Effects of ICG-001 on autistic behaviors and the development of hippocampal dendritic spine morphology in rats / 中华实用儿科临床杂志

Objective:To explore the effects of the compound ICG-001 on autism-like behaviors and the morphological development of dendritic spines in hippocampal pyramidal neurons of rats.Methods:Healthy Wistar rats were mated.The offspring were divided into the saline-treated group, ICG-001 control group, Sodium valproate (VPA) group and ICG-001 treatment group by using the random number table method.Each group had 12 rats.Social interaction, repetitive, compulsive and anxiety-like behaviors in rodents were assessed by three-chambered social approach, marble burying, open-field and elevated plus maze tests.The number of neuronal nuclei (NeuN)-positive neurons in the hippocampal CA1 region was calculated by the immunofluorescence method.Golgi staining was carried out to detect the density and morphological changes of dendritic spines in hippocampal pyramidal neurons of rats.The expression of phosphorylated LIM kinase 1(LIMK1), phosphorylated actin binding protein(Cofilin), fibros actin (F-actin) and developmentally-regulated brain protein A (Drebrin A) was examined by Western blot.The univariate analysis was made to examine whether the difference was statistically significant, and the data between groups were compared by the Tukey method. Results:(1) In the three-chambered social approach test, the rats in the saline-treated group, ICG-001 control group, VPA group and ICG-001 treatment group spent (219.42±5.38) s, (218.67±10.12) s, (126.58±5.02) s, and (218.58±6.63) s in the chamber, respectively.The corresponding preference score of the said 4 groups were 0.43±0.05, 0.43±0.04, 0.22±0.01 and 0.42±0.04, respectively.Compared with the VPA group, the ICG-001 treatment group spent longer time in the chamber and had a higher preference score (all P<0.05). (2) In the marble burying experiment, the number of marbles buried in said 4 groups were 9.13±0.52, 9.08±0.64, 15.13±0.82 and 9.42±0.86, respectively.ICG-001-treated rats buried markedly less marbles than VPA-exposed rats ( P<0.05). (3) In the open-field test, the rats in the said 4 groups spent (82.33±1.83) s, (81.32±4.19) s, (45.51±3.02) s and (81.44±3.19) s in the center area, respectively.Administration of ICG-001 significantly increased the time that VPA-exposed rats spent in the center area ( P<0.05). (4)In the elevated plus maze trial, the rats in the said 4 groups spent (107.75±7.23) s, (106.08±7.50) s, (63.42±1.91) s and (106.67±7.07) s in open arms, respectively.ICG-001 treatment notably increased the time that VPA-exposed rats spent in open arms ( P<0.05). (5) Immunofluorescence analysis results revealed that the number of NeuN-positive cells in the hippocampal CA1 region of said 4 groups was (41.83±1.17)×10 4/μm 2, (41.00±0.77)×10 4/μm 2, (27.17±0.95)×10 4/μm 2 and (40.00±0.90)×10 4/μm 2, respectively.ICG-001 treatment normalized the alteration in the number of NeuN-containing neurons in VPA-exposed rats ( P<0.05). (6) Golgi staining showed that the density of dendritic spines in hippocampal CA1 pyramidal neurons of said 4 groups was (0.74±0.04)/μm, (0.73±0.03)/μm, (0.49±0.03)/μm and (0.70±0.02) /μm, respectively.Of all types of dendritic spines, mushroom spines accounted for (0.49±0.02)%, (0.49±0.02)%, (0.33±0.02)% and (0.43±0.02) % in said 4 groups.Thin spines accounted for (0.27±0.02)%, (0.26±0.02)%, (0.34±0.01)% and (0.26±0.01) % in said 4 groups, respectively.Compared with the VPA group, the ICG-001 treatment group showed a significant increase in the density of dendritic spines in hippocampal CA1 pyramidal neurons ( P<0.05). After ICG-001 treatment, the number of mushroom spines greatly increased and the number of thin spines sharply decreased in VPA-exposed rats (all P<0.05). (7) According to Western blot test results, the phosphorylated LIMK1/LIMK1 ratio of the hippocampus in said 4 groups were 100.33±2.30, 99.34±2.28, 57.76±4.10 and 99.13±1.90, respectively.The phosphorylated Cofilin /Cofilin ratio were 100.18±2.43, 100.18±1.70, 57.12±1.88 and 99.53±1.69, respectively.The F-actin/globular actin(G-actin) ratio were 100.07±0.86, 99.99±1.72, 51.19±1.23 and 99.28±3.17, respectively.The expression level of Drebrin A were 100.79±1.19, 100.12±2.04, 52.86±3.26 and 99.97±2.44, respectively.Administration of ICG-001 effectively prevented the decrease of phosphorylated LIMK1, phosphorylated Cofilin, F-actin and Drebrin A in the hippocampus of VPA-exposed rats (all P<0.05). Conclusions:ICG-001 regulates the LIMK1/Cofilin signaling pathway, promotes the generation of F-actin, increases the expression of Drebrin A, and thereby alleviates autistic-associated symptoms.

