DUSP10 alleviates ischemic stroke-induced neuronal damage by restricting p38/JNK pathway.

Neuronal apoptosis is considered one of the hallmarks of ischemic stroke. Dual specificity phosphatase 10 (DUSP10), a member of the dual-specificity phosphatase family, which is involved in the regulation of apoptosis process. This study aimed to investigate the effect of on apoptosis in primary cortical neurons exposed to oxygen-glucose deprivation and reoxygenation (OGD/R) and mice suffered from transient middle cerebral artery occlusion and reperfusion (MCAO/R). The results showed that DUSP10 overexpression improved survival and reduced apoptosis in neurons subjected to OGD/R, which was manifested by decreased apoptotic proteins (cleaved caspase 3 and bax) and TUNEL+ cells, as well as increased the anti-apoptotic protein (bcl-2). DUSP10 overexpression inhibited the p38/JNK signaling pathway after OGD/R treatment, whilst DUSP10 knockdown had opposite effects. In addition, the p38 inhibitor SB203580 or JNK inhibitor SP600125 attenuated the increased apoptosis of OGD/R-stimulated neurons treated with DUSP10 silencing. Consistently, DUSP10 knockdown exacerbated infarct volume in MCAO/R injury. The data of Nissl staining and TUNEL-NeuN double staining revealed that DUSP10 interference aggravated neuronal damage in the ischemic penumbra of mice. Furthermore, DUSP10 inhibition activated the p38/JNK axis accompanied by enhanced phosphorylation of p38 and JNK in vivo. In summary, DUSP10 is a neuroprotective agent against ischemic stroke-induced neuronal damage via suppressing the p38/JNK signaling pathway.

Protein Kinase CK2 Modulates the Calcium Sensitivity of Type 3 Small-conductance Calcium-activated Potassium Channels in Colonic Platelet-derived Growth Factor Receptor Alpha-positive Cells From Streptozotocin-induced Diabetic Mice.

Background/Aims: The gastrointestinal symptom of diabetes mellitus, chronic constipation, seriously affects patients' life. Whereas, the mechanism of chronic constipation is still ambiguous, resulting in a lack of effective therapies for this symptom. As a part of the smooth muscle cells, interstitial cells of Cajal, and platelet-derived growth factor receptor alpha-positive (PDGFRα+) cells syncytium (SIP syncytium), PDGFRα+ cells play an important role in regulating colonic motility. According to our previous study, in PDGFRα+ cells in colons of diabetic mice, the function of the P2Y1 purinergic receptor/type 3 small-conductance calcium-activated potassium (SK3) channel signaling pathway is strengthened, which may lead to colonic dysmotility. The purpose of this study is to investigate the changes in SK3 channel properties of PDGFRα+ cells in diabetic mice. Methods: Whole-cell patch clamp, Western blotting, superoxide dismutase activity measurement, and malondialdehyde measurement were main methods in the present study. Results: The present study revealed that when dialysed with low calcium ion (Ca2+) solution, the SK3 current density was significantly decreased in PDGFRα+ cells from diabetic mice. However, the SK3 current density in PDGFRα+ cells was enhanced from diabetic mice when dialysed with high Ca2+ solution. Moreover, hydrogen peroxide-treatment mimicked this phenomenon in SK3 transgenic HEK293 cells. The subunit of SK3 channels, protein kinase CK2, was up-regulated in colonic muscle layers and hydrogen peroxide-treated HEK293 cells. Additionally, protein phosphatase 2A, the subunit of SK3 channels, was not changed in streptozotocin-treated mouse colons or hydrogen peroxide-treated HEK293 cells. Conclusion: The diabetic oxidative stress-induced upregulation of CK2 contributed to modulating SK3 channel sensitivity to Ca2+ in colonic PDGFRα+ cells, which may result in colonic dysmotility in diabetic mice.

Sodium houttuyfonate against cardiac fibrosis attenuates isoproterenol-induced heart failure by binding to MMP2 and p38.

