[Bioinformatics analysis of the microRNA expression profile in the peripheral blood lymphocytes of Kazakh patients with essential hypertension in Xinjiang].

This study aimed to investigate the differential expression profiles of microRNAs (miRNAs) in peripheral blood lymphocytes between patients with essential hypertension and healthy individuals in Xinjiang Kazakh and to provide insight into the mechanism involved in the pathogenesis of hypertension in this ethnic group. From April 2016 to May 2019, 30 Kazakh patients with essential hypertension in the inpatient and outpatient departments of Cardiology, First Affiliated Hospital of Shihezi University were used as the hypertension group; 30 healthy Kazakh patients were used as the control group. The miRNA expression profiles in peripheral blood lymphocytes of 6 Kazakh hypertensive patients and 6 matched healthy individuals were compared, and the differentially expressed miRNAs were analyzed by cluster analysis, GSEA enrichment analysis, target gene prediction, target gene annotation and other bioinformatics analyses. In addition, qRT-PCR was used to verify the differentially expressed miRNAs. The results showed that compared with the control group, 73 differentially expressed miRNAs were identified in the hypertension group, of which 39 miRNAs were up-regulated and 34 miRNAs were down-regulated. A total of 11 miRNAs related to hypertension were screened by GSEA enrichment analysis, including hsa-miR-100-5p, hsa-miR-150-5p, hsa-miR-299-5p, hsa-miR-299-3p, hsa-miR-296-5p, hsa-miR-196b-5p, hsa-miR-503-5p, hsa-miR-628-5p, hsa-miR-874-3p, hsa-miR-543 and hsa-miR-940. qRT-PCR test found that the expression of hsa-miR-100-5p, hsa-miR-299-5p, hsa-miR-299-3p, hsa-miR-196b-5p, hsa-miR-503-5p, hsa-miR-628-5p and hsa-miR-543 was up-regulated, while the expression of hsa-miR-150-5p, hsa-miR-296-5p, hsa-miR-874-3p and hsa-miR-940 was down-regulated in the hypertension group compared with the control group. The expression trend in the gene chip was consistent with the results verified by qRT-PCR. Using online database to predict target genes of 11 miRNAs related to hypertension, we found that a total of 1 647 target genes might be regulated by these 11 miRNAs. GO function enrichment showed that (a) in biological processes, the predicted hypertension related target genes are mainly relevant to nervous system development, cellular localization, regulation of cellular metabolic process, generation of neurons and positive regulation of biological process; (b) In terms of cellular components, they are mainly related to membrane-bounded organelle, cytoplasm, intracellular membrane-bounded organelle, synapse part, neuron part, and nucleoplasm; (c) In terms of molecular function, they are mainly related to protein binding, transcription regulatory region DNA binding, RNA polymerase II regulatory region DNA binding, transcription regulator activity, and ion binding. KEGG enrichment analysis showed that the p53 signaling pathway, adrenergic signaling in cardiomyocytes, cAMP signaling pathway, TGF-ß signaling pathway, endocrine and other factor-regulated calcium reabsorption, mTOR signaling pathway, and aldosterone-regulated sodium reabsorption may be related to the occurrence and development of hypertension. In conclusion, there are significant differences in the expression of miRNAs in peripheral blood lymphocytes between Kazakh patients with essential hypertension and healthy people. The differentially expressed miRNAs may be related to the occurrence and development of essential hypertension in Kazakh. However, the underlying mechanism needs to be further explored and verified.

Inhibition of KIR2.1 decreases pulmonary artery smooth muscle cell proliferation and migration.

The investigation of effective therapeutic drugs for pulmonary hypertension (PH) is critical. KIR2.1 plays crucial roles in regulating cell proliferation and migration, and vascular remodeling. However, researchers have not yet clearly determined whether KIR2.1 participates in the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) and its role in pulmonary vascular remodeling (PVR) also remains elusive. The present study aimed to examine whether KIR2.1 alters PASMC proliferation and migration, and participates in PVR, as well as to explore its mechanisms of action. For the in vivo experiment, a PH model was established by intraperitoneally injecting Sprague­Dawley rats monocrotaline (MCT). Hematoxylin and eosin staining revealed evidence of PVR in the rats with PH. Immunofluorescence staining and western blot analysis revealed increased levels of the KIR2.1, osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) proteins in pulmonary blood vessels and lung tissues following exposure to MCT, and the TGF­ß1/SMAD2/3 signaling pathway was activated. For the in vitro experiments, the KIR2.1 inhibitor, ML133, or the TGF­ß1/SMAD2/3 signaling pathway blocker, SB431542, were used to pre­treat human PASMCs (HPASMCs) for 24 h, and the cells were then treated with platelet­derived growth factor (PDGF)­BB for 24 h. Scratch and Transwell assays revealed that PDGF­BB promoted cell proliferation and migration. Immunofluorescence staining and western blot analysis demonstrated that PDGF­BB upregulated OPN and PCNA expression, and activated the TGF­ß1/SMAD2/3 signaling pathway. ML133 reversed the proliferation and migration induced by PDGF­BB, inhibited the expression of OPN and PCNA, inhibited the TGF­ß1/SMAD2/3 signaling pathway, and reduced the proliferation and migration of HPASMCs. SB431542 pre­treatment also reduced cell proliferation and migration; however, it did not affect KIR2.1 expression. On the whole, the results of the present study demonstrate that KIR2.1 regulates the TGF­ß1/SMAD2/3 signaling pathway and the expression of OPN and PCNA proteins, thereby regulating the proliferation and migration of PASMCs and participating in PVR.