Clinical efficacy of endovascular stent therapy for treatment of lower extremity atherosclerotic occlusive disease in 40 patients / 中国基层医药

Objective:To investigate the short-term and long-term efficacy of endovascular stent therapy for lower extremity atherosclerotic occlusive disease.Methods:Eighty patients with lower extremity atherosclerotic occlusive disease who received treatment in Lishui Central People's Hospital from January 2020 to January 2021 were included in this study. They were randomly divided into control and observation groups, with 40 patients in each group. The control group received lower extremity artery bypass grafting, and the observation group received endovascular stent therapy. Clinical efficacy, ankle-brachial index, claudication distance, blood flow dynamics of dorsalis artery, nerve conduction velocity of the lower extremities, and postoperative complications were compared between the two groups in the short-term and 1-year follow-ups.Results:Total response rate in the observation group was 87.5% (35/40), which was significantly higher than 67.5% (27/40) in the control group ( Z = 2.00, P < 0.05). At 1-year follow-up, total response rate in the observation group was 70.0% (28/40), which was slightly, but not significantly, higher than 47.5% (19/40) in the control group ( Z = 1.77, P > 0.05). After treatment, the ankle-brachial index and claudication distance in the observation group were significantly higher than those in the control group ( t = 3.34, 8.30, both P < 0.001). The diameter, peak velocity and blood flow of dorsal foot artery in the observation group were significantly superior to those in the control group ( t = 6.98, 4.46, 5.95, all P < 0.001). Lower extremity nerve conduction velocity in the observation group was significantly higher than that in the control group ( t = 3.01, 3.70, both P < 0.05). The incidence of postoperative complications in the observation group was slightly, but not significantly, lower than that in the control group [5.0% (2/40) vs. 15.0% (6/40), P > 0.05]. Conclusion:Compared with lower extremity artery bypass grafting, endovascular stent therapy has good short-term and long-term efficacy in the treatment of lower extremity atherosclerotic occlusive disease. Endovascular stent therapy can increase ankle-brachial index and claudication distance, improve the hemodynamic indexes of dorsalis pedis artery, increase lower extremity nerve conduction velocity and has a few complications.

Effect of the regulator of G-protein signaling 2 on the proliferation and invasion of oral squamous cell carcinoma cells and its molecular mechanism / 华西口腔医学杂志

OBJECTIVES@#This study aims to investigate the effect of the regulator of G-protein signaling 2 (RGS2) on the proliferation and invasion of oral squamous cell carcinoma (OSCC) cells and its potential molecular mechanism. Metho⁃ds The expression status and clinical significance of RGS2 in head and neck squamous cell carcinomas and matched adjacent normal tissues were evaluated using TCGA database. Three OSCC cell lines (i.e., SCC-9, Cal27, and Fadu) were overexpressed with RGS2, and the effect of RGS2 on cell proliferation and invasion was determined using the Transwell, clone formation, and cell counting kit (CCK)-8 assays. Moreover, the yeast two-hybrid scree-ning and co-immunoprecipitation (Co-IP) assays were conducted to detect the correlation of RGS2, four and a half LIM domains protein 1 (FHL1), and damage DNA-binding protein 1 (DDB1).@*RESULTS@#The expression level of RGS2 in OSCC was significantly lower than that in matched adjacent normal tissues (@*CONCLUSIONS@#RGS2 plays an important role in the inhibition of OSCC proliferation and invasion. The structure stability of RGS2 is competitively regulated by FHL1 and DDB1.

The Role of ISL1 and LHX5 LIM Homeobox Genes in Bladder Tumourigenesis

@#Introduction: Lin-11, Isl-1 and Mec-3 domains (LIM) homeobox genes are among the most important sub-families of homeobox genes. These genes are thought to play an important role in cancer. In this study, the protein expression of these genes was examined in urothelial carcinoma of the bladder. The expression pattern of Islet-1 (ISL1) and LIM homeobox 5 (LHX5) across different cancer stages and grades, as well as the association between the protein expression of these genes and patient demographics and clinicopathological features, were examined. Methods: A total of 100 formalin-fixed paraffin-embedded urothelial carcinoma tissues were selected from the Department of Pathology, Hospital Kuala Lumpur and the protein expression of ISL1 and LHX5 was determined using immunohistochemistry. Results: Positive expression of ISL1 and LHX5 was detected in 94% and 98% of the samples, respectively. There were no distinct LHX5 expression patterns associated with different cancer stages, but the proportion of high-expressing tumours was higher in high-grade tumours. In addition, there was a significant association between the expression of LHX5 and tumour grade. The proportion of tumours expressing high levels of ISL1 was found to be highest in later stage tumours. Conclusion: The high percentage of tumours expressing both these genes suggests that ISL1 and LHX5 play an important role in bladder tumourigenesis across multiple stages.

