Quercetin attenuates cisplatin-induced mitochondrial apoptosis via PI3K/Akt mediated inhibition of oxidative stress in pericytes and improves the blood labyrinth barrier permeability.

Cisplatin (CDDP) is broadly employed to treat different cancers, whereas there are no drugs approved by the Food and Drug Administration (FDA) for preventing its side effects, including ototoxicity. Quercetin (QU) is a widely available natural flavonoid compound with anti-tumor and antioxidant properties. The research was designed to explore the protective effects of QU on CDDP-induced ototoxicity and its underlying mechanisms in male C57BL/6 J mice and primary cultured pericytes (PCs). Hearing changes, morphological changes of stria vascularis, blood labyrinth barrier (BLB) permeability and expression of apoptotic proteins were observed in vivo by using the auditory brainstem response (ABR) test, HE staining, Evans blue staining, immunohistochemistry, western blotting, etc. Oxidative stress levels, mitochondrial function and endothelial barrier changes were observed in vitro by using DCFH-DA probe detection, flow cytometry, JC-1 probe, immunofluorescence and the establishment in vitro BLB models, etc. QU pretreatment activates the PI3K/AKT signaling pathway, inhibits CDDP-induced oxidative stress, protects mitochondrial function, and reduces mitochondrial apoptosis in PCs. However, PI3K/AKT specific inhibitor (LY294002) partially reverses the protective effects of QU. In addition, in vitro BLB models were established by coculturing PCs and endothelial cells (ECs), which suggests that QU both reduces the CDDP-induced apoptosis in PCs and improves the endothelial barrier permeability. On the whole, the research findings suggest that QU can be used as a novel treatment to reduce CDDP-induced ototoxicity.

The effects of noise exposure on hippocampal cognition in C57BL/6 mice via transcriptomics.

BACKGROUND: Noise is an important environmental stressor in the industrialized world and has received increasing attention in recent years. Although epidemiological research has extensively demonstrated the relationship between noise and cognitive impairment, the specific molecular mechanisms and targets remain to be fully explored and understood. METHODS: To address this issue, 5-month-old C57BL/6 mice were divided into two groups, with one group exposed to white noise at 98 dB. The effects of noise on cognition in mice were investigated through molecular biology and behavioral experiments. Subsequently, transcriptomic sequencing of the hippocampus in both groups of mice was performed and enrichment analysis of differentially expressed genes (DEGs) was conducted using KEGG and GO databases. Furthermore, LASSO analysis was used to further narrow down the relevant DEGs, followed by enrichment analysis of these genes using KEGG and GO databases. The DEGs were further validated by rt-qPCR. RESULTS: Following noise exposure, the hippocampus levels of inflammation-related factors increased, the phosphorylation of Tau protein increased, the postsynaptic density protein decreased, the number of Nissl bodies decreased, and cell shrinkage in the hippocampus increased. Moreover, the behavioral experiments manifest characteristics indicative of a decline in cognitive.A total of 472 DEGs were identified through transcriptomic analysis, and seven relevant genes were screened by the LASSO algorithm, which were further validated by PCR to confirm their consistency with the omics results. CONCLUSION: In conclusion, noise exposure affects cognitive function in mice through multiple pathways, and the omics results provide new evidence for the cognitive impairment induced by noise exposure.

A network pharmacology- and transcriptomics-based investigation reveals an inhibitory role of ß-sitosterol in glioma via the EGFR/MAPK signaling pathway.

Glioma is characterized by rapid cell proliferation, aggressive invasion, altered apoptosis and a poor prognosis. ß-Sitosterol, a kind of phytosterol, has been shown to possess anticancer activities. Our current study aims to investigate the effects of ß-sitosterol on gliomas and reveal the underlying mechanisms. Our results show that ß-sitosterol effectively inhibits the growth of U87 cells by inhibiting proliferation and inducing G2/M phase arrest and apoptosis. In addition, ß-sitosterol inhibits migration by downregulating markers of epithelial-mesenchymal transition (EMT). Mechanistically, network pharmacology and transcriptomics approaches illustrate that the EGFR/MAPK signaling pathway may be responsible for the inhibitory effect of ß-sitosterol on glioma. Afterward, the results show that ß-sitosterol effectively suppresses the EGFR/MAPK signaling pathway. Moreover, ß-sitosterol significantly inhibits tumor growth in a U87 xenograft nude mouse model. ß-Sitosterol inhibits U87 cell proliferation and migration and induces apoptosis and cell cycle arrest in U87 cells by blocking the EGFR/MAPK signaling pathway. These results suggest that ß-sitosterol may be a promising therapeutic agent for the treatment of glioma.

