[Clinical application of retrosigmoid approach for BONEBRIDGE implantation after auricle reconstruction using expanded postauricular flap].

Objective: To explore the safety and reliability of retrosigmoid approach BONEBRIDGE implantation in patients with auricle reconstruction using skin expansion flap. Methods: A retrospective analysis was conducted on 43 congenital aural atresia cases (43 ears) who underwent BONEBRIDGE implantation from September 2019 to January 2023 in Beijing Tongren Hospital. 30 males and 13 females were included in this work. The implantation age was 9-36 years old (median age=10 y/o). All cases underwent auricle reconstruction surgery using the posterior ear flap expansion method, with 36 cases using the single expanded postauricular flap method and 7 cases using two-flap method. BONEBRIDGE implant surgery was performed during the third stage of auricle reconstruction or after all stages. The hearing improvements were evaluated by comparing the changes in pure tone hearing threshold and speech recognition rate of patients before and after BONEBRIDGE implantation. Routine follow-up was conducted to observe the hearing results and complications. SPSS 14.0 software was applied for data statistical analysis. Results: All 43 patients healed well and had no surgical complications when discharge. The average bone conduction hearing threshold after surgery was (8.2±6.6) dBHL, and there was no statistically significant difference compared to the preoperative [(8.1±5.7) dBHL] (P=0.95). After surgery, the threshold of hearing assistance with power on was significantly lower than that without hearing assistance [(32.8±4.6) dBHL vs (60.5±5.5) dBHL], and the difference was statistically significant (P<0.001). The speech recognition rate of monosyllable words, disyllabic words and short sentences in quiet environment increased to 72%, 84%, and 98% respectively. The differences were statistically significant (P<0.001). The speech recognition rate of monosyllabic words, disyllabic words, and short sentences in noise environment was significantly increased by 70%, 80%, and 92% respectively (P<0.001). After a follow-up of 4 to 47 months (median=24 months), the hearing results were stable and the aesthetic outcomes were satisfying. One patient had delayed hematoma around coil of the implant. After aspiration and compressed dressing for one week, hematoma was not recurrent. Conclusion: For patients after auricle reconstruction using expanded postauricular flap, the preference of retrosigmoid approach is a good choice in terms of safety and reliability of operation, as well as aesthetic appearance.

Meta-analysis of the effects of furosemide combined with hydration therapy on contrast-induced acute kidney injury after coronary intervention.

OBJECTIVE: A meta-analysis was performed to evaluate the effect of furosemide combined with hydration therapy on the incidence and prognosis of contrast-induced acute kidney injury (CI-AKI) in patients after coronary intervention. MATERIALS AND METHODS: Through the PubMed, EMBASE, Cochrane Library and Web of Science databases, all relevant literature from database establishment until October 1, 2020, was retrieved and screened. Quality evaluation was performed using the risk of bias evaluation tool recommended by the Cochrane Collaboration network, data extraction was performed based on pre-selected effect indicators, and statistics were calculated using Review Manager 5.3 analysis software. RESULTS: A total of 2084 patients in 9 studies were included in the meta-analysis. The results showed that furosemide combined with hydrotherapy had no effect on the incidence of CI-AKI (OR = 0.85, 95% CI [0.46, 1.60], p = 0.62) and can significantly decrease the incidence of major adverse cardiovascular events (MACEs) (OR = 0.43, 95% CI [0.27, 0.67], p = 0.0003) and mortality (OR = 0.24, 95% CI [0.08, 0.79], p = 0.02) in patients. However, it had no significant impact on the need for postoperative dialysis treatment, postoperative creatinine level or length of hospital stay. CONCLUSIONS: Furosemide combined with hydration therapy has no significant effect on the incidence of CI-AKI in patients after coronary intervention but can reduce the incidence of MACEs and mortality, thereby providing clinical benefits.

Crown ether-based anion exchange membranes with highly efficient dual ion conducting pathways.

