ROCK1 Protein Expression Research Articles

MiR-129–2-3p binds SEMA4C to regulate HCC development and inhibit the EMT

BackgroundAmong primary liver cancers, HCC is the most prevalent. Small noncoding RNAs called miRNAs control the expression of downstream target genes to take part in a variety of physiological and pathological processes, including those related to cancer. MethodsmiR-129–2–3p and SEMA4C expression levels were assessed using RT-qPCR. The CCK-8, invasion, and wound healing assays were used to confirm the capacity of HCC cells for proliferation, invasion and migration respectively. Serum SEMA4C levels were detected via ELISA. The RIP and dual-luciferase reporter assays were used to confirm the existence of intergenic binding sites. Cell apoptosis assay and cell cycle assay were performed to detect the apoptosis rate and cycle distribution of cells, and WB was performed to detect the protein expression of SEMA4C, RhoA, ROCK1, E-cadherin, N-cadherin, and vimentin. Furthermore, cancer-inhibiting role of miR-129–2–3p were further confirmed by animal tests. ResultsmiR-129–2–3p expression was reduced in HCC tissues and cells. Overexpression of miR-129–2–3p decreased the proliferation, invasion, migration, and EMT in HCC cells, whereas inhibition of miR-129–2–3p had the opposite effects. Our research also showed that SEMA4C was increased in HCC tissues, serum and cells, and that SEMA4C knockdown prevented HCC cell invasion, migration, proliferation, and EMT. Overexpression of SEMA4C reversed the inhibitory effect of miR-129–2–3p on HCC. ConclusionsOverall, we discovered that through binding to SEMA4C, miR-129–2–3p regulates HCC cell proliferation, invasion, migration, and EMT.

Effects of inhibition of Rho/ROCK pathway on proliferation and migration of airway smooth muscle cells and related mechanisms

To investigate the effects and molecular mechanisms of inhibition of the Ras homolog gene (Rho)/Rho-associated coiled-coil forming protein kinase (ROCK) pathway on the proliferation and migration of airway smooth muscle cells involving myocardin (MYOCD). Human airway smooth muscle cells were infected with the adenoviral vector Ad-ZsGreen-shRNA-hROCK1 in vitro. The cells were randomly divided into four groups: ROCK1 gene silencing control (shNC) group, shNC + arachidonic acid (AA, Rho/ROCK pathway activator) group, ROCK1 gene silencing (shROCK1) group, and shROCK1 + AA group (n=3 each). Quantitative real-time polymerase chain reaction and Western blot were used to detect the expression levels of ROCK1 and MYOCD mRNA and protein. ELISA was employed to measure the levels of globular actin and filamentous actin, while immunofluorescent staining and scratch assays were utilized to assess cell proliferation and migration. Compared to the shNC + AA group, the shROCK1 + AA group exhibited decreased levels of ROCK1 and MYOCD mRNA and protein expression, reduced expression levels of globular actin and filamentous actin, and diminished cell proliferation and migration capabilities (P<0.05). Inhibition of the Rho/ROCK pathway suppresses the proliferation and migration of airway smooth muscle cells, which may be associated with the downregulation of MYOCD.

SLIT3 promotes cardiac fibrosis and differentiation of cardiac fibroblasts by RhoA/ROCK1 signaling pathway.