PGC-1α affects cochlear pericytes migration in noise-exposed mice.

OBJECTIVE: The study aimed to observe the effects of noise exposure on the pericytes of the cochlear stria vascularis (SV) in mice and to investigate its molecular mechanism. METHOD: Male C57BL/6J mice aged 6-8 weeks were used as the subjects. Auditory Brainstem Response (ABR) was used to assess hearing loss. Hematoxylin and Eosin (HE) staining was conducted to observe morphological alterations in the SV. Immunofluorescence combined with transmission electron microscopy (TEM) was used to scrutinize changes in pericytes following acoustic injury. Western blotting (WB) was used to assess the expression variations of the migration-related protein Osteopontin (OPN). Evans Blue assay was performed to evaluate the permeability of the blood labyrinth barrier (BLB). 4-Hydroxynonenal (4-HNE) staining, in conjunction with measurements of Superoxide Dismutase (SOD), Malondialdehyde (MDA), and Catalase (CAT) content, was used to ascertain whether oxidative stress injury occurred in the SV. WB, combined with immunofluorescence, was used to examine alterations in the expression of proliferator-activated receptor-gamma coactivator 1α (PGC-1α) in the SV and pericytes. RESULTS: Noise exposure resulted in permanent hearing loss in C57BL/6J mice, accompanied by SV swelling, migration of pericytes from their vascular attachments, BLB leakage, elevated oxidative stress levels in the SV, and reduced expression of PGC-1α on both the SV and migrating pericytes. CONCLUSION: Noise exposure may potentially increase oxidative stress levels in the SV, downregulate the expression levels of PGC-1α, promote pericytes migration, and subsequently lead to an elevation in BLB permeability.

Noise exposure increase apoptosis in the hippocampus of AD mice through the upregulation of CTSS.

In recent years, it has become an acknowledged fact that noise exposure can lead to cognitive impairments, and researchers have shown increasing interest in this area. However, the detrimental impact of noise exposure on Alzheimer's disease (AD) animal models might be considerably greater than on ordinary model mice, yet the mechanisms by which noise exposure affects the hippocampus in these models have been scarcely investigated. This study we used 4D Label-free proteomics to identify distinctive differentially expressed proteins in the hippocampus of AD model mice following noise exposure. Among these proteins, the presence of Cathepsin S(CTSS) cannot be disregarded. Utilizing experimental techniques such as Western blot, immunofluorescence, and rt-qPCR, we confirmed the expression of CTSS in the hippocampus of APP/PS1 mice after noise exposure. Additionally, we examined downstream molecules including P53,BCL-2, BAX, and CASPASE3 using KEGG pathway analysis. The results indicated an elevation in CTSS expression, a reduction in the anti-apoptotic gene BCL-2, and an increase in the expression of BAX and cleaved CASPASE3. Based on these findings, we hypothesize that noise exposure potentially heightens apoptosis within the hippocampus through upregulating CTSS expression, subsequently posing a threat to AD model animals.

Effect and mechanism of chlorogenic acid on cognitive dysfunction in mice by lipopolysaccharide-induced neuroinflammation.