BACKGROUND: Heart failure (HF), caused by stress cardiomyopathy, is a major cause of mortality. Cardiac fibrosis is an essential structural remodeling associated with HF; therefore, preventing cardiac fibrosis is crucial to decelerating the progression of HF. Sodium houttuyfonate (SH), an extract of Houttuynia cordata, has a potent therapeutic effect on hypoxic cardiomyocytes in a myocardial infarction model. PURPOSE: To investigate the preventative and therapeutic effects of SH during isoproterenol (ISO)-induced HF and explore the pharmacological mechanism of SH in alleviating HF. METHODS: We analyzed the overlapping target genes between SH and cardiac fibrosis or HF using a network pharmacology analytical method. We verified the suppressive effect of SH on ISO-induced proliferation and activation of cardiac fibroblasts by immunohistochemical staining and histological analysis in an isoproterenol-induced HF mouse model. Additionally, we investigated the effect of SH by evaluating fibrosis and cardiac remodeling markers. To further decipher the pharmacological mechanism of SH against cardiac fibrosis and HF, we performed a molecular docking analysis between SH and hub common target genes. RESULTS: There were 20 overlapping target genes between SH and cardiac fibrosis and 32 overlapping target genes between SH and HF. The 16 common target genes of SH against cardiac fibrosis and HF included MMP2 (matrix metalloproteinase 2), and p38. SH significantly inhibited the ISO- or TGF-ß-induced expression of Col1α (collagen 1), α-SMA (smooth muscle actin), MMP2, TIMP2 (tissue inhibitor of metalloproteinase 2), TGF-ß (transforming growth factor), and Smad2 phosphorylation. Moreover, both ISO- and TGF-ß-induced p38 phosphorylation was inhibited. Molecular docking analysis showed that SH forms a stable complex with MMP2 and p38. CONCLUSIONS: In addition to protecting cardiomyocytes, SH directly inhibits cardiac fibroblast activation and proliferation by binding to MMP2 and p38, subsequently delaying cardiac fibrosis and HF progression. Our prevention- and intervention-based approaches in this study showed that SH inhibited the development of stress cardiomyopathy-mediated cardiac fibrosis and HF when SH was administered before or after the initiation of cardiac stress.

Long non-coding RNA Opa interacting protein 5-antisense RNA 1 promotes mitochondrial autophagy and protects SH-SY5Y cells from 1-methyl-4-phenylpyridine-induced damage by binding to microRNA-137 and upregulating NIX.

Parkinson's disease (PD) is a leading cause of disability. Long noncoding RNA (LncRNA) OIP5-AS1 alleviates the accumulation and toxicity of 1-methyl-4-phenylpyridine (MPP+ )/-induced α-synuclein in human neuroblastoma SH-SY5Y cells, which may be involved in the pathological process of PD. This study explored the neuroprotective effect of lncRNA OIP5-AS1 on MPP+ /-induced SH-SY5Y cell model of PD, so as to provide a theoretical basis for PD treatment. The PD cell model was established (MPP+ group). The overexpression vector oe-OIP5-AS1 was constructed and transfected into MPP+/-induced SH-SY5Y cells, which were further transfected with miR-137 mimic or si-NIX plasmids. The localization of OIP5-AS1 and its binding sites with miR-137 were predicted by subcellular isolation and fluorescence in situ hybridization analysis. The targeting relationships between OIP5-AS1 and miR-137, and miR-137 and NIX were detected by dual-luciferase reporter assays. The mitochondrial membrane potential (Δψm) and total reactive oxygen species (ROS) levels, and expressions of α-synuclein, inflammatory cytokines, and microglia-activated chemokines, cell activity, and apoptosis were assessed. OIP5-AS1 was downregulated in MPP+ cells. After OIP5-AS1 overexpression, miR-137 was downregulated and NIX was upregulated in MPP+ cells, inflammatory factors and chemokines were downregulated. There were target relationships between OIP5-AS1 and miR-137, and miR-137 and NIX. After OIP5-AS1 overexpression, miR-137 overexpression or NIX downregulation inhibited mitochondrial autophagy and ROS levels and aggravated mitochondrial vacuolation; and partially reversed the effect of OIP5-AS1 overexpression on promoting mitochondrial autophagy and protection on MPP+ cells. Collectively, lncRNA OIP5-AS1 promoted NIX expression through competitively binding to miR-137, and promoted mitochondrial autophagy, thus protecting neurons from degeneration which might be seen in patients with PD.