Slit guidance ligand 3 (SLIT3) has been identified as a potential therapeutic regulator against fibroblast activity and fibrillary collagen production in an autocrine manner. However, this research aims to investigate the potential role of SLIT3 in cardiac fibrosis and fibroblast differentiation and its underlying mechanism. C57BL/6 mice (male, 8-10 weeks, n=47) were subcutaneously infused with Ang II (2.0 mg/kg/day) for 4 weeks. One to two-day-old Sprague-Dawley (SD) rats were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (60 mg/kg) and ketamine (50 mg/kg) and the cardiac fibroblast was isolated aseptically. The mRNA and protein expression were analyzed using RT-qPCR and Western blotting. The SLIT3 expression level was increased in Ang II-induced mice models and cardiac fibroblasts. SLIT3 significantly increased migrated cells and α-smooth muscle actin (α-SMA) expression in cardiac fibroblasts. Ang II-induced increases in mRNA expression of collagen I (COL1A1), and collagen III (COL3A1) was attenuated by SLIT3 inhibition. SLIT3 knockdown attenuated the Ang II-induced increase in mRNA expression of ACTA2 (α-SMA), Fibronectin, and CTGF. SLIT3 suppression potentially reduced DHE expression and decreased malondialdehyde (MDA) content, and the superoxide dismutase (SOD) and catalase (CAT) levels were significantly increased in cardiac fibroblasts. Additionally, SLIT3 inhibition markedly decreased RhoA and ROCK1 protein expression, whereas ROCK inhibitor Y-27632 (10 μM) markedly attenuated the migration of cardiac fibroblasts stimulated by Ang II and SLIT3. The results speculate that SLIT3 could significantly regulate cardiac fibrosis and fibroblast differentiation via the RhoA/ROCK1 signaling pathway.

Silencing geranylgeranyltransferase I inhibits the migration and invasion of salivary adenoid cystic carcinoma through RhoA/ROCK1/MLC signaling and suppresses proliferation through cell cycle regulation.

Geranylgeranyltransferase type I (GGTase-I) significantly affects Rho proteins, such that the malignant progression of several cancers may be induced. Nevertheless, the effect and underlying mechanism of GGTase-I in the malignant progression of salivary adenoid cystic carcinoma (SACC) remain unclear. This study primarily aimed to investigate the role and mechanism of GGTase-I in mediating the malignant progression of SACC. The level of GGTase-I gene in cells was stably knocked down by short hairpin RNA-EGFP-lentivirus. The effects of GGTase-I silencing on the migration, invasion, and spread of cells were examined, the messenger RNA levels of GGTase-I and RhoA genes of SACC cells after GGTase-I knockdown were determined, and the protein levels of RhoA and RhoA membrane of SACC cells were analyzed. Moreover, the potential underlying mechanism of silencing GGTase-I on the above-mentioned aspects in SACC cells was assessed by examining the protein expression of ROCK1, MLC, p-MLC, E-cadherin, Vimentin, MMP2, and MMP9. Furthermore, the underlying mechanism of SACC cells proliferation was investigated through the analysis of the expression of cyclinD1, MYC, E2F1, and p21CIP1/WAF1 . Besides, the change of RhoA level in SACC tissues compared with normal paracancer tissues was demonstrated through quantitative reverse-transcription polymerase chain reaction and western blot experiments. Next, the effect after GGTase-I silencing was assessed through the subcutaneous tumorigenicity assay. As indicated by the result of this study, the silencing of GGTase-I significantly reduced the malignant progression of tumors in vivo while decreasing the migration, invasion, and proliferation of SACC cells and RhoA membrane, Vimentin, ROCK1, p-MLC, MMP2, MMP9, MYC, E2F1, and CyclinD1 expression. However, the protein expression of E-cadherin and p21CIP1/WAF1 was notably upregulated. Subsequently, no significant transform of RhoA and MLC proteins was identified. Furthermore, RhoA expression in SACC tissues was significantly higher than that in paracancerous tissues. As revealed by the results of this study, GGTase-I shows a correlation with the proliferation of SACC through the regulation of cell cycle and may take on vital significance in the migration and invasion of SACC by regulating RhoA/ROCK1/MLC signaling pathway. GGTase-I is expected to serve as a novel exploration site of SACC.