MicroRNA-374c-5p enhances sensitivity of human breast cancer MDAMB-231/DDP cells to cisplatin by targeting LIMK1 / 肿瘤

Objective: To investigate the effect of Homo sapiens (hsa)-microRNA (miRNA, miR)-374c-5p on the sensitivity of human breast cancer MDA-MB-231/DDP cells to cisplatin and its mechanism. Methods: The expressions of hsa-miR-374c-5p and Lim domain kinase 1 (LIMK1) in breast cancer MCF-10A, MDA-MB-231 and MDA-MB-231/DDP cells were detected by real-time fluorescent quantitative PCR. Then the hsa-miR-374c-5p mimics and inhibitor, LIMK1 siRNA and LIMK1 overexpressed plasmid were transfected into cisplatin-resistant human breast cancer MDA-MB-231/DDP cells, respectively. The relative expression levels of hsa-miR374c-5p and LIMK1 mRNA were detected by real-time fluorescent quantitative PCR, the expression level of LIMK1 protein was detected by Western blotting, the cell viability was detected by CCK-8 method, the apoptosis was detected by flow cytometry, and the clone formation ability was tested by plate clone forming assay. Finally, the binding relationship between hsa-miR-374c-5p and LIMK1 was verified by luciferase reporter gene assay. Results: The expression levels of hsa-miR-374c-5p and LIMK1 were reversed in breast cancer MDA-MB-231/DDP cells. The overexpression of hsa-miR-374c-5p or the knockdown of LIMK1 decreased cell viability (both P < 0.05) and clone forming ability (both P < 0.05), increased apoptotic rate (both P < 0.05), and increased the sensitivity of MDA-MB-231/ DDP cells to cisplatin (both P < 0.05); while the effects of hsa-miR-374c-5p knockdown on proliferation, clone formation, apoptosis and cisplatin-sensitivity of MDA-MB-231/DDP cells were reversed (all P < 0.05); and the overexpression of LIMK1 reversed the phenotype changes of MDA-MB-231/DDP cells transfected with hsa-miR-374c-5p mimics (all P < 0.05). Hsa-miR-374c-5p targeted LIMK1 mRNA directly and regulated the expression of LIMK1. Conclusion: Hsa-miR-374c-5p enhances the sensitivity of human breast cancer MDAMB-231/DDP cells to cisplatin by regulating the expression of LIMK1.

Beadex, a homologue of the vertebrate LIM domain only protein, is a novel regulator of crystal cell development in Drosophila melanogaster

Haematopoiesis is a complex process in which the regulatory mechanisms of several implicated transcription factors remain uncertain. Drosophila melanogaster is an excellent model to resolve the unanswered questions about the blood cell development. This study describes the role of Beadex, a Drosophila homologue of LIM domain only 2 (LMO2), in haematopoiesis. Mutants of Beadex were analysed for blood cell abnormalities. Crystal cells, a subset of haemocytes, were significantly more in Beadex hypermorphic flies. Similarly, Beadex misexpression in prohemocytes altered the crystal cell numbers. Stage-specific misexpression analyses demonstrated that Beadex functions after the prohemocytes enter the crystal cell lineage. We also discovered that Pannier–U-shaped complex is a negative regulator of the crystal cell differentiation and is possibly negatively regulated by Beadex through its interaction with Pannier. We, therefore, suggest the mechanism of two novel regulators of crystal cell specification—Beadex and Pannier—during Drosophila haematopoiesis.

LIM kinase 1 acts as a profibrotic mediator in permanent atrial fibrillation patients with valvular heart disease

Atrial fibrillation (AF) is the most frequently diagnosed cardiac arrhythmia worldwide. Patients with permanent atrialfibrillation are at an increased risk of developing valvular heart disease. Atrial fibrosis occurs in this pathophysiologicalsetting. LIM kinase 1 (LIMK1) is a serine/threonine kinase that regulates microtubule stability and actin polymerization infibroblasts. LIMK1 has been implicated in the pathogenesis of atrial fibrillation. Clinical data and biopsies of the right atrialappendage were collected from 50 patients with valvular heart disease who underwent heart valve replacement surgery.Data from patients with permanent atrial fibrillation (AF) and patients with sinus rhythm (SR) were compared. We foundthat AF patients had upregulated expression of LIMK1 as well as higher fibrosis. Transforming growth factor-b (TGF-b)stimulation induced the differentiation of cardiac fibroblasts into myofibroblasts as well as upregulated expression ofLIMK1. Downregulation of LIMK1 by siRNA inhibited TGF-b induced fibroblast-myofibroblast transition, as evidencedby the downregulation of the expression of several differentiation markers, namely alpha-smooth muscle actin and type Iand III collagen. Our findings revealed that increased LIMK1 protein levels may contribute to atrial fibrosis, and suggestedthat LIMK1 might be involved in AF development by promoting fibrogenesis associated with TGF-b.