Reverse relationship between autophagy and apoptosis in an in vitro model of cortical neuronal injury.

Autophagy and apoptosis are intertwined, and their relationship involves complex cross-talk. Whether the activation and inhibition of autophagy protect or damage neurons in the central nervous system has been a matter of longstanding controversy. We investigated the effect of autophagy on the apoptosis of cortical neurons after oxygen- and glucose-deprivation/reoxygenation (OGD/R) injury in vitro and found that protective mechanism activation was the predominant response to enhanced autophagy activation and increased autophagic flux. After successful establishment of an OGD/R model with cortical neurons, the autophagy activator rapamycin (Rap) or the late-autophagy inhibitor bafilomycin A1 (BafA1) was added to cell groups according to the experimental design. Cell viability was determined by Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) assays, and the apoptosis rate was measured by analysing Annexin V-FITC/PI-stained cells. The protein and mRNA expression levels of the apoptosis factors Caspase8 and Caspase3 and autophagy-associated proteins LC3 and p62 were measured by Western blotting and RT-qPCR. The extent of autophagic flux was determined by measuring the intensity of double immunofluorescence labelled protein after cells were transfected with RFP-GFP-LC3-expressing virus, and the ultrastructures of autophagosomes were observed by transmission electron microscopy (TEM). The results showed that cell viability decreased and that cells underwent autophagy and apoptosis after OGD/R. After the addition of Rap, cell viability was increased, and the apoptosis rate was decreased significantly. In addition, the level of the autophagic flux protein LC3II was increased, and the level of p62 was decreased. The number of autophagosomes and the ratio of autophagosomes to lysosomes were increased significantly. After BafA1 intervention, however, these results were reversed, with decreased cell viability, a significantly increased apoptosis rate, and disrupted autophagic flux. In conclusion, enhanced autophagy activation or autophagic flux exerted a significant protective effect on neurons after OGD/R injury in vitro.

[Effects of probenecid on the migration and proliferation ability of platelet derived growth factor-BB induced pulmonary artery smooth muscle cells in rats].

OBJECTIVE: To investigate whether probenecid (PROB) could improve the proliferation and migration ability of rats' pulmonary artery smooth muscle cells induced by platelet-derived growth factor-BB (PDGF-BB). METHODS: Primary pulmonary artery smooth muscle cells (PASMCs) of SD rats were cultured in vitro, and were randomly divided into control group (CON group), PDGF-BB group (10 ng/ml PDGF-BB treatment for 24 h) and PDGF-BB+PROB group (10 ng/ml PDGF-BB and 200 µmol/L PROB treatment for 24 h, PROB is a specific blocker of pannexin-1). CCK-8 method was used to select the suitable intervention concentrations of PROB and PDGF-BB, and to detect the proliferation of PASMCs in each group. The migration ability of PASMCs was detected by TranswellTM assay and cell scratch test. Immunofluorescence cytochemistry and Western blot were used to detect the protein expressions and distribution of osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) in PASMCs. RESULTS: Compared with CON group, the migration and proliferation ability of PASMCs in PDGF-BB group were enhanced (P＜0.05). After treated with PROB, the migration and proliferation ability of PASMCs in PDGF-BB+PROB group were decreased significantly (P＜0.05). Compared with CON group, the expression and protein levels of OPN and PCNA in PDGF-BB group were increased significantly (P＜0.05), while the expression and protein levels of OPN and PCNA in PDGF-BB+PROB were decreased significantly (P＜0.05). CONCLUSION: Probenecid inhibits the migration and proliferation of PDGF-BB-induced PASMCs by blocking Pannexin-1.

17ß-Estradiol promotes angiogenesis of stria vascular in cochlea of C57BL/6J mice.

It is widely accepted that the stria vascularis (SV) in cochlea plays a critical role in the generation of endocochlear potential (EP) and the secretion of the endolymph. 17ß-estradiol (E2) is the most potent and abundant endogenous estrogen during the premenopausal period, thus, considered as the reference estrogen. This study aimd to investigate the protective effect of E2 by promoting the expression of vascular endothelial growth factor (VEGF) and thus promoting the vascular regeneration of the SV in elderly mice. After being treated with E2 either in vivo or in vitro, the hearing threshold changes of C57BL/6J elder mice continuously reduced, endothelial cell morphology improved, the number of endothelial cells (ECs) tubular nodes increased significantly, the ability of tubular formation enhanced significantly and the expression of VEGF increased. In vitro, cell model in conjunction with in vivo ovariectomized model was established to demonstrate for the first time that E2 promotes angiogenesis by promoting the secretion of VEGF through the phosphatidylinositol 3-kinase (PI3K)/AKT pathway (PI3K/AKT). In conclusion, E2 demonstrated potent angiogenesis properties with significant protection against Age-Related Hearing Loss (ARHL), which provides a new idea for the improvement of ARHL.

Neuritin attenuates oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal injury by promoting autophagic flux.