Background: Neuroinflammation is an important factor causing numerous neurodegenerative pathologies. Inflammation can lead to abnormal neuronal structure and function and even death, followed by cognitive dysfunction. There is growing evidence that chlorogenic acid has anti-inflammatory effects and immunomodulatory activity. Purpose: The aim of this study was to elucidate the potential targets and molecular mechanisms of chlorogenic acid in the treatment of neuroinflammation. Methods: We used the lipopolysaccharide-induced neuroinflammation mouse model and the lipopolysaccharide-stimulated BV-2 cells in vitro model. Behavioral scores and experiments were used to assess cognitive dysfunction in mice. HE staining and immunohistochemistry were used to assess neuronal damage in the mouse brain. Immunofluorescence detected microglia polarization in mouse brain. Western blot and flow cytometry detected the polarization of BV-2 cells. The migration of BV-2 cells was detected by wound healing assay and transwell assay. Potential targets for chlorogenic acid to exert protective effects were predicted by network pharmacology. These targets were then validated using molecular docking and experiments. Results: The results of in vivo experiments showed that chlorogenic acid had an obvious ameliorating effect on neuroinflammation-induced cognitive dysfunction. We found that chlorogenic acid was able to inhibit BV-2 cells M1 polarization and promote BV-2 cells M2 polarization in vitro while also inhibiting the abnormal migration of BV-2 cells. Based on the network pharmacology results, we identified the TNF signaling pathway as a key signaling pathway in which chlorogenic acid exerts anti-neuroinflammatory effects. Among them, Akt1, TNF, MMP9, PTGS2, MAPK1, MAPK14, and RELA are the core targets for chlorogenic acid to function. Conclusion: Chlorogenic acid can inhibit microglial polarization toward the M1 phenotype and improve neuroinflammation-induced cognitive dysfunction in mice by modulating these key targets in the TNF signaling pathway.

Activation of Kir2.1 improves myocardial fibrosis by inhibiting Ca 2+ overload and the TGF-ß1/Smad signaling pathway.

The inwardly rectifying potassium channel Kir2.1 is closely associated with many cardiovascular diseases. However, the effect and mechanism of Kir2.1 in diabetic cardiomyopathy remain unclear. In vivo, we use STZ to establish the model, and ventricular structural changes, myocardial inflammatory infiltration, and myocardial fibrosis severity are detected by echocardiography, histological staining, immunohistochemistry, and western blot analysis, respectively. In vitro, a myocardial fibrosis model is established with high glucose. The Kir2.1 current amplitude, intracellular calcium concentration, fibrosis-related proteins, and TGF-ß1/Smad pathway proteins are detected by whole-cell patch clamp, calcium probes, western blot analysis, and immunofluorescence, respectively. The in vivo results show that compared to diabetic cardiomyopathy, zacopride (a Kir2.1 selective agonist) significantly reduces the left ventricular systolic diameter and diastolic diameter, increases the left ventricular ejection fraction and left ventricular short-axis shortening, improves the degree of cell necrosis, and reduces the expression of myocardial interstitial fibrosis protein and collagen fibre deposition area. The in vitro results show that the current amplitude and protein expression of Kir2.1 are both decreased in the high glucose-induced myocardial fibrosis model. Additionally, zacopride significantly upregulates the expression of Kir2.1 and inhibits the expressions of the fibrosis-related proteins α-SMA, collagen I, and collagen III. Activation of Kir2.1 reduces the intracellular calcium concentration and inhibits the protein expressions of TGF-ß1 and p-Smad 2/3. Activation of Kir2.1 can improve myocardial fibrosis induced by diabetic cardiomyopathy, and the possible mechanism may be related to inhibiting Ca 2+ overload and the TGF-ß1/Smad signaling pathway.

Methyl Ferulic Acid Alleviates Neuropathic Pain by Inhibiting Nox4-induced Ferroptosis in Dorsal Root Ganglia Neurons in Rats.