Mechanism of Zhenwu Decoction in improving renal inflammatory injury in mice with DN of spleen-kidney Yang deficiency syndrome by regulating ROCK/IKK/NF-κB pathway

To investigate the intervention effect and mechanism of Zhenwu Decoction on diabetic nephropathy(DN) mice of spleen-kidney Yang deficiency syndrome based on the Rho-associated coiled-coil kinase(ROCK)/IκB kinase(IKK)/nuclear factor-κB(NF-κB) pathway. Ninety-five 7-week-old db/db male mice and 25 7-week-old db/m male mice were fed adaptively for one week. The DN model of spleen-kidney Yang deficiency syndrome was induced by Dahuang Decoction combined with hydrocortisone by gavage, and then the model was evaluated. After modeling, they were randomly divided into a model group, high-dose, medium-dose, and low-dose Zhenwu Decoction groups(33.8, 16.9, and 8.45 g·kg~(-1)·d~(-1)), and an irbesartan group(25 mg·kg~(-1)·d~(-1)), with at least 15 animals in each group. The intervention lasted for eight weeks. After the intervention, body weight and food intake were measured. Serum crea-tinine(Scr), blood urea nitrogen(BUN), fasting blood glucose(FBG), urinary albumin(uALb), and urine creatinine(Ucr) were determined. The uALb/Ucr ratio(ACR) and 24 h urinary protein(UTP) were calculated. Renal pathological morphology was evaluated by HE staining and Masson staining. The levels of key molecular proteins in the ROCK/IKK/NF-κB pathway were detected by Western blot. Enzyme-linked immunosorbent assay(ELISA) was used to detect interleukin-1β(IL-1β), interleukin-6(IL-6), interleukin-8(IL-8), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Compared with the blank group, the model group showed increased content of BUN, uALb, and SCr, increased values of 24 h UTP and ACR, decreased content of Ucr(P&lt;0.05), enlarged glomeruli, thickened basement membrane, mesangial matrix proliferation, inflammatory cell infiltration, and collagen fiber deposition. The protein expression of ROCK1, ROCK2, IKK, NF-κB, phosphorylated IKK(p-IKK), phosphorylated NF-κB(p-NF-κB), and phosphorylated inhibitor of NF-κB(p-IκB) increased(P&lt;0.05), while the protein expression of inhibitor of NF-κB(IκB) decreased(P&lt;0.05). The levels of inflammatory factors IL-1β, IL-6, IL-8, and TNF-α increased(P&lt;0.05), while the level of IL-10 decreased(P&lt;0.05). Compared with the model group, the groups with drug treatment showed decreased levels of BUN, uALb, SCr, 24 h UTP, and ACR, increased level of Ucr(P&lt;0.05), and improved renal pathological status to varying degrees. The high-and medium-dose Zhenwu Decoction groups and the irbesartan group showed reduced protein expression of ROCK1, ROCK2, IKK, NF-κB, p-IKK, p-NF-κB, and p-IκB in the kidneys(P&lt;0.05), increased protein expression of IκB(P&lt;0.05), decreased levels of serum inflammatory factors IL-1β, IL-6, IL-8, and TNF-α(P&lt;0.05), and increased level of IL-10(P&lt;0.05). Zhenwu Decoction can significantly improve renal function and renal pathological damage in DN mice of spleen-kidney Yang deficiency syndrome, and its specific mechanism may be related to the inhibition of inflammatory response by down-regulating the expression of key molecules in the ROCK/IKK/NF-κB pathway in the kidney.

Downregulation of the expression of galanin impairs erectile function in hypoandrogenic rats