The autophagy/apoptosis interaction has always been a focus of study in pathogenicity models. Neuritin is a neurotrophic factor that is highly expressed primarily in the central nervous system. Our previous study revealed that it protects against apoptosis in cortical neurons subjected to oxygen-glucose deprivation (OGD)/reoxygenation (OGD/R), and later animal experiments revealed that it can increase the expression of the autophagy-related protein LC3. Whether this neuroprotective effect is closely related to autophagy is still unclear. In this study, we hypothesized that neuritin can promote autophagic flux to protect nerve cells after OGD/R. To verify this hypothesis, we induced OGD/R in primary cortical neurons and assessed cell viability by the CCK8 and LDH assays. Cell apoptosis was assessed by Annexin V-FITC/PI, staining, and the contents and mRNA abundances of the autophagy-related proteins LC3 and p62, the apoptotic protein Caspase3 were quantified by Western blotting and RT-PCR. Autophagic flux was assessed by immunofluorescence after RFP-GFP-LC3 virus transfection, and ultrastructural changes in autophagosomes were observed by transmission electron microscopy (TEM). The results showed that cell viability was decreased, apoptosis was increased and autophagy was enhanced after OGD/R. Neuritin significantly increased cell viability, decreased apoptosis, further increased the expression of the autophagic flux-related protein LC3, further decreased p62 expression, and significantly increased the autophagosome number and autophagosome to lysosome ratio. Bafilomycin A1 (BafA1) is a late autophagy inhibitor, aggravated cell damage and apoptosis and counteracted the enhancement of autophagy activation and protective effects of neuritin. In conclusion, neuritin may promote the completion of autophagic flux by ameliorating neuronal damage after OGD/R.

The promoting role of Cx43 on the proliferation and migration of arterial smooth muscle cells for angiotensin II-dependent hypertension.

BACKGROUND: Recent studies have shown that endothelin-1 and angiotensin II (AngII) can increase gap junctional intercellular communication (GJIC) by activating Mitogen-activated protein kinases (MAPKs) pathway. However, not only the precise interaction of AngII with Connexin43(Cx43) and the associated functions remain unclear, but also the regulatory role of Cx43 on the AngII-mediated promotion proliferation and migration of VSMCs is poorly understood. MATERIAL AND METHODS: Our research applicated pressure myography measurements, immunofluorescence and Western blot analyses to investigate the changes in physiological indicators in spontaneously hypertensive rats (SHRs) and AngII-stimulated proliferation and migration of A7r5 SMCs(Rat vascular smooth muscle cells). The aim was to elucidate the role of CX43 in hypertension induced by AngII. RESULTS: Chronic ramipril (angiotensin converting enzyme inhibitor) management for SHRs significantly attenuated blood pressure and blood vessel wall thickness, also reduced contraction rate in the cerebral artery. The cerebral artery contraction rates, mRNA and protein expression of Cx43, osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) protein expression in the SHR + ramipril and SHR + ramipril + carbenoxolone (CBX, Cx43 specific blocker) groups were significantly lower than those in the SHR group. Cx43 protein expression and Ser368 phosphorylated Cx43 protein levels increased significantly in AngII-stimulated A7r5 cells. However, the levels of phosphorylated Cx43 decreased after pre-treatment with candesartan (AT1 receptor blocker), GF109203X (protein kinase C (PKC) blocker) and U0126 (mitogen-activated protein kinases/extracellular signal-regulated kinase1/2(MEK/ERK1/2)-specific blocker) in AngII-stimulated A7r5 cells. Cx43 was widely distributed in the cell membrane, nucleus, and cytoplasm of the SMCs. Furthermore, pre-treatment of the AngII- stimulated A7r5 cells with Gap26 (Cx43 blocker) significantly inhibited cell migration and decreased the expression levels of MEK1/2, ERK1/2, P-MEK1/2, and P-ERK1/2. CONCLUSION: Our research confirms that Cx43 plays an important role in the regulation of proliferation and migration of VSMCs via MEK/ERK and PKC signal pathway in AngII-dependent hypertension.

Detection of autophagic flux in primary cerebral cortical neurons after oxygen glucose deprivation/reperfusion (OGD/R) using various methods.

The current research hot spot in the field of autophagic flux is to explain and alleviate disease from the perspective of autophagy. A highly sophisticated, sensitive, quantifiable and comprehensive method is required to accurately determine the dynamic process of autophagic flux. There are very few methods in neuroscience that specifically examine autophagic flux. Therefore, primary cortical neurons were divided into oxygen glucose deprivation/reperfusion (OGD/R) (group A) and OGD/R plus bafilomycin A1 (BafA1) (group B) groups. ① Transfection of the LC3 gene with the RFP-GFP tandem fluorescent label was performed. ② Direct quantification was performed using transmission electron microscopy (TEM). ③ Autophagy-related tools were used to detect the transformation of LC3I/II. ④ SQSTM1/P62 combined with the LC3 protein flip test was performed to comprehensively evaluate autophagic flux. Using method one, the ratio of autophagolysosomes to autophagosomes in group A was significantly increased based on fluorescence microscopy analysis. Using method two, the autophagy process in group A was more continuous and unobstructed based on TEM analysis, while only some partial processes were observed in group B, and the number of autophagosomes and autophagy lysosomes in group A was significantly greater more than that in group B. The LC3II/I ratio measured in method three was analysed in detail to explain the autophagic flux. The ratio of soluble p62 combined with the ratio of LC3II/I detected using method four reflected the activation of autophagy. In summary, each method has its own advantages, and different methods and indicators can be used to monitor different stages of autophagy. An understanding of these advantages and mastery of these methods, is a very promising strategy to systematically and objectively study central nervous system diseases, facilitate the rational use of drugs, and formulate effective treatment plans from the perspective of autophagy.