Neuropathic pain is a disease that has become one of the major public health problems and a global burden. Nox4-induced oxidative stress can lead to ferroptosis and neuropathic pain. Methyl ferulic acid (MFA) can inhibit the Nox4-induced oxidative stress. This study aimed to estimate whether methyl ferulic acid alleviates neuropathic pain by inhibiting the expression of Nox4 and its induction of ferroptosis. Adult male Sprague-Dawley rats were subjected to spared nerve injury (SNI) model to induce neuropathic pain. After the establishment of the model, methyl ferulic acid was given 14 days by gavage. Nox4 overexpression was induced by microinjection of the AAV-Nox4 vector. All groups measured paw mechanical withdrawal threshold (PMWT), paw thermal withdrawal latency (PTWL), and paw withdrawal cold duration (PWCD). The expression of Nox4, ACSL4, GPX4, and ROS was investigated by Western blot and immunofluorescence staining. The changes in iron content were detected by a tissue iron kit. The morphological changes in mitochondria were observed by transmission electron microscopy. In the SNI group, the paw mechanical withdrawal threshold, the paw withdrawal cold duration decreased, the paw thermal withdrawal latency did not change, the Nox4, ACSL4, ROS, and iron content increased, the GPX4 decreased, and the number of abnormal mitochondria increased. Methyl ferulic acid can increase PMWT and PWCD but does not affect PTWL. Methyl ferulic acid can inhibit Nox4 protein expression. Meanwhile, ferroptosis-related protein ACSL4 expression was decreased, GPX4 expression was increased, ROS, iron content and abnormal mitochondrial number were decreased. By overexpressing Nox4, the PMWT, PWCD, and ferroptosis of rats were more severe than those of the SNI group, but they could be reversed after treatment with methyl ferulic acid. In conclusion, methyl ferulic acid can alleviate neuropathic pain, which is related to Nox4-induced ferroptosis.

[Role of Cav1.2 in cisplatin induced apoptosis of cochlear spiral ganglion neurons in C57BL/6J mice].

Objective: To investigate the role of Cav1.2 and its possible mechanism in the apoptosis of cochlear spiral ganglion neurons(SGNs) induced by cisplatin (CDDP) in C57BL/6J mice. Methods: Animal experiment: 8-week-old male C57BL/6J mice were randomly divided into the following two groups (10 mice/group) : normal saline group (Control group) and Cisplatin group (Cisplatin group). The Control group received daily intraperitoneal injections of normal saline, Cisplatin group was injected with cisplatin intraperitoneally at a dose of 3 mg/kg at the first 4 days of each cycle, and normal saline was injected daily at the last 10 days,repeat for 3 cycles. After administration, auditory threshold was detected by auditory brainstem response (ABR). Blood samples were collected from inner canthus of mice, and cochlea was cut off from neck. SOD and MDA kits were used to detect SOD activity and MDA content in serum and cochlea tissues. The expressions of apoptosis proteins in cochlear tissues were detected by Western blot. Morphological changes of spiral ganglion in mouse cochlea were observed by hematoxylin-eosin (HE) staining. TUNEL staining was used to observe the apoptosis of SGNs in cochlea of mice. The distribution and expression of Cav1.2 in SGNs of cochlea were observed by immunofluorescence. Cell experiment: Primary cultured SGNs were randomly divided into: control group (Control), solvent group (DMSO), Cav1.2 blocker group (N), cisplatin group, cisplatin and Cav1.2 blocker co-incubation group (Cisplatin+N). 5 µmol/L cisplatin was selected to treat SGNs based on the results of CCK8. Western blot was used to detect the protein expressions of Cav1.2.and apoptotic proteins. Hoechst33342 staining was used to observe the apoptosis of each group. Flow cytometry was used to detect the apoptosis rate of each group. Mitochondrial superoxide indicator (MitoSOXTM-Red) was used to detect the ROS release of mitochondria. Results: Animal experiments: Compared to the Control group, the hearing threshold was increased in Cisplatin group (P<0.01), the content of MDA in serum and cochlea tissues, apoptosis protein Cleaved caspase-3, Bax protein level, TUNEL positive rate, Cav1.2 protein expression level were increased significantly (P<0.05, P<0.01); the activity of SOD in serum and cochlear tissue, anti-apoptotic protein bcl-2 protein level and SGCs density in cochlear tissue were decreased significantly (P<0.05, P<0.01). Cell tests: Compared with the Control group, the expression of Cav1.2, apoptosis rate, Cleaved caspase-3, Bax protein level, intracellular calcium ion concentration, and ROS release were increased significantly only in Cisplatin group (P<0.05, P<0.01). The levels of bcl-2 protein and mitochondrial membrane potential were decreased significantly (P<0.01). Cav1.2 blockers could partially reverse the above changes (P<0.05). Conclusion: Cisplatin may increase intracellular Ca2+ concentration through up-regulation of Cav1.2, and then damage mitochondria, causing oxidative stress injury of SGNs and inducing neuronal apoptosis.