Anion exchange membranes (AEMs) are a crucial constituent for alkaline fuel cells. As the core component of fuel cells, the low performance AEMs restrict the development and application of the fuel cells. Herein, the trade-off between the OH- conductivity and dimensional stability was solved by constructing AEMs with adequate OH- conductivity and satisfactory alkali resistance using Tröger's base (TB) poly (crown ether)s (PCEs) as the main chain, the embedded quaternary ammonium (QA) and Na+-functionalized crown ether units as the cationic group. Crown ether is an electron donator, and can capture Na+ to form Na+-functionalized crown ether units to conveniently transfer OH- and significantly promote the alkaline stability of the AEMs. The influence of the Na+-functionalized crown ether units on the performance of AEMs was studied in detail. The PCEs based AEMs show an obvious hydrophobic-hydrophilic microphase separation. These features make them ideal platforms for the OH- conduction applications. As expected, the as-prepared PCEs-QA-100% (100% is the degree of cross-linking) AEM with an ionic exchange capacity (IEC) of 2.07 meq g-1 has a high OH- conductivity of 159 mS cm-1 at 80 °C. Furthermore, the membrane electrode assemblies fabricated using the PCEs-QA-100% AEM possess a maximum power density of 291 mW cm-2 under the current density of 500 mA cm-2.

Molecular mechanism concerning conformational changes of CDK2 mediated by binding of inhibitors using molecular dynamics simulations and principal component analysis.

Cyclin-dependent kinase 2 (CDK2) has been regarded as a promising drug target for anti-tumour agents. In this study, molecular dynamics (MD) simulations and principal component (PC) analysis were used to explore binding mechanism of three inhibitors 1PU, CDK, 50Z to CDK2 and influences of their bindings on conformational changes of CDK2. The results show that bindings of inhibitors yield obvious impacts on internal dynamics, movement patterns and conformational changes of CDK2. In addition, molecular mechanics generalized Born surface area (MM-GBSA) was applied to calculate binding free energies between three inhibitors and CDK2 and evaluate their binding ability to CDK2. The results show that CDK has the strongest binding to CDK2 among the current three inhibitors. Residue-based free energy decomposition method was further utilized to decode the contributions of a single residue to binding of inhibitors, and it was found that three inhibitors not only produce hydrogen bonding interactions and hydrophobic interactions with key residues of CDK2, which promotes binding of three inhibitors to CDK2, but also share similar binding modes. This work is expected to be helpful for design of efficient drugs targeting CDK2.

[Rib cartilage framework supporting combined with local flap grafting for correction of cryptotia].

Objective: To investigate the curative effect of rib cartilage framework supporting combined with local flap grafting for correction of cryptotia. Methods: Fourteen cases (nineteen ears) were corrected by rib cartilage framework supporting combined with local flap grafting method from January 2017 to March 2019. Part of the 7th rib cartilage was carved into a scalloped cartilage piece, which was implanted on the retroauricular cartilage to release and expand the deformed cartilage. A "M" incision was designed on the retroauricular skin to make the local flap grafting. Results: All corrected auricles were followed up for four months to three year and abtained satisfactory and stable appearance. In one case, the edge of the helix was broken two weeks after the operation, and well healed after repairing. Conclusions: The rib cartilage framework supporting combined with local flap grafting method can fully correct the deformity of cryptotia, and the long-term effect is satisfied. It can be an important complement to the classic methods.

[Bonebridge implantation combined with simultaneous bilateral auricle reconstruction for bilateral congenital aural atresia].

Objective: To investigate the feasibility and safety of auricle reconstruction combined with Bonebridge implantation for bilateral aural atresia patients. Methods: A retrospective analysis was conducted for 36 cases(72 ears) who underwent Bonebridge implantation combined with bilateral auricle reconstruction from February 1, 2017 to January 15, 2020. All cases were bilateral congenital aural atresia and underwent Nagata auricle reconstruction for both sides simultaneously. Bonebridge implantations were performed during the second stage of auricle reconstruction. Results: All 36 patients healed well and had no surgical complications when discharged. The preoperative average bone conduction threshold of the patients was(8.5±5.8) dB HL and postoperative bone conduction threshold was (8.4±5.2) dB HL. There was no significant change after the implantation (P=0.724). The preoperative average air conduction threshold of was(64.9±7.4)dB HL and postoperative air conduction threshold was (24.0±5.3) dB HL, which had a significant change after the implantation (P<0.001). The hearing threshold with Bonebridge significantly decreased by 40.9 dB HL compared with the preoperative air conduction threshold(P<0.001). The speech recognition rate of monosyllable words, disyllabic words and short sentences in quiet environment increased by 62.5%, 63.5% and 72.2% respectively. The differences were statistically significant (P<0.001). The speech recognition rate of monosyllabic words, disyllabic words and short sentences in noise environment were significantly increased by 55.9%, 58.9% and 69.9% respectively (P<0.001). After a follow-up of 18.3 months in average, the hearing results were stable and the aesthetic outcomes were satisfied. One patient had implant rupture and healed after revision surgery. Conclusions: With an integrated surgical procedure, patients with bilateral congenital aural atresia can complete bilateral auricle reconstruction and hearing implantation within six months. This integrated surgical procedure is safe and efficient, with a stable hearing improvement and good appearance.

