LBPs, but further mechanistic and clinical studies are warranted to establish the dose-response relationships and safety profiles of LBPs.

[Study of relationship between stress hyperglycemia and insulin-resistance related factors].

OBJECTIVE: To observe related factors in the stress hyperglycemia (SHG) of critical illness and to investigate possible pathogenesis of insulin-resistance (IR). METHODS: Blood glucose (BG), insulin (INS), C-peptide (C-P), cortisol (Cor), somatostatin (SS), glucagon (Gluc), tumor necrosis factor-alpha (TNF-alpha),soluble tumor necrosis factor receptor I (sTNFRI) and sTNFRII were determined respectively by radioimmunoassay (RIA) or enzyme linked immunoadsorbent assay (ELISA) in 47 SHG patients with critical illness and 15 healthy volunteers serving as normal controls. Their insulin sensitivity index (ISI) was calculated. RESULTS: (1)Eleven of 47 patients died, while 36 cases survived. Mean acute pathology and chronic health evaluation II (APACHEII) was (13.89+/-6.29) scores within 24 hours after admission to intensive care unit (ICU), mean days of stay in ICU was (5.5+/-6.3) days,and mean duration of mechanical ventilation (MV) was (51.49+/-66.01) hours. (2)The concentrations of INS, ISI, C-P, Cor, Gluc, TNF-alpha, sTNFRI and sTNFRII in 47 SHG patients with critical illness were significantly higher than those in normal controls, except for SS, the differences among groups were significant (P<0.05 or P<0.01). (3)The results of analysis of severity of SHG showed that the more severe SHG was, the higher C-P and INS were, and the less prominent ISI was. (4)Analysis of scores of APACHEII in 47 cases of SHG showed that BG was not increased, but duration of MV, Cor, Gluc, SS, TNF-alpha, sTNFRI and sTNFRII were significantly increased with higher scores of APACHEII. (5)The effect of SHG was significant on MV (F=10.438,P<0.01), but not significant for outcome and days of stay in ICU. (6)The main correlative factors of BG were respectively concentrations of INS (r=0.674, P<0.01), C-P(r=0.552,P<0.01), ISI (r=-0.787, P<0.01), APACHE II(r=0.267,P<0.05) and sTNFRI(r=0.465, P<0.01). CONCLUSION: These results show that main reason of SHG in critical illness is IR. There is no strong significant correlation between acute stress hormones and the level of SHG. sTNFRI has an influence on SHG. However, the over release of TNF-alpha and sTNFRII could be the results of seriousness of the critical illness. There is closely correlation between BG and MV, but not with the age, outcome and days of stay in ICU. The strategy of control and therapy of SHG should be alleviation of stress and improve the utilization of BG in the tissue, and increase sensitivity of INS in the tissue.