Identification of Blood miR-216a, miR-377 and Their Target Genes ANGPTL4, GAP-43 and Serum of PPARG as Biomarkers for Diabetic Peripheral Neuropathy of Type 2 Diabetes.

BACKGROUND: Diabetic peripheral neuropathy (DPN) is one of the most common and complex chronic complications of diabetes, but it is clinically lacking effective means for early diagnosis and early treatment. MicroRNA, in the occurrence and development of the disease, has an important regulatory role. Its role in diabetes has been reported more. However, specific research on microRNA in DPN is rare. METHODS: Based on the results of bioinformatics screening, miR-377 and miR-216a, their respective target molecules growth association protein 43 (GAP-43) and angiopoietin-like 4 protein (ANGPTL4), and related pathways peroxisome proliferator activated receptor gamma (PPARG) and chemerin were tested by RT-qPCR and ELISA in blood samples of DPN to analyze the correlation between these differentially expressed molecules and clinico-pathological factors of DPN. RESULTS: In this study, we found that miR-377, miR-216a, GAP-43, ANGPTL4, and PPARG were significantly differentially expressed genes for DPN. The correlation analysis showed that they were closely related to the clinical indicators of DPN suggesting that they may be involved in the development of DPN. In addition, receiver operating characteristic (ROC) curves generated for miR216a, miR377, ANGPTL4, GAP43, PPARG revealed that they can be used as new molecular diagnostic markers of DPN. CONCLUSIONS: miR-216a, miR-377, ANGPTL4, GAP-43, and PPARG could potentially be biomarkers of DPN.

GPR120 Regulates Pancreatic Polypeptide Secretion From Male Mouse Islets via PLC-Mediated Calcium Mobilization.

The free fatty acid receptor G protein-coupled receptor 120 (GPR120) is expressed in pancreatic islets, but its specific cell distribution and function have not been fully established. In this study, a GPR120-IRES-EGFP knockin (KI) mouse was generated to identify GPR120-expressing cells with enhanced green fluorescence proteins (EGFP). EGFP-positive cells collected from KI mouse islets by flow cytometry had a significantly higher expression of pancreatic polypeptide (PP) evidenced by reverse transcriptase (RT)-quantitative polymerase chain reaction (qPCR). Single-cell RT-PCR and immunocytochemical double staining also demonstrated the coexpression of GPR120 with PP in mouse islets. The GPR120-specific agonist TUG-891 significantly increased plasma PP levels in mice. TUG-891 significantly increased PP levels in islet medium in vitro, which was markedly attenuated by GPR120 small interfering RNA treatment. TUG-891-stimulated PP secretion in islets was fully blocked by pretreatment with YM-254890 (a Gq protein inhibitor), U73122 (a phospholipase C inhibitor), or thapsigargin (an inducer of endoplasmic reticulum Ca2+ depletion), respectively. TUG-891 triggered the increase in intracellular free Ca2+ concentrations ([Ca2+]i) in PP cells, which was also eliminated by YM-254890, U73122, or thapsigargin. GPR120 gene expression was significantly reduced in islets of high-fat diet (HFD)-induced obese mice. TUG-891-stimulated PP secretion was also significantly diminished in vivo and in vitro in HFD-induced obese mice compared with that in normal-chow diet control mice. In summary, this study demonstrated that GPR120 is expressed in mouse islet PP cells and GPR120 activation stimulated PP secretion via the Gq/PLC-Ca2+ signaling pathway in normal-chow diet mice but with diminished effects in HFD-induced obese mice.

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.

Dimethylation of Histone 3 Lysine 9 is sensitive to the epileptic activity, and affects the transcriptional regulation of the potassium channel Kcnj10 gene in epileptic rats.

Potassium channels can be affected by epileptic seizures and serve a crucial role in the pathophysiology of epilepsy. Dimethylation of histone 3 lysine 9 (H3K9me2) and its enzyme euchromatic histone­lysine N­methyltransferase 2 (G9a) are the major epigenetic modulators and are associated with gene silencing. Insight into whether H3K9me2 and G9a can respond to epileptic seizures and regulate expression of genes encoding potassium channels is the main purpose of the present study. A total of 16 subtypes of potassium channel genes in pilocarpine­modelled epileptic rats were screened by reverse transcription­quantitative polymerase chain reaction, and it was determined that the expression ATP­sensitive inward rectifier potassium channel 10 (Kcnj10) increased in hippocampus and insular cortex, while the expression of most of the other subtypes decreased. The total level of H3K9me2 decreased in the model group compared with the control. The Kcnj10 gene encoding the Kir4.1 channel was selected to analyse changes in H3K9me2 in the promoter region by the chromatin immuno­precipitation method. Anti­H3K9me2 and anti­G9a antibodies were used to identify the modified DNAs. Five primers were designed across the promoter region of the Kcnj10 gene. In epileptic hippocampi, the relative abundance of H3K9me2 and G9a in the promoter region of Kcnj10 decreased markedly. Removal of the H3K9me2 repressive mark resulted in decreased transcriptional inhibition of the Kcnj10 gene and therefore increased its expression. In the cultured C6 cells, specific inhibition of the enzymatic activity of G9a by 2­(Hexahydro­4­methyl­1H­1,4­diazepin­1­yl)­6,7­di­ methoxy­N­(1­(phenyl­methyl)­4­piperidinyl)­4­quinazolinamine tri­hydrochloride hydrate (bix01294) resulted in upregulation of the expression of Kir4.1 proteins. The present study demonstrated that H3K9me2 and G9a are sensitive to epileptic seizure activity during the acute phase of epilepsy and can affect the transcriptional regulation of the Kcnj10 channel.