Improving NKCC1 Function Increases the Excitability of DRG Neurons Exacerbating Pain Induced After TRPV1 Activation of Primary Sensory Neurons.

Background Our aim was to investigate the effects of the protein expression and the function of sodium, potassium, and chloride co-transporter (NKCC1) in the dorsal root ganglion (DRG) after activation of transient receptor potential vanilloid 1 receptor (TRPV1) in capsaicin-induced acute inflammatory pain and the possible mechanism of action. Methods Male Sprague-Dawley rats were randomly divided into control, capsaicin, and inhibitor groups. The expression and distribution of TRPV1 and NKCC1 in rat DRG were observed by immunofluorescence. Thermal radiation and acetone test were used to detect the pain threshold of heat and cold noxious stimulation in each group. The expressions of NKCC1 mRNA, NKCC1 protein, and p-NKCC1 in the DRG were detected by PCR and western blotting (WB). Patch clamp and chloride fluorescent probe were used to observe the changes of GABA activation current and intracellular chloride concentration. After intrathecal injection of protein kinase C (PKC) inhibitor (GF109203X) or MEK/extracellular signal-regulated kinase (ERK) inhibitor (U0126), the behavioral changes and the expression of NKCC1 and p-ERK protein in L4 - 6 DRG were observed. Result: TRPV1 and NKCC1 were co-expressed in the DRG. Compared with the control group, the immunofluorescence intensity of NKCC1 and p-NKCC1 in the capsaicin group was significantly higher, and the expression of NKCC1 in the nuclear membrane was significantly higher than that in the control group. The expression of NKCC1 mRNA and protein of NKCC1 and p-NKCC1 in the capsaicin group were higher than those in the control group. After capsaicin injection, GF109203X inhibited the protein expression of NKCC1 and p-ERK, while U0126 inhibited the protein expression of NKCC1. In the capsaicin group, paw withdrawal thermal latency (WTL) was decreased, while cold withdrawal latency (CWL) was prolonged. Bumetanide, GF109203X, or U0126 could reverse the effect. GABA activation current significantly increased in the DRG cells of the capsaicin group, which could be reversed by bumetanide. The concentration of chloride in the DRG cells of the capsaicin group increased, but decreased after bumetanide, GF109203X, and U0126 were administered. Conclusion Activation of TRPV1 by exogenous agonists can increase the expression and function of NKCC1 protein in DRG, which is mediated by activation of PKC/p-ERK signaling pathway. These results suggest that DRG NKCC1 may participate in the inflammatory pain induced by TRPV1.

[Down-regulation of the Expression of Senescence Proteins P16 and P21 by Activating Connexin 43 in the Smooth Muscle of Spiral Modiolar Artery of Guinea Pigs].

OBJECTIVE: To analyze the correlation between connexin 43 (Cx43) and the expression of P16 and P21, aging-related proteins, and to investigate the possible role of Cx43 in the development of cell senescence with an aging model prepared by D-galactose (D-gal) intervention in the vascular smooth muscle cells (VSMCs) of guinea pig spiral modiolar artery (SMA). METHODS: The VSMCs of guinea pig SMA were cultured with the adhesion method, and the markers of VSMCs were detected with immunofluorescence technique. The experiment has a control group, a D-gal group, and a group that received D-gal and gap junction agonist AAP10 intervention, hereafter referred to as the AAP10 group. Cell Counting Kit-8 (CCK-8) was used to check VSMC activity and to determine the concentration and duration of D-gal intervention. The mRNA expression of Cx43 in each group was checked with qRT-PCR. The expression of Cx43, P16 and P21 proteins in each group was examined with the Western blot. The expression and distribution of P16 and P21 proteins were examined with immunofluorescence assay. RESULTS: Immunofluorescence results showed that the positive expression rate of cell actin (α-SM-actin) was over 90%. CCK-8 results showed that the optimal concentration of D-gal intervention was 30 mg/mL and the intervention duration was 48 h. qRT-PCR test showed that the mRNA expression of Cx43 in VSMCs in the D-gal group was significantly lower than that in the control group ( P<0.01), while it is higher in the AAP10 group than that of the D-gal group ( P<0.01); Western blot assay showed that the Cx43 expression level in VSMCs in the D-gal group was significantly lower than that in the control group ( P<0.01) and the expression of P16 and P21 was significantly higher than that in the control group ( P<0.01), the expression of Cx43 protein in AAP10 group was significantly up-regulated compared with that in the D-gal group ( P<0.01), while the expression of P16 and P21 was down-regulated significantly ( P<0.01); The results of immunofluorescence showed that P16 and P21 were mainly expressed in the cell nucleus. Semi-quantitative analysis of fluorescence intensity showed that the level of P16 and P21 protein in the D-gal group was significantly higher than that in the control group, and the fluorescence intensity of AAP10 group was significantly lower than that in the D-gal group ( P<0.01). CONCLUSION: Up-regulation of Cx43 expression can reverse the D-gal-induced abnormal expression of P16 and P21, two aging-related proteins, in SMA. It is suggested that Cx43 on SMA may be involved in D-gal-induced cell senescence, which provides a theoretical basis and possible intervention target for the delay of cell senescence.

[Expression of ANO1 during cardiac fibroblasts differentiation and its electrophysiological characteristics as calcium-activated chloride channel protein].