Diagnostic significance of serum miR-26b and miR-21 expressions in ovarian cancer and their associations with clinicopathological characteristics and prognosis of patients.

OBJECTIVE: The aim of this study was to detect the expressions of serum micro-ribonucleic acid (miR)-26b and miR-21 in ovarian cancer patients, and to explore their associations with the diagnosis, clinicopathological parameters and prognosis of ovarian cancer. PATIENTS AND METHODS: A total of 86 patients diagnosed with ovarian cancer in our hospital from January 2014 to January 2015 were enrolled in the observation group. Meanwhile, another 86 subjects receiving physical examination in our hospital during the same period were enrolled in the control group. The expressions of serum miR-26b and miR-21 in both groups were detected via Real Time fluorescence-quantitative Polymerase Chain Reaction (RT-qPCR). Receiver operating characteristic (ROC) curves were plotted. Later, the clinical diagnostic value of combined detection of miR-26b and miR-21 in ovarian cancer was analyzed. Moreover, the associations of serum miR-26b and miR-21 expressions with clinicopathological characteristics and prognosis of ovarian cancer patients were explored. RESULTS: The expression of serum miR-26b in ovarian cancer patients was significantly lower than that of healthy subjects, while miR-21 expression was markedly higher in ovarian cancer patients (p<0.05). The area under the ROC curve (AUC), the sensitivity and specificity of miR-26b detection, miR-21 detection and combined detection in the diagnosis of ovarian cancer were 0.753 vs. 0.826 vs. 0.916, 47.2 vs. 76.3 vs. 87.6 and 78.5 vs. 85.6 vs. 90.4, respectively. Therefore, it could be observed that both the sensitivity and specificity of combined detection were remarkably higher than those of single detection (p<0.05). In addition, the expressions of serum miR-26b and miR-21 were associated with clinical stage and lymph node metastasis of ovarian cancer patients, whereas it was not correlated with age and histological type. The 3-year survival rate of patients with high expression of serum miR-26b was significantly higher than that in those with low expression of serum miR-26b. However, the 3-year survival rate of patients with low expression of serum miR-21 was higher than that in those with high expression. CONCLUSIONS: MiR-21 is highly expressed, while miR-26b is lowly expressed in the serum of ovarian cancer patients. Both of them may be involved in the incidence and development of ovarian cancer. Furthermore, combined monitoring of serum miR-26b and miR-21 has a certain value in the clinical diagnosis and treatment of ovarian cancer.

Highly expressed miR-182-5p can promote preeclampsia progression by degrading RND3 and inhibiting HTR-8/SVneo cell invasion.