Expression of LDB2 in lung cancer tissues and its correlation with S1PR1 / 临床检验杂志

Objective@#To investigate the expression of LIM domain binding 2 (LDB2) in lung cancer tissues and its correlation with sphingosine-1 phosphate receptor 1 (S1PR1). @*Methods@#Lung cancer tissues and the corresponding adjacent tissues from 52 patients in Nantong Tumor Hospital during April 2010 and May 2011 were collected as the experimental group and the control group, respectively. The expression levels of LDB2 and S1PR1 were detected by the real-time PCR (qRT-PCR). The expression results of LDB2 gene were further verified by the Oncomine database, and its correlations with clinicopathological parameters were analyzed. The ROC curve was drawn to evaluate the diagnosis value of LDB2 expression in lung cancer. The correlation of LDB2 expression with the prognosis of lung cancer was analyzed by the “Kaplan-Meier Plotter” database. In addition, the relationship between LDB2 and S1PR1 was also analyzed. @*Results@#The expression levels of LDB2 in lung cancer tissues (0.158 [0.062,0.383]) were significantly lower than that in the adjacent tissues (0.403 [0.261,0.711], U=700.0, P＜ 0.01). A total of 9 eligible studies were retrieved from the Oncomine database, and their expressions of LDB2 were also low (P＜0.01). The expressions of LDB2 in lung cancer tissues were not related to gender, age, smoking history, pathological type, tumor size, TNM staging and lymphatic metastasis (P＞0.05). The results of ROC curve showed that when the area under the ROC curve (AUC ROC ) was 0.741 (95% CI:0.643-0.839) and the cut-off value was 0.247, the sensitivity and specificity of LDB2 in the diagnosis of lung cancer were 80.8% and 61.5%, respectively. The Kaplan-Meier survival analysis showed that the 5-year overall survival time of the patients with low expression of LDB2 was shorter than that of the patients with high expression of LDB2(P＜0.01). In addition, the expression levels of S1PR1 in lung cancer tissues (0.710[0.337,1.523]) were significantly lower than that in the adjacent tissues (1.582[0.913,3.533],U=780.0, P＜0.01), and the expression levels of S1PR1 in lung cancer tissues were positively correlated with that of LDB2(r=0.827,P＜0.01). @*Conclusion@#The expressions of LDB2 and S1PR1 in lung cancer tissues are down-regulated, and have a positive correlation, and they may play an important role in the occurrence and development of lung cancer.

Impact of LDB3 gene polymorphisms on clinical presentation and implantable cardioverter defibrillator (ICD) implantation in Chinese patients with idiopathic dilated cardiomyopathy / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

OBJECTIVE@#Mutations in LIM domain binding 3 (LDB3) gene cause idiopathic dilated cardiomyopathy (IDCM), a structural heart disease with a complicated genetic background. However, the association of polymorphisms in the LDB3 gene with susceptibility to IDCM in Chinese populations remains unexplored as dose the impact on clinical presentation.@*METHODS@#We sequenced all exons and the adjacent part of introns of the LDB3 gene in 159 Chinese Han IDCM patients and 247 healthy controls. Then we detected the distribution of polymorphisms in the LDB3 gene in all participants and assessed their associations with risk of IDCM. Additionally, we conducted a stratified genotype-phenotype correlation analysis.@*RESULTS@#The A allele of rs4468255 was significantly associated with IDCM (P<0.01). The rs4468255, rs11812601, rs56165849, and rs3740346 were also associated with diastolic blood pressure (DBP) and left ventricular ejection fraction (LVEF) (P<0.05). Notably, a higher frequency of rs4468255 polymorphism was observed in implantable cardioverter defibrillator (ICD) recipients under a recessive model (P<0.01), whereas the significant association disappeared after adjusting for potential confounders. However, in the dominant model, notable correlations could only be observed after adjusting for multi parameters.@*CONCLUSIONS@#The rs4468255 was significantly correlated with IDCM of Chinese Han population. A allele of rs4468255 is higher in IDCM patients with ICD implantation, suggesting the influence of genetic background in the generation of this response. In addition, rs11812601, rs56165849, and rs3740346 in LDB3 show association with brain natriuretic peptide, DBP, and LVEF levels in patients with IDCM but did not show any association with IDCM susceptibility.