[Capsaicin inhibits proliferation and migration of cerebral basilar artery smooth muscle cells in spontaneously hypertensive rats].

Objective To investigate whether capsaicin (CAP) can improve the proliferation and migration of cerebral basilar artery smooth muscle cells (BASMCs) in spontaneously hypertensive rats (SHR). Methods Primary BASMCs of SHR and Wistar-Kyoto (WKY) rats were cultured in vitro, randomly divided into control group (WKY group), SHR group and capsaicin treatment group (CAP group). The intervention concentration of CAP was determined by CCK-8 assay; TranswellTM chamber assay and scratch test were used to detect the migration ability of BASMCs; the expression and distribution of osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) in BASMCs were detected by immunofluorescence assay, and Western blot analysis was used to detect the protein levels of OPN and PCNA in BASMCs. Results Compared with WKY group, the proliferation and migration ability of BASMC in SHR group were enhanced, while the CAP treatment undermined the proliferation and migration of BASMCs. OPN was expressed in the cytoplasm and nucleus of BASMCs, while PCNA was mainly expressed in the nuclei. Compared with WKY group, the expression and protein level of OPN and PCNA were increased in SHR group, and decreased significantly after CAP treatment. Conclusion Capsaicin can reduce the proliferation and migration of SHR derived BASMCs.

Emodin activates BK channel in vascular smooth muscle cells and relaxes the interlobar renal artery of rat.

AIM: The purpose of this study was to investigate the mechanical and electrophysiological effects of emodin on BK channels in the IRASMCs, of the rat. METHODS: Isolated interlobar renal artery was used for vascular reactivity measurements using a pressure myograph system. Electrophysiological measurements of single vascular smooth muscle cells were conducted using whole-cell and cell-attached patch-clamp recording. Laser scanning confocal microscope technology was used to measure cytosolic calcium ion signals. KEY RESULTS: Emodin relaxed the interlobar renal artery and enhanced the outward currents amplitude of IRASMCs in a concentration-dependent manner, and IbTX inhibited these emodin-induced outward currents. Incubation of IRASMCs in a calcium ion free medium for 30 min decreased the observed effects of emodin on IRASMCs membrane currents. Furthermore, the application of nimodipine, an L-Type calcium ion channel blocker, ryanodine, a ryanodine receptor modifier, and heparin, an IP3 receptor blocker, decreased the emodin-induced BK channel currents, respectively. BAPTA-AM, a selective calcium ion chelator, abolished the emodin-induced BK channel currents. Emodin repolarized cytomembrane and enhanced BK channel open probabilities and elevated cytosolic calcium ion concentration. CONCLUSION: The vasorelaxant effect of emodin on vessels is mediated through the activation of BK channels.

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.

[Emodin inhibits the proliferation and migration of human pulmonary artery smooth muscle cells by blocking SMAD2/3 signaling pathway].

Objective To investigate the effect of emodin on the proliferation and migration of human pulmonary artery smooth muscle cells (HPASMCs) induced by transforming growth factor ß1 (TGF-ß1). Methods HPASMCs were cultured in vitro, and HPASMCs in logarithmic growth phase were divided into control group, TGF-ß1 group, and TGF-ß1 combined with emodin group. The activity of HPASMCs was detected by CCK-8 assay, the migration ability of HPASMCs was detected by TranswellTM chamber assay and scratch assay, and the expressions of osteopontin (OPN) and proliferating cell nuclear antigen (PCNA) were detected by immunofluorescence assay. The protein levels of OPN and PCNA and the phosphorylation of SMAD family member 2 (SMAD2) and SMAD family member 2 (SMAD3) in HPASMCs were detected by Western blot. Results Compared with those in the control group, in TGF-ß1 group, the protein expressions of OPN and PCNA, the proliferation and migration of HPASMCs, and the phosphorylation of SMAD2 and SMAD3 were increased. Compared with those in the TGF-ß1 group, in the TGF-ß1 combined with emodin group, the proliferation and migration of HPASMCs, the expressions of OPN and PCNA, and the phosphorylation of SMAD2 and SMAD3 were decreased. Conclusion Emodin inhibits the up-regulation of OPN and PCNA and the proliferation and migration of PASMCs induced by TGF-ß1, which may be related to the blocking of SMAD2/3 signaling pathway.