OBJECTIVE: The role of miR-182-5p in preeclampsia was studied, and its mechanism was also explored. PATIENTS AND METHODS: Fifty patients with preeclampsia were assigned to the study group and 50 normal pregnant women to the control group. The age, weight, blood pressure, urinary protein, and weight of newborns were compared between the two groups. The placental tissues of the above-mentioned subjects were collected, and quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR) assay was used to detect the expression of miR-182-5p. MiR-182-5p was overexpressed or knocked down using a cell transfection assay in HTR-8/SVneov cell, which is a kind of human chorionic trophoblast cell. Changes in cell migration and invasiveness before and after transfection were determined by wound healing test and transwell assay, respectively. Western blot was performed to analyze the change of RND3 protein level before and after transfection. The biological prediction of the relationship between miR-182-5p and RND3 was performed and a dual luciferase reporter gene experiment was designed to verify the results. Finally, a rescue experiment was conducted to investigate whether RND3 could affect the role of miRNA-182-5p in the capacity of cell migration and invasion. RESULTS: Preeclampsia patients had higher systolic blood pressure, diastolic blood pressure, and urinary protein than normal pregnant women, while neonatal weight decreased compared with normal pregnant women. MiRNA-182-5p was highly expressed in placental tissues of patients with preeclampsia. After miRNA-182-5p was overexpressed, the migration and invasion of HTR-8/SVneo cells were significantly attenuated, and the mRNA and protein levels of RND3 were markedly downregulated, and vice versa. The dual luciferase reporting assay confirmed that miRNA-182-5p could bind to 3'UTR of RND3. In addition, the results of rescue experiment showed that overexpressing miRNA-182-5p could markedly inhibit the migration and invasion of HTR-8/SVneo cells; however, when RND3 was simultaneously overexpressed, the inhibitory effect of miRNA-182-5p was partially reversed. CONCLUSIONS: The highly expressed miRNA-182-5p in patients with preeclampsia promoted the development of preeclampsia, the possible mechanism of which might be that the increased miRNA-182-5p expression could inhibit the migratory and invasive ability of trophoblast cells through targeted degrading RND3 protein.

LINC01116 promotes the progression of epithelial ovarian cancer via regulating cell apoptosis.

OBJECTIVE: To explore the biological function of LINC01116 in epithelial ovarian cancer (EOC) and its underlying mechanism. PATIENTS AND METHODS: The expression level of LINC01116 in 60 EOC tissues, 30 normal ovarian tissues and EOC cells were detected by qRT-PCR (quantitative real-time polymerase chain reaction). Disease-free survival (DFS) and overall survival (OS) of enrolled EOC patients were recorded. The correlation between LINC01116 expression, DFS and OS of EOC patients was analyzed using ROC curve. Influencing factors for DFS and OS were analyzed by univariable and multivariable Cox regression model. For in vitro experiments, the effect of LINC01116 knockdown on proliferation, invasion and apoptosis of EOC cells were detected by CCK-8 (cell counting kit-8), transwell assay and flow cytometry, respectively. Protein expressions of apoptosis-related genes in EOC cells transfected with pc-DNA-LINC01116 or si-LINC01116 were detected by Western blot. RESULTS: LINC01116 was overexpressed in EOC tissues than that of paracancerous tissues. DFS and OS in EOC patients with higher expression of LINC01116 were remarkably shorter than those with lower expression. FIGO (International Federation of Gynecology and Obstetrics) clinical stage and LINC01116 expression were the independent factors that affected DFS and OS of EOC patients. Besides, LINC01116 expression was positively correlated to the diagnostic sensitivity of EOC patients. In vitro experiments found that LINC01116 overexpression promoted proliferation and invasion of EOC cells. Overexpressed LINC01116 resulted in upregulated Bcl-2, and downregulated cleaved Caspase-3 and cleaved Caspase-9 in EOC cells. CONCLUSIONS: Overexpressed LINC01116 promotes EOC progression via increasing proliferation and migration, and inhibiting cell apoptosis.

Dunye Guanxinning Improves Acute Myocardial Ischemia-Reperfusion Injury by Inhibiting Neutrophil Infiltration and Caspase-1 Activity.

Acute myocardial infarction is the most serious manifestation of cardiovascular disease, and it is a life-threatening condition. Dunye Guanxinning (DG) is a protective traditional Chinese patent herbal medicine with high clinical efficacy and suitable for the treatment of myocardial infarction. However, the mechanism through which it is beneficial is unclear. In this study, we hypothesized that DG improves acute myocardial ischemia-reperfusion injury by inhibiting neutrophil infiltration and caspase-1 activity. We found that DG administration decreased infarct size and cardiomyocyte apoptosis and improved left ventricular ejection fraction, fractional shortening, end-systolic volume index, end-systolic diameter, and carotid arterial blood flow output in rats. DG administration also improved hemorheological parameters, myocardial damage biomarkers, and oxidative stress indexes. The findings showed that DG administration inhibited neutrophil infiltration and reduced the serum interleukin-1 beta (IL-1ß) level and myocardial IL-1ß maturation. Moreover, DG administration inhibited caspase-1 activity and activated adenosine monophosphate-activated protein kinase (AMPK) phosphorylation in rat hearts. These results suggested that DG administration inhibits inflammasome activity and IL-1ß release through the AMPK pathway. Our findings support the clinical efficacy of DG and partially reveal its mechanism, which is beneficial for understanding the therapeutic effects of this protective traditional Chinese patent drug.