Abstract Background The relationship between galanin and erectile function under low androgen levels is still unclear. Aim To explore whether a low testosterone level damages the erection of a rat by regulating the expression of galanin and GalR in penile cavernous tissue. Methods Thirty-six male Sprague-Dawley rats, 8 weeks of age, were randomly grouped as follows (n = 6): control, castration, castration + testosterone replacement, control + transfection, castration + transfection, and castration + empty transfection. At 4 weeks after castration, rats in the transfection group were injected with lentivirus carrying the targeting galanin gene (2 × 108 TU/mL, 10 μL) in the corpus cavernosum. After 1 week of injection, the intracavernosal pressure (ICP), mean arterial blood pressure (MAP), nitric oxide (NO), serum testosterone concentration, galanin, GalR1-3, ROCK1, ROCK2, and p-eNOS/eNOS in the rat penile tissues were evaluated. Outcomes ICPmax/MAP and the expression of galanin in the corpus cavernosum in castrated rats were obviously decreased as compared with those in the control rats. Results The castrated rats showed remarkably lower ICPmax/MAP, galanin, GalR1-3, p-eNOS/eNOS, and NO content and markedly higher ROCK1 and ROCK2 in penile tissues than the control group (P &amp;lt; .05). The transfected rats administrated with LV Gal had obviously higher ICPmax/MAP, p-eNOS/eNOS, and NO content and less ROCK1 and ROCK2 protein expression in the corpus cavernosum when compared with the castration group (P &amp;lt; .05). Clinical Translation Upregulating the expression of galanin in the penile corpus cavernosum might be a novel method of treating erectile dysfunction caused by a low androgen level. Strengths and Limitations The conclusions obtained in the animal experiments need to be confirmed in human data. Conclusion The erectile function of hypoandrogen rats might be inhibited by downregulating the level of galanin and GalR1-3, upregulating ROCK1 and ROCK2 levels, and inhibiting the eNOS/NO signaling pathway in penile corpus cavernosum.

IncRNA MALAT1 Regulates the Proliferation, Apoptosis, Migration, and Invasion of Osteosarcoma Cells by Targeting miR-873-5p/ROCK1.

The malignant bone tumor osteosarcoma (OS) was one of the most aggressive tumors. Despite breakthroughs in treatment options for OS recently, the survival rate of patients with metastasis or reoccurring disease has remained unchanged over the last 25 years, at around 20%. lncRNA expression dysregulation is linked to carcinogenesis, advancement, and metastasis. Additionally, the fundamental mechanism of lncRNAs in regulating OS cell biological activity and progression is still being investigated. The expression of miR-873-5p and MALAT1 were detected by quantitative real-time polymerase chain reaction (qRT-PCR) in OS. The relationship between the expression level of MALAT1 and the survival rate of OS individuals was evaluated by the Kaplan-Meier plotter. The tumor cell's capability of proliferation was determined using the CCK-8. Transwell was used to test the migratory and invasive properties of tumor cells. ROCK1 protein expression was analyzed by western blot, while qRT-PCR was used to detect ROCK1 mRNA expression. Targeted genes of MALAT1 or miR-873-5p were predicted by StarBase2.0. The target association among miR-873-5p and MALAT1 or ROCK1 was confirmed using the luciferase assay. The relationship between ROCK1 and MALAT1 or miR-873-5p expression in OS was investigated using Spearman's correlation analysis. MALAT1 was up-regulated and was linked to a lower survival rate of patients in OS. The malignant behaviors of cells were inhibited by down-regulated MALAT1 in vitro. Dual-luciferase gene experiments confirmed the presence of MALAT1/miR-873-5p/ROCK1 axis. The up-regulated miR-873-5p blocked the promoted effects of MALAT1 on cell behaviors. Over-expressed MALAT1 promoted the malignant behaviors of cells by miR-873-5p/ROCK1 axis in OS.

Protective Effect of Rho-Associated Protein Kinase (ROCK) Activated by Bone Marrow Mesenchymal Stem Cells on Bone Strength and Osteoblasts in Rats with Osteoporosis

This study assesses BMSCs’ effect on bone strength and osteoblast activity in rats with osteoporosis. 40 SD female rats were assigned into normal (A) group, model (B) group, puerarin (C) group, and BMSCs (D) group followed by analysis of bone strength by small animal bone strength analyzer and the expression of ROCK1 protein in tibia by immunohistochemistry. The bone strength of group B was lower than group A (P &lt; 0.05), and higher in groups C and D than group B (P &lt; 0.05) with further higher in group D than group C (P &lt; 0.05). Group A showed obvious mesh cancellous bone trabecular bone and continuity, compact structure, and group B cells appeared loose and irregular distribution, parts cancellous bone trabeculae decrease and part of the fracture, and arranged in sparse irregular distribution. The decrease of trabecular bone in group C and D is not more obvious than group B and the morphology in group D got better improved than in group C. Group B showed lower proliferation of osteoblasts and ALP activity than group A (P &lt; 0.05) and the proliferation and ALP activity in groups C and D was increased significantly compared with group B (P &lt; 0.05). ROCK1 expression was significantly lower in group B than group A (P &lt; 0.05) higher in groups C and D than group B (P &lt; 0.05). Bone marrow mesenchymal stem cells (BMSCs) can effectively increase the bone strength and activity of osteoblasts in rats with osteoporosis, and promote the activation of ROCK signal.