OBJECTIVE: To investigate the expression and electrophysiological characteristics of calcium-activated chlorine channel anoctamin-1 (ANO1) protein during the differentiation of cardiac fibroblasts (CFs) into myofibroblasts (MFs), and to elucidate the role of ANO1 in myocardial fibrosis. METHODS: The primary CFs from neonatal rats were isolated and the cells differentiated into MFs by subculture. The Ca2+-activated Cl- current (ICl(Ca)) in CFs and MFs were measured by whole-cell patch clamp, and the expressions of ANO1, α-smooth muscle actin(α-SMA)and vimentin in CFs and MFs were detected by immunofluorescence assay and Western blot, respectively. RESULTS: The current density in the early adherent CFs was stronger than that in MFs. ANO1 was expressed preferentially within and around the nuclei, and a small amount of ANO1 was expressed on the cell membrane. Moreover, ANO1 expression was weak in the early adherent CFs and displayed stronger expression in the MFs with proliferation tendency. CONCLUSION: The expression of ANO1 is closely related to the differentiation of MFs and it may be involved in modulation myocardial fibrosis.

Upregulation of Nav1.7 by endogenous hydrogen sulfide contributes to maintenance of neuropathic pain.

Nav1.7 is closely associated with neuropathic pain. Hydrogen sulfide (H2S) has recently been reported to be involved in numerous biological functions, and it has been shown that H2S can enhance the sodium current density, and inhibiting the endogenous production of H2S mediated by cystathionine ß­synthetase (CBS) using O­(carboxymethyl)hydroxylamine hemihydrochloride (AOAA) can significantly reduce the expression of Nav1.7 and thus the sodium current density in rat dorsal root ganglion (DRG) neurons. In the present study, it was shown that the fluorescence intensity of H2S was increased in a spared nerve injury (SNI) model and AOAA inhibited this increase. Nav1.7 is expressed in DRG neurons, and the expression of CBS and Nav1.7 were increased in DRG neurons 7, 14 and 21 days post­operation. AOAA inhibited the increase in the expression of CBS, phosphorylated (p)­MEK1/2, p­ERK1/2 and Nav1.7 induced by SNI, and U0126 (a MEK blocker) was able to inhibit the increase in p­MEK1/2, p­ERK1/2 and Nav1.7 expression. However, PF­04856264 did not inhibit the increase in CBS, p­MEK1/2, p­ERK1/2 or Nav1.7 expression induced by SNI surgery. The current density of Nav1.7 was significantly increased in the SNI model and administration of AOAA and U0126 both significantly decreased the density. In addition, AOAA, U0126 and PF­04856264 inhibited the decrease in rheobase, and the increase in action potential induced by SNI in DRG neurons. There was no significant difference in thermal withdrawal latency among each group. However, the time the animals spent with their paw lifted increased significantly following SNI, and the time the animals spent with their paw lifted decreased significantly following the administration of AOAA, U0126 and PF­04856264. In conclusion, these data show that Nav1.7 expression in DRG neurons is upregulated by CBS­derived endogenous H2S in an SNI model, contributing to the maintenance of neuropathic pain.

[Effects of estrogen on apoptosis of spiral ganglion cells in cochlea of aged C57BL/6J mice].

Objective: To observe the effects of estrogen on cochlear spiral ganglia cell apoptosis in aged C57BL/6J mice, and to explore the possible mechanism of estrogen's protective effects on senile deafness. Methods: Forty C57BL/6J mice were divided into the following four groups (10 mice/group): 3 m group (3 months old group), 12 m group (12 months old sham operation group); In the 12 m OVX group (ovariectomized at 12 months), bilateral oophorectomy was performed at the age of 9 months and normal feeding was performed until the age of 12 months.The 12m OVX+E2 group (estrogen intervention group) underwent bilateral oophorectomy at 9 months of age. After the one-month washout period, mice in the other groups were treated with estrogen at the dose of 100 µg/(kg·d) by subcutaneous injection, lasting 2 months to 12 months old. Mice in the other groups were fed normally.Blood samples were collected from the tail vein at the end of the treatment in 12 m OVX+E2 group. Enzyme-linked immunosorbent assays (ELISAs) was used to determine the serum estrogen levels. Auditory brainstem response (ABR) was used to detect the changes of hearing threshold in each group.Mice were anesthetized with 2% pentobarbital sodium. Bilateral cochlea was extracted after neck amputation and paraffin-embedded sections were performed.Hematoxylin eosin (HE) staining was used to observe the morphological changes in the cochlea spiral ganglion neurons (SGN), and TUNEL staining was used to observe the apoptosis of SGN. The expression levels of Caspase-3, Bax and Bcl-2 mRNA of the apoptotic proteins in cochlear spiral ganglion were measured by real-time fluorescence quantitative PCR (QRT-PCR). Results: Compared with the 3 m group, the hearing threshold of the 12 m group was improved, the loss of spiral ganglion cells was aggravated, and the apoptosis of the cells was increased(P＜0.01). After removal of the ovaries, the hearing threshold of the mice in the 12 m OVX group was higher than that in the 12 m control group (P＜0.01), and this increased threshold was accompanied by an increased loss of spiral ganglion cells, and increased apoptosis (P＜0.01). Meanwhile, the mRNA levels of apoptotic protein Caspase-3 and Bax were increased (P＜0.01), while the mRNA level of anti-apoptotic protein Bcl-2 was decreased (P＜0.01). After exogenous estrogen was given to the 12 m OVX+E2 group, the hearing threshold was lower than that in 12 m OVX group(P＜0.01). At the same time, the apoptosis of helical ganglion cells was reduced, the mRNA levels of Caspase-3 and Bax were decreased (P＜0.01), and the Bcl-2 mRNA level was increased (P＜0.01). Conclusion: Estrogen inhibited apoptosis of cochlear spiral ganglion cells in aged C57BL/6J mice ,thus achieving a protective effect on presbycusis.