Molecular Mechanism of Naringenin Against High-Glucose-Induced Vascular Smooth Muscle Cells Proliferation and Migration Based on Network Pharmacology and Transcriptomic Analyses.

Although the protective effects of naringenin (Nar) on vascular smooth muscle cells (VSMCs) have been confirmed, whether it has anti-proliferation and anti-migration effects in high-glucose-induced VSMCs has remained unclear. This study aimed to clarify the potential targets and molecular mechanism of Nar when used to treat high-glucose-induced vasculopathy based on transcriptomics, network pharmacology, molecular docking, and in vivo and in vitro assays. We found that Nar has visible anti-proliferation and anti-migration effects both in vitro (high-glucose-induced VSMC proliferation and migration model) and in vivo (type 1 diabetes mouse model). Based on the results of network pharmacology and molecular docking, vascular endothelial growth factor A (VEGFA), the proto-oncogene tyrosine-protein kinase Src (Src) and the kinase insert domain receptor (KDR) are the core targets of Nar when used to treat diabetic angiopathies, according to the degree value and the docking score of the three core genes. Interestingly, not only the Biological Process (BP), Molecular Function (MF), and KEGG enrichment results from network pharmacology analysis but also transcriptomics showed that phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) is the most likely downstream pathway involved in the protective effects of Nar on VSMCs. Notably, according to the differentially expressed genes (DEGs) in the transcriptomic analysis, we found that cAMP-responsive element binding protein 5 (CREB5) is a downstream protein of the PI3K/Akt pathway that participates in VSMCs proliferation and migration. Furthermore, the results of molecular experiments in vitro were consistent with the bioinformatic analysis. Nar significantly inhibited the protein expression of the core targets (VEGFA, Src and KDR) and downregulated the PI3K/Akt/CREB5 pathway. Our results indicated that Nar exerted anti-proliferation and anti-migration effects on high-glucose-induced VSMCs through decreasing expression of the target protein VEGFA, and then downregulating the PI3K/Akt/CREB5 pathway, suggesting its potential for treating diabetic angiopathies.

Hypoxia Induces Apoptosis of Microglia BV2 by Upregulating Kir2.1 to Activate Mitochondrial-Related Apoptotic Pathways.

Aim: To explore the role of Kir2.1 in hypoxia-induced microglial apoptosis. Methods: BV2 microglial cell lines were cultured and treated with ML133 hydrochloride, a Kir2.1 channel blocker, for 23 h and with 500 µmol/L of CoCl2 for 8 h. Cells were divided into the control, CoCl2 (hypoxia-induced model), and CoCl2+ML133 (hypoxia-induced model established after ML133 pretreatment) groups. Cell activity was assessed using the CCK-8 technique. The membrane potential and Kir2.1 current of BV2 were evaluated with the whole-cell patch-clamp technique. The protein levels and mRNA levels of Kir2.1, apoptotic proteins Bax and caspase-3, and antiapoptotic protein Bcl-2 in BV2 cells were evaluated via immunofluorescence, Western blot analysis, and real-time quantitative reverse transcription. The apoptosis rate of BV2 cells was detected via flow cytometry. Results: CCK-8 analysis showed that the cell activity of each group increased initially and then decreased. The 2 h intervention group had the highest cell activity, and that of the 8 h group was >90%. Hence, there was a significant difference in the results (P < 0.05). Western blot analysis revealed that the expression of cleaved caspase-3 significantly increased in the 8 h group compared with the 0 h group. Compared with the control group, the expression of Kir2.1 and mRNA in the CoCl2 group increased. Thus, hypoxia could upregulate the expression of Kir2.1. The whole-cell patch-clamp results showed that the Kir2.1 channel current amplitude of the CoCl2 group increased compared with that of the control group. Therefore, hypoxia could enhance Kir2.1 function. The apoptosis rate of the CoCl2 group was significantly higher than that of the control group. Further, the ML133 group had a significantly lower apoptosis rate than the CoCl2 group. The expression of apoptotic proteins Bax and cleaved caspase-3 increased in the CoCl2 group, and that of the antiapoptotic protein Bcl-2 decreased. The expression of apoptotic proteins Bax and cleaved caspase-3 reduced in the CoCl2+ML133 group, whereas that of the antiapoptotic protein Bcl-2 increased. Conclusion: Hypoxia can induce microglia BV2 apoptosis accompanied by the upregulation of Kir2.1 and mRNA expression levels and an increase in the Kir2.1 current. Moreover, ML133 can inhibit hypoxia-induced BV2 cell apoptosis. Hence, Kir2.1 may be involved in the process of hypoxia-induced BV2 cell apoptosis.