Two novel missense mutations of ATP2A2 in two Chinese patients with sporadic Darier disease.

Darier disease (DD) is a rare autosomal dominant skin disorder with characteristic abnormal keratinization and acantholysis. The causative gene, ATP2A2, is located on chromosome 12, and encodes a sarco/endoplasmic reticulum calcium pump ATPase (SERCA2). Two Chinese patients with sporadic DD participated in this study. Genomic sequence analysis identified two novel missense mutations (c.742C>A and c.2098A>G) in the ATP2A2 gene. Our findings provide an additional ATP2A2 mutation causative for DD development, and new lines of evidences for the understanding of genotype-phenotype correlations.

Protein-protein interaction network analysis of osteoarthritis-related differentially expressed genes.

The purpose of this study was to identify genes and pathways for osteoarthritis (OA) diagnosis and therapy. We downloaded the gene expression profile of OA from Gene Expression Omnibus (GEO) database including 10 early OA, 9 late OA, and 5 normal control samples. Next, we screened differentially expressed genes (DEGs) between early- and late-stage OA samples comparing with healthy control samples. Then, the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) software was used to construct protein-protein interaction (PPI) network, which was to predict the proteins that may interact with DEGs. The Gene Ontology (GO)-enrichment method was used to analyze the function of genes in the PPI networks. Meanwhile network module analysis was performed using Cytoscape. A total of 24 and 29 DEGs were identified for the early and late OA, respectively. TAC1 showed the highest degree in the PPI network. Functional annotation of the TAC1 network module indicated that this gene is associated with the G protein-coupled signal transduction pathway. In summary, TAC1, together with G protein-coupled receptors, appear to play a role in the biogenesis and progress of OA. Further analysis of this gene and pathway could therefore provide a potential target for the diagnosis and treatment of OA.

The flavonoid from Polygonum perfoliatum L. inhibits herpes simplex virus 1 infection.

Herpes simplex virus 1 (HSV-1) is one of the most prevalent human pathogens in both industrialized and developing countries. This study was performed to analyze the antiviral activity of purified flavonoid from Polygonum perfoliatum L. against HSV-1 infection in vitro and in vivo. Flavonoid showed no inhibitory effect, when treated before virus infection, but it strongly inhibited viral replication and cell-to-cell spread which was vital for the virus's propagation. The therapeutic effect of the flavonoid in treating HSV-1 induced encephalitis was also investigated in mice. A dose-dependent increase of survival rate and mean survival time (MST) were observed in the flavonoid-treated mice. These results suggested that the flavonoid may be a viable therapeutic option for recurrent HSV-1 infection.

The fibrotic role of phosphatidylinositol-3-kinase/Akt pathway in injured skeletal muscle after acute contusion.

Transforming growth factor ß (TGF-ß) is a multifunctional cytokine with fibrogenic properties. Previous studies demonstrated that Phosphatidylinositol 3-Kinase (PI3K)/Akt/ mammalian target of Ramycin (mTOR), a non-Smad TGF-ß pathway, plays an important role in the fibrotic pathogenesis of different organs such as the lung, kidney, skin and liver. However, the role of PI3k-Akt pathway in fibrosis in injured skeletal muscle is still unclear. In this study, we determined the fibrotic role of PI3K-Akt pathway in injured skeletal muscle. We established a mouse model for acute muscle contusion. Western blotting analysis showed that TGF-ß, phosphorylated Akt and phosphorylated mTOR were increased in muscles after acute contusion, which indicated that the PI3K-Akt- mTOR pathway was activated in skeletal muscle after acute contusion. The pathway was inhibited by a PI3K inhibitor, LY294002. Moreover, the expression of fibrosis markers vimentin, α SMA and collagen I and the area of scar decreased in injured skeletal muscle after PI3K pathway was blocked. The muscle function improved in terms of both fast-twitch and tetanic strength after PI3K/Akt pathway was inhibited in injured skeletal muscle. In conclusion, activation of PI3K-Akt-mTOR pathway might promote collagen production and scar formation in the acute contused skeletal muscle. Blocking of PI3K-Akt-mTOR pathway could improve the function of injured skeletal muscle.