[Effects of T16Ainh-A01 on the apoptosis and senescence of endothelial cells in the cochlea stria vascularis].

Objective: Primary cultured cochlear stria vascularis endothelial cells (ECs) of guinea pig were used to investigate the expression changes of TMEM16A and its effect on apoptosis and senescence of ECs in the cochlear stria vascularis. Methods: Primary cultured ECs in the cochlear stria vascularis were used to establish aging models according to CCK-8 and SA-ß-galactosidase. Senescent cells were randomly divided into senescent group (P12), DMSO group (P12+DMSO), T16Ainh-A01 group (P12+T16Ainh-A01). Immunofluorescence and Western blot were used to detect the expression of TMEM16A in ECs. Flow cytometry was used to detect the apoptotic rate. Western blot was used to detect the protein expressions of Bax, Bcl-2 and cleaved casepase-3 in each group. Results: The positive rate of primary cultured cochlear stria vascularis ECs was above 95%, and the 12th generation cochlear stria vascularis ECs were determined as the senescence group, and the expression of TMEM16A in protein and fluorescence was increased (P＜0.05). After intervention with T16Ainh-A01 for 24 h, the protein expressions of Bax and cleaved casepase-3 were down-regulated (P＜0.01), the protein expression of Bcl-2 was increased (P＜0.05), the apoptotic rate and the positive rate of SA-ß-gal were down-regulated (P＜0.01). Conclusion: It was found that apoptosis and TMEM16A expression were increased in cochlear stria vascularis senescent ECs, TMEM16A specific blocker T16Ainh-A01 could reduce the apoptosis and senescence in ECs of the cochlear stria vascularis. These results suggest that TMEM16A may participate in apoptosis and senescence of ECs in the cochlear stria vascularis.

Expression and effect of sodium-potassium-chloride cotransporter on dorsal root ganglion neurons in a rat model of chronic constriction injury.

Sodium-potassium-chloride cotransporter 1 (NKCC1) and potassium-chloride cotransporter 2 (KCC2) are associated with the transmission of peripheral pain. We investigated whether the increase of NKCC1 and KCC2 is associated with peripheral pain transmission in dorsal root ganglion neurons. To this aim, rats with persistent hyperalgesia were randomly divided into four groups. Rats in the control group received no treatment, and the rat sciatic nerve was only exposed in the sham group. Rats in the chronic constriction injury group were established into chronic constriction injury models by ligating sciatic nerve and rats were given bumetanide, an inhibitor of NKCC1, based on chronic constriction injury modeling in the chronic constriction injury + bumetanide group. In the experiment measuring thermal withdrawal latency, bumetanide (15 mg/kg) was intravenously administered. In the patch clamp experiment, bumetanide (10 µg/µL) and acutely isolated dorsal root ganglion neurons (on day 14) were incubated for 1 hour, or bumetanide (5 µg/µL) was intrathecally injected. The Hargreaves test was conducted to detect changes in thermal hyperalgesia in rats. We found that the thermal withdrawal latency of rats was significantly decreased on days 7, 14, and 21 after model establishment. After intravenous injection of bumetanide, the reduction in thermal retraction latency caused by model establishment was significantly inhibited. Immunohistochemistry and western blot assay results revealed that the immune response and protein expression of NKCC1 in dorsal root ganglion neurons of the chronic constriction injury group increased significantly on days 7, 14, and 21 after model establishment. No immune response or protein expression of KCC2 was observed in dorsal root ganglion neurons before and after model establishment. The Cl- (chloride ion) fluorescent probe technique was used to evaluate the change of Cl- concentration in dorsal root ganglion neurons of chronic constriction injury model rats. We found that the relative optical density of N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (a Cl- fluorescent probe whose fluorescence intensity decreases as Cl- concentration increases) in the dorsal root ganglion neurons of the chronic constriction injury group was significantly decreased on days 7 and 14 after model establishment. The whole-cell patch clamp technique revealed that the resting potential and action potential frequency of dorsal root ganglion neurons increased, and the threshold and rheobase of action potentials decreased in the chronic constriction injury group on day 14 after model establishment. After bumetanide administration, the above indicators were significantly suppressed. These results confirm that CCI can induce abnormal overexpression of NKCC1, thereby increasing the Cl- concentration in dorsal root ganglion neurons; this then enhances the excitability of dorsal root ganglion neurons and ultimately promotes hyperalgesia and allodynia. In addition, bumetanide can achieve analgesic effects. All experiments were approved by the Institutional Ethics Review Board at the First Affiliated Hospital, College of Medicine, Shihezi University, China on February 22, 2017 (approval No. A2017-169-01).

Carbenoxolone decreases monocrotaline­induced pulmonary inflammation and pulmonary arteriolar remodeling in rats by decreasing the expression of connexins in T lymphocytes.