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.

Nitric oxide participates in cold-inhibited Camellia sinensis pollen germination and tube growth partly via cGMP in vitro.

Nitric oxide (NO) plays essential roles in many biotic and abiotic stresses in plant development procedures, including pollen tube growth. Here, effects of NO on cold stress inhibited pollen germination and tube growth in Camellia sinensis were investigated in vitro. The NO production, NO synthase (NOS)-like activity, cGMP content and proline (Pro) accumulation upon treatment with NO scavenger cPTIO, NOS inhibitor L-NNA, NO donor DEA NONOate, guanylate cyclase (GC) inhibitor ODQ or phosphodiesterase (PDE) inhibitor Viagra at 25°C (control) or 4°C were analyzed. Exposure to 4°C for 2 h reduced pollen germination and tube growth along with increase of NOS-like activity, NO production and cGMP content in pollen tubes. DEA NONOate treatment inhibited pollen germination and tube growth in a dose-dependent manner under control and reinforced the inhibition under cold stress, during which NO production and cGMP content promoted in pollen tubes. L-NNA and cPTIO markedly reduced the generation of NO induced by cold or NO donor along with partly reverse of cold- or NO donor-inhibited pollen germination and tube growth. Furthermore, ODQ reduced the cGMP content under cold stress and NO donor treatment in pollen tubes. Meanwhile, ODQ disrupted the reinforcement of NO donor on the inhibition of pollen germination and tube growth under cold condition. Additionally, Pro accumulation of pollen tubes was reduced by ODQ compared with that receiving NO donor under cold or control condition. Effects of cPTIO and L-NNA in improving cold-treated pollen germination and pollen tube growth could be lowered by Viagra. Moreover, the inhibitory effects of cPTIO and L-NNA on Pro accumulation were partly reversed by Viagra. These data suggest that NO production from NOS-like enzyme reaction decreased the cold-responsive pollen germination, inhibited tube growth and reduced Pro accumulation, partly via cGMP signaling pathway in C. sinensis.

Characterization of the expression profiles of calpastatin (CAST) gene in chicken.

The calpain system, a Ca(2+)-activated protease family, plays an important role in postmortem tenderization of skeletal muscle due to its involvement in the degradation of important myofibrillar and associated proteins, as well as in cytoskeletal remodeling and regulation of muscle growth. In this study, we quantified the expression of calpastatin (CAST) in two Chinese chicken breeds (mountainous black-bone chicken breed (MB) and a commercial meat type chicken breed (S01)), to discern the tissue and age-related specific expression pattern and its potential role on muscle tissue metabolism. Real-time quantitative PCR (RT-qPCR) assay was developed for accurate measurement of CAST mRNA levels in various tissues from chicken with different ages (0, 2, 4, 6, 8, 10, and 12 week). CAST mRNA was detected in collected organs. The heart and leg muscle tissues had the highest expression of CAST than other tissues from the same chicken (P < 0.01). Age-related expression pattern of CAST gene was evident in breast muscle, liver, and brain tissues (P < 0.05), but not in heart and leg muscle tissues (P > 0.05). Overall, the CAST mRNA level exhibited a "rise-decline-rise-decline" developmental change in breast muscle and liver, with the highest expression at 2 weeks and the lowest expression at 8 weeks. The S01 chicken had significantly higher expression of CAST in breast muscle and heart than the MB chicken (P < 0.05) at 10 weeks. Our results suggested the CAST expression may be related to muscle fiber development.

Tissue-specific expression of the chicken calpain2 gene.

We quantified chicken calpain 2 (CAPN2) expression in two Chinese chicken breeds (mountainous black-bone chicken breed [MB] and a commercial meat type chicken breed [S01]) to discern the tissue and ontogenic expression pattern and its effect on muscle metabolism. Real-time quantitative PCR assay was developed for accurate measurement of the CAPN2 mRNA expression in various tissues from chickens of different ages (0, 2, 4, 6, 8, 10, and 12 weeks). Results showed that the breast muscle and leg muscle tissues had the highest expression of CAPN2 compared to the other tissues from the same individual (P < .05). Overall, the CAPN2 mRNA level exhibited a "rise" developmental change in all tissues. The S01 chicken had a higher expression of the CAPN2 mRNA in all tissues than the MB chicken. Our results suggest that chicken CAPN2 expression may be related to chicken breeds and tissues.

[Genetic analysis of 32 microsatellite loci in 13 families of Wuzhishan pig by multiplex PCR and gene scanning technique].

Wuzhishan pig is one of the rare and endangered breeds in china. They have the following characteristics such as :light body weight and small size, early sexually maturity, high meat quality and genetic identification with 6 approximately 8 pares litter size,body weight of born 0.3 approximately 0.4 kg, 15 approximately 16 kg at 6 month old, 35 kg at 2 years old, and so on. They may be used for laboratory utilization, comparative studies on human medical model, embryonic engineering, nutrition metabolism, sensitivity test on virus and bacteria, skin brut and tranfer, removing lipid, teeth and mouth cavity diseases, studies on cardiovascular model and evaluation of new medicine products. The polymorphisms of 32 microsatellites in 13 families of Wuzhishan pig in Hainan were Analysed. Number of alleles in each family was counted, mean heterozygosity and polymorphism Information content(PIC) were calculated. The results showed that number of alleles was 13.66, mean heterozygosity was 0.559 while polymorphism information content was 0.731. This revealed that genetic diversity is abundant in Wuzhishan pig in Hainan. These results have instructional significance for preserving breeds, selection and breeding, development and utilization of Wuzhishan pig in Hainan.