MicroRNA-148a induces apoptosis and prevents angiogenesis with bevacizumab in colon cancer through direct inhibition of ROCK1/c-Met via HIF-1α under hypoxia.

Angiogenesis and antiapoptosis effects are the major factors influencing malignancy progression. Hypoxia induces multiple mechanisms involving microRNA (miRNA) activity. Vascular endothelial growth factor (VEGF) is correlated with angiogenesis. An antiapoptotic factor, myeloid leukemia 1 (Mcl-1) is the main regulator of cell death. This study examined the role of miR-148a in inhibiting VEGF and Mcl-1 secretion by directly targeting ROCK1/c-Met by downregulating HIF-1α under hypoxia. The protein expression of ROCK1 or Met/HIF-1α/Mcl-1 in HCT116 and HT29 cells (all P < 0.05) was significantly reduced by miR-148a. The tube-formation assay revealed that miR-148a significantly suppressed angiogenesis and synergistically enhanced the effects of bevacizumab (both P < 0.05). The MTT assay revealed the inhibitory ability of miR-148a in HCT116 and HT29 cells (both P < 0.05). miR-148a and bevacizumab exerted synergistic antitumorigenic effects (P < 0.05) in an animal model. Serum miR-148a expression of metastatic colorectal cancer (mCRC) patients with a partial response was higher than that of mCRC patients with disease progression (P = 0.026). This result revealed that miR-148a downregulated HIF-1α/VEGF and Mcl-1 by directly targeting ROCK1/c-Met to decrease angiogenesis and increase the apoptosis of colon cancer cells. Furthermore, serum miR-148a levels have prognostic/predictive value in patients with mCRC receiving bevacizumab.

Mechanical Stretch Promotes Macrophage Polarization and Inflammation via the RhoA-ROCK/NF-κB Pathway.

Macrophages play an essential role in the pathogenesis of most inflammatory diseases. Recent studies have shown that mechanical load can influence macrophage function, leading to excessive and uncontrolled inflammation and even systemic damage, including cardiovascular disease and knee osteoarthritis. However, the molecular mechanism remains unclear. In this study, murine RAW264.7 cells were treated with mechanical stretch (MS) using the Flexcell-5000T Tension System. The expression of inflammatory factors and cytokine release were measured by RT-qPCR, ELISA, and Western blotting. The protein expression of NF-κB p65, Iκb-α, p-Iκb-α, RhoA, ROCK1, and ROCK2 was also detected by Western blotting. Then, Flow cytometry was used to detect the proportion of macrophage subsets. Meanwhile, Y-27632 dihydrochloride, a ROCK inhibitor, was added to knockdown ROCK signal transduction in cells. Our results demonstrated that MS upregulated mRNA expression and increased the secretion levels of proinflammatory factors iNOS, IL-1β, TNF-α, and IL-6. Additionally, MS significantly increased the proportion of CD11b+CD86+ and CD11b+CD206+ subsets in RAW264.7 macrophages. Furthermore, the protein expression of RhoA, ROCK1, ROCK2, NF-κB p65, and IκB-α increased in MS-treated RAW264.7 cells, as well as the IL-6 and iNOS. In contrast, ROCK inhibitor significantly blocked the activation of RhoA-ROCK and NF-κB pathway, decreased the protein expression of IL-6 and iNOS, reduced the proportion of CD11b+CD86+ cells subpopulation, and increased the proportion of CD11b+CD206+ cell subpopulation after MS. These data indicate that mechanical stretch can regulate the RAW264.7 macrophage polarization and enhance inflammatory responses in vitro, which may contribute to activation the RhoA-ROCK/NF-κB pathway.