Mechanism of LMO3 in tumor metastasis / 国际外科学杂志

The LIM domains proteins are of significance proteins of human tumors. LMO3, a member of LIM-only subclass of LIM proteins, participates in the occurrence and development in many kinds of tumors, including neuroblastoma, meningioma, lung cancer and hepatocellular carcinoma, etc. LMO3 functions as interacting with neuronal transcription factor, HUA enhancer 2, interacting with upstream gene and inhibits its transcriptional activity or directly interacting with gene signaling. As an incomparable role in processes of tumors, LMO3 contributes to huge opportunities for therapeutic targeting for the future of the field.This article focuses on the molecular mechanism of LMO3 in the process of tumor development and metastasis, and reviews its recent progress.

Expression and potential role of ajuba LIM protein in oral squamous cell carcinoma / 中国肿瘤临床

Objective: To assess the expression of ajuba LIM protein (AJUBA) in oral squamous cell carcinoma (OSCC) and to determine its role in cell proliferation, migration, and invasion in OSCC. Methods: The expression of AJUBA at mRNA and protein levels in OSCC was determined by quantitative polymerase chain reaction (qPCR) and immunohistochemical approaches, respectively. This was fol-lowed by analysis of the correlations between AJUBA levels and clinicopathological features of OSCC. The effects of AJUBA on cell pro-liferation, migration, and invasion in OSCC were assessed by MTT, wound healing, and transwell migration assays, respectively. West-ern blot assays were performed to check for the potential regulation of the Snail/E-cadherin pathway by AJUBA. Results: The expres-sion of AJUBA was significantly higher in OSCC tissues compared to that in adjacent normal tissues and correlated with T stage, cell dif-ferentiation, lymph node metastasis, and recurrence in OSCC. Elevated AJUBA levels indicated poor prognosis in patients with OSCC. Depletion of AJUBA impaired cell proliferation, migration, and invasion abilities of OSCC cells. Data from Western blot assays showed that AJUBA facilitated the expression of Snail but inhibited that of E-cadherin. Conclusions: AJUBA is overexpressed in OSCC and may influence cell proliferation and invasion in OSCC by modulating the Snail/E-cadherin pathway.

Mechanism of LMO3 in tumor metastasis / 国际外科学杂志

The LIM domains proteins are of significance proteins of human tumors.LMO3,a member of LIM-only subclass of LIM proteins,participates in the occurrence and development in many kinds of tumors,including neuroblastoma,meningioma,lung cancer and hepatocellular carcinoma,etc.LMO3 functions as interacting with neuronal transcription factor,HUA enhancer 2,interacting with upstream gene and inhibits its transcriptional activity or directly interacting with gene signaling.As an incomparable role in processes of tumors,LMO3 contributes to huge opportunities for therapeutic targeting for the future of the field.This article focuses on the molecular mechanism of LMO3 in the process of tumor development and metastasis,and reviews its recent progress.

Impact of LDB3 gene polymorphisms on clinical presentation and implantable cardioverter defibrillator (ICD) implantation in Chinese patients with idiopathic dilated cardiomyopathy / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Objective: Mutations in LIM domain binding 3 (LDB3) gene cause idiopathic dilated cardiomyopathy (IDCM), a structural heart disease with a complicated genetic background. However, the association of polymorphisms in the LDB3 gene with susceptibility to IDCM in Chinese populations remains unexplored as dose the impact on clinical presentation. Methods: We sequenced all exons and the adjacent part of introns of the LDB3 gene in 159 Chinese Han IDCM patients and 247 healthy controls. Then we detected the distribution of polymorphisms in the LDB3 gene in all participants and assessed their associations with risk of IDCM. Additionally, we conducted a stratified genotypephenotype correlation analysis. Results: The A allele of rs4468255 was significantly associated with IDCM (P<0.01). The rs4468255, rs11812601, rs56165849, and rs3740346 were also associated with diastolic blood pressure (DBP) and left ventricular ejection fraction (LVEF) (P<0.05). Notably, a higher frequency of rs4468255 polymorphism was observed in implantable cardioverter defibrillator (ICD) recipients under a recessive model (P<0.01), whereas the significant association disappeared after adjusting for potential confounders. However, in the dominant model, notable correlations could only be observed after adjusting for multi parameters. Conclusions: The rs4468255 was significantly correlated with IDCM of Chinese Han population. A allele of rs4468255 is higher in IDCM patients with ICD implantation, suggesting the influence of genetic background in the generation of this response. In addition, rs11812601, rs56165849, and rs3740346 in LDB3 show association with brain natriuretic peptide, DBP, and LVEF levels in patients with IDCM but did not show any association with IDCM susceptibility.