Flatness Prediction of Cold Rolled Strip Based on Deep Neural Network with Improved Activation Function.

With the improvement of industrial requirements for the quality of cold rolled strips, flatness has become one of the most important indicators for measuring the quality of cold rolled strips. In this paper, the strip production data of a 1250 mm tandem cold mill in a steel plant is modeled by an improved deep neural network (the improved DNN) to improve the accuracy of strip shape prediction. Firstly, the type of activation function is analyzed, and the monotonicity of the activation function is deemed independent of the convexity of the loss function in the deep network. Regardless of whether the activation function is monotonic, the loss function is not strictly convex. Secondly, the non-convex optimization of the loss functionextended from the deep linear network to the deep nonlinear network, is discussed, and the critical point of the deep nonlinear network is identified as the global minimum point. Finally, an improved Swish activation function based on batch normalization is proposed, and its performance is evaluated on the MNIST dataset. The experimental results show that the loss of an improved Swish function is lower than that of other activation functions. The prediction accuracy of a deep neural network (DNN) with an improved Swish function is 0.38% more than that of a deep neural network (DNN) with a regular Swish function. For the DNN with the improved Swish function, the mean square error of the prediction for the flatness of cold rolled strip is reduced to 65% of the regular DNN. The accuracy of the improved DNN is up to and higher than the industrial requirements. The shape prediction of the improved DNN will assist and guide the industrial production process, reducing the scrap yield and industrial cost.

Involvement of PAR2 in platelet-derived growth factor receptor-α-positive cell proliferation in the colon of diabetic mice.

Our previous study indicated that streptozotocin (STZ)-induced diabetes leads to colonic platelet-derived growth factor receptor-α-positive (PDGFRα+ ) cell proliferation accompanied by slow colonic transit in mice; however, the mechanism of this effect is unclear. The present study used western blotting, immunohistochemistry, and quantitative PCR to investigate whether proteinase-activated receptor 2 (PAR2) mediates PDGFRα+ cell proliferation. Our results showed that PDGFRα, PAR2, and Ki-67 coexpression was increased in the diabetic colonic muscle layer. PDGFRα and PAR2 mRNA and protein expression levels were also markedly enhanced in the diabetic colonic muscle layer. Mice treated with 2-furoyl-LIGRLO-amide (2-F-L-a), a PAR2 agonist, exhibited significant colon elongation and increased smooth muscle weight. In the 2-F-L-a-treated mice, PDGFRα, PAR2, and Ki-67 coexpression was increased and PDGFRα and PAR2 mRNA and protein expression was significantly enhanced in the colonic smooth muscle layer. 2-F-L-a also increased proliferation and PDGFRα expression in NIH/3T3 cells cultured in high glucose, while LY294002, a PI3K antagonist, decreased cell proliferation and PDGFRα expression. PI3K and Akt protein and mRNA expression and p-Akt protein expression in diabetic and 2-F-L-a-treated mice were markedly reduced in colonic smooth muscle. 2-F-L-a also reduced PI3K, Akt, and p-Akt protein expression in NIH/3T3 cells, while the PI3K antagonist LY294002 increased this expression. The results indicate that PAR2 is involved in the proliferation of PDGFRα+ cells through the PI3K/Akt signaling pathway in the colon of STZ-induced diabetic mice, which may contribute to the slow transit and constipation that are associated with diabetes.

Diabetes-induced damage of gastric nitric oxide neurons mediated by P2X7R in diabetic mice.