Inhibition of Connexin 43 reverses ox-LDL-mediated inhibition of autophagy in VSMC by inhibiting the PI3K/Akt/mTOR signaling pathway.

Background: Oxidized low-density lipoproteins (ox-LDL) may induce foam cell formation from the vascular smooth muscle cell (VSMC) by inhibiting VSMC autophagy. This process accelerates the formation of atherosclerosis (AS). Connexin 43 (Cx43), which is the most widely distributed connexin in VSMC is associated with autophagy. However, the mechanism of action and the involvement of Cx43 in ox-LDL-inhibited VSMC autophagy remain unclear. Methods: The primary VSMC were obtained and identified, before primary VSMC were pretreated with an inhibitor (Cx43-specific inhibitor Gap26 and PI3K inhibitor LY294002) and stimulated with ox-LDL. Results: Ox-LDL not only inhibited autophagy in VSMC via downregulation of autophagy-related proteins (such as Beclin 1, LC3B, p62), but also increased Cx43 protein levels. Then we added Gap26 to VSMC in the ox-LDL+Gap26 group, in which autophagy-related proteins were increased and the accumulation of lipid droplets was reduced. These result suggested that an enhanced level of autophagy and an alleviation of lipid accumulation might be caused by inhibiting Cx43 in VSMC. The phosphorylation levels of PI3K, AKT, mTOR were increased by ox-LDL, thus down-regulating autophagy-related proteins. However, this situation was partially reversed by the Gap26. Moreover, Cx43 expression were decreased by LY294002 in ox-LDL-induced VSMCs. Conclusion: Inhibiting Cx43 may activate VSMC autophagy to inhibit foam cell formation by inhibiting the PI3K/AKT/mTOR signaling pathway.

[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.

A Systematic Study of the Mechanism of Acacetin Against Sepsis Based on Network Pharmacology and Experimental Validation.

Sepsis is a dysregulated systemic response to infection, and no effective treatment options are available. Acacetin is a natural flavonoid found in various plants, including Sparganii rhizoma, Sargentodoxa cuneata and Patrinia scabiosifolia. Studies have revealed that acacetin potentially exerts anti-inflammatory and antioxidative effects on sepsis. In this study, we investigated the potential protective effect of acacetin on sepsis and revealed the underlying mechanisms using a network pharmacology approach coupled with experimental validation and molecular docking. First, we found that acacetin significantly suppressed pathological damage and pro-inflammatory cytokine expression in mice with LPS-induced fulminant hepatic failure and acute lung injury, and in vitro experiments further confirmed that acacetin attenuated LPS-induced M1 polarization. Then, network pharmacology screening revealed EGFR, PTGS2, SRC and ESR1 as the top four overlapping targets in a PPI network, and GO and KEGG analyses revealed the top 20 enriched biological processes and signalling pathways associated with the therapeutic effects of acacetin on sepsis. Further network pharmacological analysis indicated that gap junctions may be highly involved in the protective effects of acacetin on sepsis. Finally, molecular docking verified that acacetin bound to the active sites of the four targets predicted by network pharmacology, and in vitro experiments further confirmed that acacetin significantly inhibited the upregulation of p-src induced by LPS and attenuated LPS-induced M1 polarization through gap junctions. Taken together, our results indicate that acacetin may protect against sepsis via a mechanism involving multiple targets and pathways and that gap junctions may be highly involved in this process.

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.