The effects of single versus combined therapy using LIM-kinase 2 inhibitor and type 5 phosphodiesterase inhibitor on erectile function in a rat model of cavernous nerve injury-induced erectile dysfunction / 亚洲男科学杂志(英文版)

We aimed to determine whether combination of LIM-kinase 2 inhibitor (LIMK2i) and phosphodiesterase type-5 inhibitor (PDE5i) could restore erectile function through suppressing cavernous fibrosis and improving cavernous apoptosis in a rat model of cavernous nerve crush injury (CNCI). Seventy 12-week-old Sprague-Dawley rats were equally distributed into five groups as follows: (1) sham surgery (Group S), (2) CNCI (Group I), (3) CNCI treated with daily intraperitoneal administration of 10.0 mg kg-1 LIMK2i (Group I + L), (4) daily oral administration of 20.0 mg kg-1 udenafil, PDE5i (Group I + U), and (5) combined administration of 10.0 mg kg-1 LIMK2i and 20.0 mg kg-1 udenafil (Group I + L + U). Rats in Groups I + L, I + U, and I + L + U were treated with respective regimens for 2 weeks after CNCI. At 2 weeks after surgery, erectile response was assessed using electrostimulation. Penile tissues were processed for histological studies and western blot. Group I showed lower intracavernous pressure (ICP)/mean arterial pressure (MAP), lower area under the curve (AUC)/MAP, decreased immunohistochemical staining for alpha-smooth muscle (SM) actin, higher apoptotic index, lower SM/collagen ratio, increased phospho-LIMK2-positive fibroblasts, decreased protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) phosphorylation, increased LIMK2/cofilin phosphorylation, and increased protein expression of fibronectin, compared to Group S. In all three treatment groups, erectile responses, protein expression of fibronectin, and SM/collagen ratio were improved. Group I + L + U showed greater improvement in erectile response than Group I + L. SM content and apoptotic index in Groups I + U and I + L + U were improved compared to those in Group I. However, Group I + L did not show a significant improvement in SM content or apoptotic index. The number of phospho-LIMK2-positive fibroblasts was normalized in Groups I + L and I + L + U, but not in Group I + U. Akt/eNOS phosphorylation was improved in Groups I + U and I + L + U, but not in Group I + L. LIMK2/cofilin phosphorylation was improved in Groups I + L and I + L + U, but not in Group I + U. Our data indicate that combined treatment of LIMK2i and PDE5i immediate after CN injury could improve erectile function by improving cavernous apoptosis or eNOS phosphorylation and suppressing cavernous fibrosis. Rectification of Akt/eNOS and LIMK2/cofilin pathways appears to be involved in their improvement.

The effects of single versus combined therapy using LIM-kinase 2 inhibitor and type 5 phosphodiesterase inhibitor on erectile function in a rat model of cavernous nerve injury-induced erectile dysfunction / 亚洲男科学杂志(英文版)

We aimed to determine whether combination of LIM-kinase 2 inhibitor (LIMK2i) and phosphodiesterase type-5 inhibitor (PDE5i) could restore erectile function through suppressing cavernous fibrosis and improving cavernous apoptosis in a rat model of cavernous nerve crush injury (CNCI). Seventy 12-week-old Sprague-Dawley rats were equally distributed into five groups as follows: (1) sham surgery (Group S), (2) CNCI (Group I), (3) CNCI treated with daily intraperitoneal administration of 10.0 mg kg-1 LIMK2i (Group I + L), (4) daily oral administration of 20.0 mg kg-1 udenafil, PDE5i (Group I + U), and (5) combined administration of 10.0 mg kg-1 LIMK2i and 20.0 mg kg-1 udenafil (Group I + L + U). Rats in Groups I + L, I + U, and I + L + U were treated with respective regimens for 2 weeks after CNCI. At 2 weeks after surgery, erectile response was assessed using electrostimulation. Penile tissues were processed for histological studies and western blot. Group I showed lower intracavernous pressure (ICP)/mean arterial pressure (MAP), lower area under the curve (AUC)/MAP, decreased immunohistochemical staining for alpha-smooth muscle (SM) actin, higher apoptotic index, lower SM/collagen ratio, increased phospho-LIMK2-positive fibroblasts, decreased protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) phosphorylation, increased LIMK2/cofilin phosphorylation, and increased protein expression of fibronectin, compared to Group S. In all three treatment groups, erectile responses, protein expression of fibronectin, and SM/collagen ratio were improved. Group I + L + U showed greater improvement in erectile response than Group I + L. SM content and apoptotic index in Groups I + U and I + L + U were improved compared to those in Group I. However, Group I + L did not show a significant improvement in SM content or apoptotic index. The number of phospho-LIMK2-positive fibroblasts was normalized in Groups I + L and I + L + U, but not in Group I + U. Akt/eNOS phosphorylation was improved in Groups I + U and I + L + U, but not in Group I + L. LIMK2/cofilin phosphorylation was improved in Groups I + L and I + L + U, but not in Group I + U. Our data indicate that combined treatment of LIMK2i and PDE5i immediate after CN injury could improve erectile function by improving cavernous apoptosis or eNOS phosphorylation and suppressing cavernous fibrosis. Rectification of Akt/eNOS and LIMK2/cofilin pathways appears to be involved in their improvement.