The adaptive immune response mediated by T lymphocytes is a well­established factor in the pathogenesis of pulmonary inflammation. Changes in the expression of various connexins (Cxs) or disruption of connexin­mediated cellular communication in T lymphocytes contribute to inflammation or tissue remodeling. The aim of the present study was to investigate the potential therapeutic value of blocking Cxs in a monocrotaline (MCT)­induced pulmonary inflammation rat model. Carbenoxolone (CBX) was used to inhibit connexin­mediated cellular communication. An MCT rat model was established by intraperitoneal (i.p.) injection of a single dose of MCT (60 mg/kg), and CBX treatment (20 µg/kg/day, i.p.) was initiated on the day following MCT treatment for 28 days. Vehicle­treated male Sprague­Dawley rats were used as the negative control. The MCT rat model was evaluated by measuring the pulmonary artery flow acceleration time and right ventricular hypertrophy index (RVHI). Histopathological features of the lung tissues and pulmonary arteriolar remodeling were assessed. The proportions of T lymphocyte subtypes, Cx40/cx43 expression in the T cell subtypes and the cytokine levels in the plasma and the lung tissues were also analyzed. Pharmacological inhibition of Cxs using CBX attenuated MCT­induced right ventricular hypertrophy, pulmonary arteriolar remodeling, lung fibrosis and inflammatory cell infiltration by decreasing the RVHI, pulmonary arterial wall thickening, collagen deposition and pro­inflammatory cytokines production as well as CD3+ and CD4+ T cell accumulation in lung tissues of MCT­treated rats. Furthermore, flow cytometry analysis revealed that CBX may inhibit MCT­induced Cx40 and Cx43 expression in CD4+ and CD8+ T lymphocytes in lung tissues. The present study provides evidence that pharmacological inhibition of Cxs may attenuate MCT­induced pulmonary arteriolar remodeling and pulmonary inflammatory response, at least in part, by decreasing Cx expression. The results highlight the critical role of Cxs in T lymphocytes in the MCT­induced pulmonary inflammatory response and that targeting of Cxs may be a potential therapeutic method for treating pulmonary inflammatory diseases.

17ß-Estradiol Attenuates Neuropathic Pain Caused by Spared Nerve Injury by Upregulating CIC-3 in the Dorsal Root Ganglion of Ovariectomized Rats.

17ß-estradiol plays a role in pain sensitivity, analgesic drug efficacy, and neuropathic pain prevalence, but the underlying mechanisms remain unclear. Here, we investigated whether voltage-gated chloride channel-3 (ClC-3) impacts the effects of 17ß-estradiol (E2) on spared nerve injury (SNI)-induced neuropathic pain in ovariectomized (OVX) female Sprague Dawley rats that were divided into OVX, OVX + SNI, OVX + SNI + E2, OVX + SNI + E2 + DMSO (vehicle, dimethyl sulfoxide), or OVX + SNI + E2+Cltx (ClC-3-blocker chlorotoxin) groups. Changes in ClC-3 protein expression were monitored by western blot analysis. Behavioral testing used the paw withdrawal threshold to acetone irritation and paw withdrawal thermal latency (PWTL) to thermal stimulation. Immunofluorescence indicated the localization and protein expression levels of ClC-3. OVX + SNI + E2 rats were subcutaneously injected with 17ß-estradiol once daily for 7 days; a sheathed tube was implanted, and chlorotoxin was injected for 4 days. Intrathecal Cltx to OVX and OVX + SNI rats was administered for 4 consecutive days (days 7-10 after SNI) to further determine the contribution of ClC-3 to neuropathic pain. Patch clamp technology in current clamp mode was used to measure the current threshold (rheobase) dorsal root ganglion (DRG) neurons and the minimal current that evoked action potentials (APs) as excitability parameters. The mean number of APs at double-strength rheobase verified neuronal excitability. There was no difference in behaviors and ClC-3 expression after OVX. Compared with OVX + SNI rats, OVX + SNI + E2 rats showed a lower paw withdrawal threshold to the acetone stimulus, but the PWTL was not significantly different, indicating increased sensitivity to cold but not to thermal pain. Co-immunofluorescent data revealed that ClC-3 was mainly distributed in A- and C-type nociceptive neurons, especially in medium/small-sized neurons. 17ß-estradiol administration was associated with increased expression of ClC-3. 17ß-estradiol-induced increase in ClC-3 expression was blocked by co-administration of Cltx. Cltx causes hyperalgesia and decreased expression of ClC-3 in OVX rats. Patch clamp results suggested that 17ß-estradiol attenuated the excitability of neurons induced by SNI by up-regulating the expression of ClC-3 in the DRG of OVX rats. 17ß-estradiol administration significantly improved cold allodynia thresholds in OVX rats with SNI. The mechanism for this decreased sensitivity may be related to the upregulation of ClC-3 expression in the DRG.

[Effects of calcium-sensitive receptors on 11ß-hydroxysteroid dehydrogenase type 2 and cortisol in neonatal mice with persistent pulmonary hypertension].