Mechano Growth Factor Accelerates ACL Repair and Improves Cell Mobility of Mechanically Injured Human ACL Fibroblasts by Targeting Rac1-PAK1/2 and RhoA-ROCK1 Pathways

Exceeded mechanical stress leads to a sublethal injury to anterior cruciate ligament (ACL) fibroblasts, and it will hinder cell mobility and ACL regeneration, and even induce osteoarthritis. The mechano growth factor (MGF) could be responsible for mechanical stress and weakening its negative effects on cell physiological behaviors. In this study, effects of MGF on cell mobility and relevant molecules expression in injured ACL fibroblasts were detected. After an injurious mechanical stretch, the analysis carried out, at 0 and 24 h, respectively, showed that the cell area, roundness, migration, and adhesion of ACL fibroblasts were reduced. MGF (10, 100 ng/mL) treatment could improve cell area, roundness and promote cell migration and adhesion capacity compared with the injured group without MGF. Further study indicated that cell mobility-relevant molecules (PAK1/2, Cdc42, Rac1, RhoA, and ROCK1) expression in ACL fibroblasts was down-regulated at 0 or 24 h after injurious stretch (except Rac1 and RhoA at 0 h). Similarly, MGF improved cell mobility-relevant molecule expression, especially the ROCK1 expression level in ACL fibroblasts at 0 or 24 h after injurious stretch. Protein expression of ROCK1 in injured ACL fibroblasts was also reduced and could be recovered by MGF treatment. In a rabbit partial ACL transection (ACLT) model, ACL exhibited poor regenerative capacity in collagen and extracellular matrix (ECM) synthesis after partial ACLT for 2 or 4 weeks, and MGF remarkably accelerated ACL regeneration and restored its mechanical loading capacity after partial ACLT for four weeks. Our findings suggest that MGF weakens the effects of pathological stress on cell mobility of ACL fibroblasts and accelerates ACL repair, and might be applied as a future treatment approach to ACL rupture in the clinic.

Effects of BMI1 Gene on Regulating Apoptosis, Invasion, and Migration of HEC-1B Cells Induced by Ionizing Radiation.

The aim of this study was to examine the role of B lymphoma Moloney murine leukemia virus insertion region 1 (BMI1) gene in regulating the apoptosis, invasion, and migration of human endometrial adenocarcinoma cell line (HEC-1B) cells induced by ionizing radiation. The expression of BMI1 mRNA was detected by quantitative real-time polymerase chain reaction (qRT-PCR), and the positive expression of BMI1 was detected by immunohistochemistry (IHC) staining. HEC-1 B cells were randomly divided into three groups: control group, BMI1 overexpression group, and BMI1 inhibitor group. Cell proliferation was detected by cell counting kit-8 (CCK-8); cell migration and invasion were detected by Transwell test; cell apoptosis was detected by flow cytometry; and the expression of MMP2, MMP7, MMP9, Rock1, RhoA, P53, P21, and Bax protein was detected by the western blot. The results suggested that the expression of BMI1 mRNA and tissue positive in endometrial cancer tissues was increased significantly. After ionizing radiation, compared with the control group, the proliferation, cell migration, and invasion of HEC-1B cells were increased significantly in the BMI1 overexpression group, while the proliferation, cell migration, and invasion of HEC-1B cells were decreased significantly in BMI1 inhibitor group. The apoptosis rate of BMI1 overexpression group was decreased significantly, while the BMI1 inhibitor group was increased significantly. The levels of MMP2, MMP7, MMP9, Rock1, RhoA and p53, p21, Bax protein in BMI1 overexpression group were significantly increased, while the levels of MMP2, MMP7, MMP9, Rock1, RhoA and p53, p21, Bax protein in BMI1 inhibitor group were significantly decreased. BMI1 is highly expressed in endometrial cancer tissues, and inhibiting BMI1 expression can reduce the proliferation, migration, and invasion of HEC-1B cells after ionizing radiation and promote apoptosis, which offers new insights into the clinical radiotherapy of tumors.