It is generally considered that enteric neuropathy is one of the causative factors in diabetic gastroparesis. Our previous study demonstrated that there is a loss of NOS neurons in diabetic mice. However, the underlying mechanism remains unclear. The present study was designed to clarify the relationship between neuronal P2X7R and NOS neuron damage. The effect of P2X7R on diabetes-induced gastric NOS neurons damage and its mechanism were investigated by using quantitative RT-PCR，immunofluorescence, western blot, isometric force recording, intracellular calcium ([Ca2+]i) measurement and whole-cell patch clamp techniques. The immunohistochemistry and western blot results showed that nNOS expression was significantly down-regulated in diabetic mice, meanwhile, electric field stimulation-induced NOS sensitive relaxation was significantly suppressed. Myenteric neurons expressed P2X7R and pannexin1, and the mRNA and protein level of P2X7R and pannexin1 were up-regulated in diabetic mice. BzATP, a P2X7R activator, evoked [Ca2+]i increase in Hek293 cells with heterologous expression of P2X7R (Hek293-P2X7R cells) and the same dose of ATP-induced [Ca2+]i was more obvious in Hek293-P2X7R cells than in Hek293 cells. Application of BzATP activated an inward current of Hek293-P2X7R in a dose dependent manner. Hek293-P2X7R but not untransfected Hek293 cells could take up of YO-PRO-1. In addition, the uptake of YO-PRO-1 by Hek293-P2X7R was blocked by oxATP, a P2X7 antagonist and CBX, a pannexin1 inhibitor. The results suggest that the P2X7R of enteric neurons may be involved in diabetes-induced NOS neuron damage via combining with pannexin-1 to form transmembrane pores which induce macromolecular substances and calcium into the cells.

Colonic PDGFRα Overexpression Accompanied Forkhead Transcription Factor FOXO3 Up-Regulation in STZ-Induced Diabetic Mice.

BACKGROUND: Colonic transit disorder-induced constipation is a major complication in diabetic patients. PDGFRα+ (platelet-derived growth factor receptor α-positive) cells play critical roles in the inhibitory regulation of colonic motility, and FOXO3 (forkhead transcription factor 3) has a broad range of biological functions. The present study was designed to investigate the relationship between FOXO3 and PDGFRα+ cell proliferation in streptozotocin (STZ)-induced diabetic mice. METHODS: The major experimental techniques used in this paper are immunohistochemistry, quantitative RT-RCR and Western blotting for the evaluation of specific protein expression; ChIP assay for identifying the interaction between FOXO3 protein and the PDGFRα promotor; and lentiviral transfection for the overexpression of short hairpin RNAs (shRNAs) to down-regulate FOXO3. RESULTS: In proximal colonic smooth muscle tissue of STZ-induced diabetic mice, there was a significant increase in PDGFRα and Ki67 immunoreactivity. PDGFRα mRNA and protein expression levels were both significantly increased in colonic smooth muscle tissue, but PDGFRß expression was unchanged. Meanwhile, the expression of PDGF ligands, including both PDGFα and PDGFß, was significantly increased in diabetic colonic smooth muscle tissue. In whole cell and nuclear extracts, the expression of FOXO3 protein was also significantly increased; however, the expression of P-FOXO3 (phosphorylated FOXO3) protein was significantly decreased. When NIH cells were incubated with 50 mmol/L glucose for 12 h, 24 h and 48 h, the expression of PDGFRα significantly increased, and in whole cell and nuclear extracts, the expression of FOXO3 protein was significantly increased. However, the expression of P-FOXO3 protein was significantly decreased. FOXO3 could bind to a site on the PDGFRα promoter, and the basal expression of PDGFRα was significantly reduced when endogenous FOXO3 expression was knocked down with FOXO3 short hairpin RNA (shRNA) in NIH cells. The expression of phosphorylated Akt was significantly down-regulated in diabetic colonic muscle tissue. CONCLUSIONS: These results suggest that diabetes-induced colonic PDGFRα+ cell proliferation is mediated by FOXO3 up-regulation. FOXO3 up-regulation may be induced by inhibiting the PI3K/Akt signaling pathway in STZ-induced diabetic mice. PDGFRα+ cell proliferation could be a new target for clinical therapy of diabetes-induced colonic transit disorder.

[Physiological and pathophysiological meanings of gastrointestinal smooth muscle motor unit SIP syncytium].

Gastrointestinal smooth muscle layer contains two kinds of interstitial cells with special differentiation, i.e., interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor α-positive (PDGFRα+) cells. The ICC and PDGFRα+ cells contact with smooth muscle cells (SMCs) by gap junctions and regulate contractive function of the SMCs. Therefore, these three kinds of cells constitute a functional syncytium, i.e., the SMC, ICC and PDGFRα+ cells syncytium (SIP syncytium). Various neurotransmitters, humoral factors, endogenous bioactive molecules, as well as drugs regulate gastrointestinal motility through the SIP syncytium. In this review, we introduce the concept of SIP syncytium and summarize functions of the syncytium, as well as its physiological and pathological significances.