Upregulation of DLX2 confers a poor prognosis in glioblastoma patients by inducing a proliferative phenotype.

The human Distal-less Homeobox (DLX) gene family encodes homeobox transcription factors involved in the control of morphogenesis and tissue homeostasis, which is primarily expressed in embryonic development. Recently, DLX gene family was reported to have essential roles in carcinogenesis. We have profiled whole genome expressed genes in 83 glioblastoma multiforme (GBM) patients from the Chinese Glioma Genome Atlas (CGGA) Group. Two major groups of samples were identified in mRNA expression profiles (referred to as Cluster 1 (C1) and Cluster 2 (C2)). We identified 7 out of the top 10 Gene Ontology terms in the C1 group were associated with differentiation and development of neuronal cell. The most significant prognostic gene was DLX2 (P < 0.001, OR = 1.744); overexpression of DLX2 indicated poor survival in the 83 GBM patients (low DLX2 vs high DLX2, 77.6 vs 44.7 weeks, P < 0.001). Annotation of mRNA profiling data on GBM from The Cancer Genome Atlas and MD Anderson Cancer Center showed the proneural and neural subtypes highly correlated with low and high DLX2 expression, respectively. Knocking down of DLX2 in GBM cell line-LN229 results in decreased cyclin D1 expression and cell proliferation. Collectively, these data identified high expression of DLX2 as a poor prognostic marker to GBM patients.

Urodynamic study of bladder function for patients with lumbar spinal stenosis treated by surgical decompression.

Lumbar spinal stenosis usually leads to different degrees of nerve damage, presenting with back and leg pain, and/or neurogenic bladder symptoms. To determine whether lumbar decompression improved urological function, bladder dysfunction was evaluated in this retrospective study of 26 patients with lumbar spinal stenosis who had undergone lumbar decompression surgery. Urodynamic study procedures were performed pre-operatively and 6 months post-operatively. The Japanese Orthopaedic Association score rating system and Oswestry Disability Index were employed for clinical evaluation. Following surgery, post-voiding residual urine, maximum cystometric capacity and maximum flow rate improved significantly. There was no statistically significant improvement in voided volume, bladder compliance, maximum detrusor pressure or upper urinary tract damage. Urodynamic study was important in the diagnosis of neurogenic bladder dysfunction, prevention of renal deterioration and assessment of post-operative effects after surgical decompression for patients with lumbar spinal stenosis.

Activation of GABA receptors attenuates neuronal apoptosis through inhibiting the tyrosine phosphorylation of NR2A by Src after cerebral ischemia and reperfusion.

Cerebral ischemia can induce both the increase of excitation and the decrease of inhibition, which leads to neuronal excitotoxicity. Since glutamatergic and GABAergic transmissions work by each counterbalancing the function of the other, enhancing GABAergic activity should balance excessive glutamatergic excitation. But the potential mechanisms underlying these effects are obscure. Here, we used two GABA agonists, muscimol and baclofen, and performed immunoblotting, immunoprecipitation and histology analysis to evaluate the neuroprotective effects by stimulating GABA receptors in rat four-vessel occlusion (4-VO) ischemic model, and to investigate the potential mechanism. Our results indicate that whether in global cerebral ischemia in vivo, or in oxygen glucose deprivation (OGD) in vitro, coapplication of muscimol with baclofen can protect neurons from neuronal death through down-regulating the function of N-methyl-d-aspartic acid (NMDA) receptors via attenuating the tyrosine phosphorylation of NR2A subunit. We further elucidate that the phosphorylation level of Src kinase and the interaction among Src, post-synaptic density protein 95 and NR2A were also suppressed by coapplication of muscimol with baclofen. Both MK-801, a specific antagonist of NMDA receptors, and chelerythrine, an inhibitor of protein kinase C (PKC), could down-regulate the phosphorylation of NR2A via inhibiting the activation of Src and PKC respectively. These results suggest that the modified pattern of dynamic balance between excitation and inhibition by coactivation of the GABA receptors in cerebral ischemia can attenuate the excitatory NMDAR via inhibiting a novel postsynaptic NMDAR/Src-mediated signal amplification, the 'NMDAR-Ca(2+) --> PKC --> Src --> NMDAR-Ca(2+)' cycle.