Perk heterozygosity ameliorates chronic hypoxia-induced pulmonary hypertension and right ventricular hypertrophy in male rats

ABSTRACT Background Pulmonary hypertension (PH) is a rare and deadly disease characterized by remodeling of the pulmonary vasculature and increased pulmonary artery pressure. hypobaric pulmonary hypertension (HPH) is clinically classified as group 4 of pulmonary hypertension and has a poor prognosis . Previous reports showed that HPH was associated with increased endoplasmic reticulum (ER) stress. The protein kinase R-like endoplasmic reticulum kinase (PERK) is an ER-associated stress protein. However, to date, its physiological effects on HPH and RVF development remains unknown. This study aimed to assess PERK’s role in HPH and RV function using in vivo experimental model. Methods Perk-knockout male Sprague–Dawley rats were generated and were housed in either a hypobaric chamber or in a normoxic environment. After stimulation for 4 weeks, the hemodynamic parameters of the rats were measured. The heart and lungs were harvested for pathological observation. Blood was collected for the detection of inflammatory indexes. The right ventricle tissue was collected to assess phosphorylated-AKT, ROCK1, ET1, and MMP2 protein expression. Results: we firstly generated Perk+/− rats, Under normal conditions, Perk +/− rats showed no changes in mPAP(mean pulmonary artery pressure), RVHI(Right ventricular hypertrophy index), cardiomyocyte size and interstitial fibrosis, and pulmonary vascular remodeling. However, in response to chronic hypoxia, Perk +/− rats exhibited decreased in mPAP, RVHI, ventricular fibrosis, and lung remodeling compared to wild-type rats. Perk +/− rats also showed lower expression of phosphor-AKT, ROCK1, ET1, and MMP2 protein in response to chronic hypoxia. Conclusions These findings suggest that Perk heterozygosity protects against HPH and Perk may be a suitable target for treating HPH.

Dexmedetomidine alleviates cardiomyocyte apoptosis and cardiac dysfunction may be associated with inhibition of RhoA/ROCK pathway in mice with myocardial infarction.

The global incidence of myocardial infarction has been consistently high, and it is one of the main causes of poor cardiovascular prognosis. Dexmedetomidine (DEX) is a highly selective α2 receptor agonist. Recent studies have found that DEX has a protective effect on myocardial infarction, but its specific mechanism is still unclear. In this experiment, we permanently ligated the anterior descending branch of mice to explore the protective mechanism of DEX against myocardial infarction. Our study found that intraperitoneal injection of DEX for 7 days after myocardial infarction in mice can increase the reduction of ejection fraction (EF) and fractional shortening (FS) caused by myocardial infarction and significantly reduce the release of serum markers. The results of myocardial HE and Sirius red staining suggest that the changes in the myocardial structure of mice after using DEX are reduced. Immunohistochemistry shows that DEX reduces the expression of ROCK1 protein after myocardial infarction. TUNEL staining and the protein expression levels of cleaved caspase-3 and cleaved caspase-9 were used to detect cell apoptosis and results make clear that DEX can reduce the apoptosis caused by myocardial infarction. Western blot experiments showed that DEX can reduce the expression levels of ROCK1 and ROCK2 (Rho-kinase). At the same time, it was observed that DEX improved the Bcl-2/Bax ratio. The above results indicate that DEX reduces cardiomyocyte apoptosis and improves cardiac function likely through inhibiting the RhoA/ROCK signaling pathway. This study may provide new insights into the protective effect of DEX after myocardial infarction in mice.

Hirudin prevents vascular endothelial cell apoptosis and permeability enhancement induced by the serum from rat with chronic renal failure through inhibiting RhoA/ROCK signaling pathway.