Role of inhibiting LIM-kinase2 in improving erectile function through suppression of corporal fibrosis in a rat model of cavernous nerve injury / 亚洲男科学杂志(英文版)

We evaluated whether LIM-kinase 2 inhibitor (LIMK2i) could improve erectile function by suppressing corporal fibrosis through the normalization of the Rho-associated coiled-coil protein kinase 1 (ROCK1)/LIMK2/Cofilin pathway in a rat model of cavernous nerve crush injury (CNCI). Sixty 11-week-old male Sprague-Dawley rats were divided equally into five groups: sham surgery (S), CNCI (I), and CNCI treated with low-dose (L), medium-dose (M), and high-dose (H) LIMK2i. The L, M, and H groups were treated with a daily intraperitoneal injection of LIMK2i (2.5, 5.0, and 10.0 mg kg-1 body weight, respectively) for 1 week after surgery. The erectile response was assessed using electrostimulation at 1 week, postoperatively. Penile tissues were processed for Masson's trichrome staining, double immunofluorescence, and Western blot assay. Erectile responses in the H group improved compared with the I group, while the M group showed only partial improvement. A significantly decreased smooth muscle/collagen ratio and an increased content of fibroblasts positive for phospho-LIMK2 were noted in the I group. The M and H groups revealed significant improvements in histological alterations and the dysregulated LIMK2/Cofilin pathway, except for LIMK2 phosphorylation in the M group. The inhibition of LIMK2 did not affect the ROCK1 protein expression. The content of fibroblasts positive for phospho-LIMK2 in the H group returned to the level found in the S group, whereas it did not in the M group. However, the L group did not exhibit such improvements. Our data suggest that the inhibition of LIMK2, particularly with administration of 10.0 mg kg-1 body weight LIMK2i, can improve corporal fibrosis and erectile function by normalizing the LIMK2/Cofilin pathway.

Role of inhibiting LIM-kinase2 in improving erectile function through suppression of corporal fibrosis in a rat model of cavernous nerve injury / 亚洲男科学杂志(英文版)

We evaluated whether LIM-kinase 2 inhibitor (LIMK2i) could improve erectile function by suppressing corporal fibrosis through the normalization of the Rho-associated coiled-coil protein kinase 1 (ROCK1)/LIMK2/Cofilin pathway in a rat model of cavernous nerve crush injury (CNCI). Sixty 11-week-old male Sprague-Dawley rats were divided equally into five groups: sham surgery (S), CNCI (I), and CNCI treated with low-dose (L), medium-dose (M), and high-dose (H) LIMK2i. The L, M, and H groups were treated with a daily intraperitoneal injection of LIMK2i (2.5, 5.0, and 10.0 mg kg-1 body weight, respectively) for 1 week after surgery. The erectile response was assessed using electrostimulation at 1 week, postoperatively. Penile tissues were processed for Masson's trichrome staining, double immunofluorescence, and Western blot assay. Erectile responses in the H group improved compared with the I group, while the M group showed only partial improvement. A significantly decreased smooth muscle/collagen ratio and an increased content of fibroblasts positive for phospho-LIMK2 were noted in the I group. The M and H groups revealed significant improvements in histological alterations and the dysregulated LIMK2/Cofilin pathway, except for LIMK2 phosphorylation in the M group. The inhibition of LIMK2 did not affect the ROCK1 protein expression. The content of fibroblasts positive for phospho-LIMK2 in the H group returned to the level found in the S group, whereas it did not in the M group. However, the L group did not exhibit such improvements. Our data suggest that the inhibition of LIMK2, particularly with administration of 10.0 mg kg-1 body weight LIMK2i, can improve corporal fibrosis and erectile function by normalizing the LIMK2/Cofilin pathway.

Effect of LIM homeobox gene-8 on biological behavior of human ovarian cancer SKOV3 cells / 第二军医大学学报

Objective To investigate the effect of LIM homeobox gene-8 (Lhx8) on the proliferation, metastasis and invasion of human ovarian cancer SKOV3 cells. Methods Lhx8-overexpression lentivirus (LV-Lhx8) was transfected into ovarian cancer SKOV3 cells to establish Lhx8-overexpression model and the negative control lentivirus (LV-NC) was used as control. Then Lhx8-siRNA and FAM-siRNA (used as control; NC group) siRNA was transfected into SKOV3 cells using Lipofectamine 2000 to construct cell interference model. Cells in wild type (WT) group were only given equivalent transfection reagent without virus or any interference fragment. The expression of Lhx8 was detected by immunofluorescence, qPCR and Western blotting. The proliferation of cells after overexpressing or interfering Lhx8 was measured by EDU assays and cell cycle assay. The migration and invasion of cells after transfection were measured by wound scratch experiments and Transwell assay. Results The expression of Lhx8 in SKOV3 cells in the LV-Lhx8 group was significantly higher than that in the LV-NC and WT groups (P<0.01), and its mRNA and protein expressions were significantly decreased after interfering Lhx8 (P<0.01). Compared with the WT and LV-NC groups, the proliferation of SKOV3 cells was significantly decreased in the LV-Lhx8 group and was significantly increased in the Lhx8-siRNA group (P<0.01). The cell cycle assay showed that Lhx8 overexpression significantly inhibited cell proliferation by increasing the number of cells in the G0/G1 phase, while the number of cells in the S phase in the Lhx8-siRNA group was significantly higher than that in the WT and NC groups (P<0.01). The migration and invasion of SKOV3 cells and the expression of matrix metalloproteinase (MMP)-2 and MMP-9 in the LV-Lhx8 group were significantly lower than those in the WT and LV-NC groups (P<0.01), while those in the Lhx8-siRNA group were significantly higher (P<0.01). Conclusion Lhx8 can inhibit the proliferation, migration and invasion of ovarian cancer SKOV3 cells, and down-regulate the expressions of MMP-2 and MMP-9.