Ischemic preconditioning negatively regulates plenty of SH3s-mixed lineage kinase 3-Rac1 complex and c-Jun N-terminal kinase 3 signaling via activation of Akt.

Activation of Akt/protein kinase B has been recently reported to play an important role in ischemic tolerance. We here demonstrate that the decreased protein expression and phosphorylation of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) underlie the increased Akt-Ser-473 phosphorylation in the hippocampal CA1 subfield in ischemic preconditioning (IPC). Co-immunoprecipitation analysis reveals that Akt physically interacts with Rac1, a small Rho family GTPase required for mixed lineage kinase 3 (MLK3) autophosphorylation, and both this interaction and Rac1-Ser-71 phosphorylation induced by Akt are promoted in preconditioned rats. In addition, we show that Akt activation results in the disassembly of the plenty of SH3s (POSH)-MLK3-Rac1 signaling complex and down-regulation of the activation of MLK3/c-Jun N-terminal kinase (JNK) pathway. Akt activation results in decreased serine phosphorylation of 14-3-3, a cytoplasmic anchor of Bax, and prevents ischemia-induced mitochondrial translocation of Bax, release of cytochrome c, and activation of caspase-3. The expression of Fas ligand is also decreased in the CA1 region. Akt activation protects against apoptotic neuronal death as shown in TUNEL staining following IPC. Intracerebral infusion of LY294002 before IPC reverses the increase in Akt phosphorylation and the decrease in JNK signaling activation, as well as the neuroprotective action of IPC. Our results suggest that activation of pro-apoptotic MLK3/JNK3 cascade can be suppressed through activating anti-apoptotic phosphoinositide 3-kinase/Akt pathway induced by a sublethal ischemic insult, which provides a functional link between Akt and the JNK family of stress-activated kinases in ischemic tolerance.

The neuroprotective effects of K252a through inhibiting MLK3/MKK7/JNK3 signaling pathway on ischemic brain injury in rat hippocampal CA1 region.

It has been well documented that the activation of c-Jun N-terminal protein kinase (JNK) pathway and caspase-3 signal are involved in the delayed neuronal cell death in cerebral ischemia. In this study, we first detected the activation pattern of JNK signaling including mixed lineage kinase (MLK)3, mitogen-activated protein kinase kinase (MKK)7 and JNK3 in hippocampal CA1 and CA3/DG regions at various time points after 15 min of ischemia. These results indicated that cerebral ischemia induced the continuous activation of MLK3/MKK7/JNK3 cascade, which all had two active waves only in the CA1 region. We also detected the phosphorylation of JNK substrates c-Jun and Bcl-2, and the activation of a key protease of caspase-3 in CA1 region, which only had one active peak, respectively. Because K252a has recently been shown to be a potent inhibitor of MLK3 activity both in vivo and in vitro, we further examined the possible effects and mechanism of this interesting drug in cerebral ischemia. In our present paper, we found that administration of K252a 20 min prior to ischemia inhibited MLK3/MKK7/JNK3 signaling, Bcl-2 phosphorylation, the activation of c-Jun and caspase-3, but had no significant effects on these protein expressions. Additionally, pretreatment of K252a significantly increased the number of the surviving CA1 pyramidal cells at 5 days of reperfusion. Our results suggest that K252a play a neuroprotective role in ischemic injury via inhibition of the JNK pathway, involving the death effector of caspase-3. Thus, JNK signaling may eventually emerge as a prime target for novel therapeutic approaches to treatment of ischemic stroke, and K252a may serve as a potential and important neuroprotectant in therapeutic aspect in ischemic stroke.