OBJECTIVE: To investigate the effects of calcium-sensitive receptors (CaSR) on the expression of 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) and cortisol concentration in a neonatal mouse model of persistent pulmonary hypertension (PPH). METHODS: Fifty-six newborn C57BL/6 mice were randomly divided into a control group (n=14), a PPH group (n=14), an agonist group (n=14), and an inhibitor group (n=14). The mice in the PPH, agonist, and inhibitor groups were exposed to a 12% oxygen concentration, and the agonist group and inhibitor group were given CaSR agonist (GdCl3, 16 mg/kg) and CaSR antagonist (NPS2390, 1 mg/kg) intraperitoneally once a day, respectively. The mice in control group were exposed to air, and then injected with an equal volume of normal saline as those in the PPH group every day. All mice were treated for 14 days. Morphological examination of heart and lung tissues was performed using hematoxylin-eosin staining. The expression levels of 11ß-HSD2 mRNA and 11ß-HSD2 protein in lung tissues were measured by qRT-PCR and Western blot respectively. Brain natriuretic peptide (BNP) and cortisol levels in lung tissues were determined using ELISA. RESULTS: Compared with the control group, the PPH group had significantly increased pulmonary artery wall thickness (WT%), ratio of right to left ventricular thickness (RV/LV), alveolar mean linear intercept, and BNP concentration and a significantly reduced radial alveolar count (P<0.05); compared with the PPH group, the agonist group showed significant increases in WT% and BNP concentration, while the inhibitor group showed significant reductions in the two indicators (P<0.05). Compared with the control group, the PPH group showed significant reductions in the expression levels of 11ß-HSD2 mRNA and 11ß-HSD2 protein, but a significant increase in cortisol concentration (P<0.05); compared with the PPH group, the agonist group had significantly lower expression levels of 11ß-HSD2 mRNA and 11ß-HSD2 protein, but a significant higher cortisol concentration, while the inhibitor group showed opposite changes in these indicators (P<0.05). CONCLUSIONS: CaSR may control the development and progression of PPH in newborn mice by regulating the expression of 11ß-HSD2 and cortisol concentration.

[G protein-coupled estrogen receptor alleviates cerebral ischemia-reperfusion injury through inhibiting endoplasmic reticulum stress].

The aim of this study was to investigate whether G protein-coupled estrogen receptor (GPER) could alleviate hippocampal neuron injury under cerebral ischemia-reperfusion injury (CIRI) by acting on endoplasmic reticulum stress (ERS). The CIRI animal model was established by middle cerebral artery occlusion (MCAO). Female ovariectomized (OVX) Sprague-Dawley (SD) female rats were randomly divided into 4 groups: control, ischemia-reperfusion injury (MCAO), vehicle (MCAO+DMSO), and GPER-specific agonist G1 (MCAO+G1) groups. The neurobehavioral score was assessed by the Longa score method, the morphological changes of the neurons were observed by the Nissl staining, the cerebral infarction was detected by the TTC staining, and the neural apoptosis in the hippocampal CA1 region was detected by TUNEL staining. The distribution and expression of GRP78 (78 kDa glucose-regulated protein 78) in the hippocampal CA1 region were observed by immunofluorescent staining. The protein expression levels of GRP78, Caspase-12, CHOP and Caspase-3 were detected by Western blot, and the mRNA expression levels of GRP78, Caspase-12, and CHOP were detected by the real-time PCR. The results showed that the neurobehavioral score, cerebral infarct volume, cellular apoptosis index, as well as GRP78, Caspase-12 and CHOP protein and mRNA expression levels in the MCAO group were significantly higher than those of control group. And G1 reversed the above-mentioned changes in the MCAO+G1 group. These results suggest that the activation of GPER can decrease the apoptosis of hippocampal neurons and relieve CIRI, and its mechanism may involve the inhibition of ERS.

Age­associated variation in the expression and function of TMEM16A calcium­activated chloride channels in the cochlear stria vascularis of guinea pigs.

The present study was designed to investigate the expression and function of transmembrane protein 16 (TMEM16A), a calcium­activated chloride channel (CaCC), in the stria vascularis (SV) of the cochlea of guinea pigs at different ages, and to understand the role of CaCCs in the pathogenesis of presbycusis (age­related hearing loss), the most common type of sensorineural hearing loss that occurs with natural aging. Guinea pigs were divided into the following groups: 2 weeks (young group), 3 months (youth group), 1 year (adult group), D­galactose intervention (D­gal group; aging model induced by subcutaneous injection of D­galactose) and T16Ainh­A01 (intraperitoneal injection of 50 µg/kg/day TMEM16A inhibitor T16Ainh­A01 for 2 weeks). Differences in the hearing of guinea pigs between the various age groups were analyzed using auditory brainstem response (ABR), and immunofluorescence staining was performed to detect TMEM16A expression in the SV and determine the distribution. Reverse transcription­quantitative PCR and western blot analyses were conducted to detect the mRNA and protein levels of TMEM16A in SV in the different age groups. Morris water maze behavior analysis demonstrated that spatial learning ability and memory were damaged in the D­gal group. Superoxide dismutase activity and malondialdehyde content assays indicated that there was oxidative stress damage in the D­gal group. The ABR thresholds gradually increased with age, and the increase in the T16Ainh­A01 group was pronounced. Immunofluorescence analysis in the cochlear SV of guinea pigs in different groups revealed that expression of TMEM16A increased with increasing age (2 weeks to 1 year); fluorescence intensity was reduced in the D­gal model of aging. As the guinea pigs continued to mature, the protein and mRNA contents of TMEM16A in the cochlea SV increased gradually, but were decreased in the D­gal group. The findings indicated that CaCCs in the cochlear SV of guinea pigs were associated with the development of hearing in guinea pigs, and that downregulation of TMEM16A may be associated with age­associated hearing loss.