Edaravone protects against cerebral ischemia-reperfusion injury in rats by upregulation of the LMO4 expression / 国际脑血管病杂志

ObjectiveTo investigate the neuroprotective mechanism of edaravone for cerebral ischemia-reperfusion injury in rats.MethodsThirty-six healthy adult male SD rats were randomly divided into three groups: a sham operation group, an ischemic model group, and an edaravone group (n=12 in each group).A focal cerebral ischemia model was induced by the suture method.Reperfusion was resumed after 2 h of ischemia;then the animals were sacrificed at 24 h after reperfusion.Edaravone 3 mg/kg was injected intraperitoneally immediately after cerebral ischemia-reperfusion in the edaravone group.The rats in the model group were injected equal volume normal saline.HE staining was used to observe the pathological changes.TUNEL staining was used to detect apoptotic cells in the ischemic cortex.Western blot and immunofluorescent staining were used to detect the expression levels of LIM domain protein 4 (LMO4) and LMO4 positive cells.Results HE staining showed that cellular morphology was basically normal in the sham operation group;both the model group and edaravone group had cell necrosis, but the latter was less severe.The number of morphologically normal cells in the edaravone group was significantly more than that in the model group (P<0.01).TUNEL staining showed that no TUNEL positive cells in the sham operation group were observed.The TUNEL positive cells in the edaravone group was significantly less that in the model group (P<0.01).Immunofluorescence staining showed that the expression level of LMO4 in the ischemic cortex in the edaravone group was significantly higher than that in the model group (P<0.01).ConclusionsEdaravone can alleviate the cerebral ischemia-reperfusion injury and inhibit neuronal apoptosis.Its mechanism may be associated with the upregulation of LMO4 expression.

Expresssion of miRNA-1 in esophageal squamous cell carcinoma tissues and its mechanism / 肿瘤

Objective: To investigate the expression of microRNA-1 (miRNA-1) in human esophageal squamous cell carcinoma (ESCC) tissues and its possible mechanism. Methods: The expression levels of miRNA-1 and LIM and SH3 domain protein 1 (LASP1) mRNA and LASP1 protein in 55 cases of ESCC and its adjacent para-cancerous tissues were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The potential target gene of miRNA-1 was predicted by online bioinformatics software. The target gene of miRNA-1 after Eca109 cells co-transfection with recombination dual luciferase reporter vector psiCHECK-2-LASP1 containing 3'-untranslated region (3'-UTR) with miRNA-1 binding site of LASP1 gene and miRNA-1 mimic was verified by dual-luciferase reporter assay system. The expressions of miRNA-1 and LASP1 protein in Eca109 cells after transfection with miRNA-1 mimic or miRNA-1 inhibitor were detected by real-time fluorescent quantitative-PCR and Western blotting, respectively. Results: The expression level of miRNA-1 in ESCC tissues was lower than that in adjacent para-cancerous tissues (P < 0.01), and the expression levels of LASP1 mRNA and protein were opposite (all P < 0.05). The expressions of miRNA-1 and LASP1 were associated with lymph metastasis, TNM staging and histological grade (all P < 0.05). The miRNA-1 expression in ESCC tissues was positively associated with LASP1 (r = +0.45, P < 0.05). LASP1 was target gene of miRNA-1. miRNA-1 could directly target the LASP1 3'-UTR. The expression level of miRNA-1 was up-regulated in Eca109 cells after transfection with miRNA-1 mimic (P < 0.01), and the expression level of LASP1 protein was down-regulated (P < 0.05). The expression level of miRNA-1 was down-regulated in Eca109 cells after transfection with miRNA-1 inhibitor (P < 0.05), and the expression level of LASP1 protein was up-regulated (P < 0.05). Conclusion: The expression level of miRNA-1 is low in ESCC tissues, and its mechanism may be associated with regulation of target gene LASP1.