Endothelial cells injury and activation contribute to arteriovenous fistula (AVF) stenosis. Hirudin (Hiru) can inhibit the activity of thrombin, which was reported to enhance endothelial cell permeability and promote vascular inflammatory responses. RhoA/ROCK signaling pathway is also important in regulating vascular endothelial permeability. This study aimed to investigate the role of Hiru on the viability and permeability of human umbilical vein endothelial cells (HUVECs) following stimulation of serum from rat with chronic renal failure (CRF) and illustrated the effects of Hiru on RhoA/ROCK signaling. Wistar rats were randomly divided into control group and CRF group. Serum from each group was collected to stimulate HUVECs. Proliferation capability was estimated with Cell Count Kit-8 (CCK-8) assay. Transwell assay was performed to determine permeability. Cell apoptosis was examined using Tunel staining. Telomere length and telomerase activity were determined by qPCR. Moreover, the expression of RhoA, ROCK1 and ROCK2 was estimated via western blot. Results showed that the serum from CRF rat significantly inhibited cell viability while enhanced cell permeability and apoptosis. Different concentrations of Hiru prevented the above effects caused by CRF serum. Additionally, Hiru recovered the CRF serum-induced decreased telomere length and telomerase activity. Hiru also inhibited the protein expression of RhoA, ROCK1 and ROCK2, which were activated by CRF serum. Moreover, the ROCK inhibitor, Y27632, exhibited similar effects with Hiru. In conclusion, Hiru-restored HUVECs cell viability, telomere length and telomerase activity, suppressed permeability and apoptosis in the presence of CRF serum might depend on inactivating the RhoA/ROCK signaling.

Effects of Different Doses of Calcium on the Mitochondrial Apoptotic Pathway and Rho/ROCK Signaling Pathway in the Bone of Fluorosis Rats.

For this study, we investigate more deeply the effect calcium (Ca) develops on the mechanism underlying fluoride-triggered osteocyte apoptosis. We detected the morphology of osteocytes by HE staining, mitochondrial microstructure by using the transmission electron microscope, and the biochemical indexes related to bone metabolism and the expression of apoptosis-related genes. These results showed that NaF brought out the reduced osteocytes and ruptured mitochondrial outer membrane, with a significantly increased StrACP activity by 10.414IU/L at the 4th week (P < 0.05), markedly upregulating the mRNA expression of Bax, Cyto-C, Apaf-1, caspase-7, ROCK-1, BMP-2, and BGP (P < 0.01), as well as caspase-6 (P< 0.05), while downregulating Bcl-2 by 61.3% (P < 0.01). Through immunohistochemical analysis, we also found that NaF notably increased the protein expression of ROCK-1 (P < 0.05) and Cyto-C, BMP-2, and BGP (P < 0.01), suggesting that NaF triggered the activation of the mitochondrial apoptotic pathway and Rho/ROCK signaling pathway. Nevertheless, 1% Ca supplementation in diet notably enhanced the mRNA expression of Bcl-2 by 39.3% (P < 0.01), thus blocking the increment of the expression of mitochondrial apoptotic pathway-related genes and ROCK-1. Meanwhile, Ca could attenuate the StrACP activity by 10.741IU/L at the 4th week (P < 0.05) and protect the integrity of the mitochondrial outer membrane. These findings strongly suggest that 1% Ca abated the mitochondrial apoptosis pathway by increasing the anti-apoptotic gene Bcl-2 expression, and effectively inhibited the hyper-activation of ROCK-1, dually protecting the structural integrity of the mitochondrial outer membrane and maintaining normal cellular metabolic function.

Low androgen status inhibits erectile function by up-regulating the expression of P2X receptors in rat corpus cavernosum.

The aim of our study was to investigate whether low androgen level inhibits the erectile function of rats by regulating the expression of P2X receptors. Thirty-six 8-week-old male SD rats were randomly divided into six groups: sham-operated groups (4w-sham, 8w-sham), castration groups (4w-cast, 8w-cast) and androgen replacement after castration groups (4w-cast+T, 8w-cast+T). The maximum intracavernous pressure/mean arterial pressure (ICPmax/MAP), the levels of serum testosterone (T) and nitric oxide (NO), and the expression of P2X1, P2X2, P2X3, eNOS, p-eNOS, ROCK1 and ROCK2 in the cavernous tissue of rats were determined. The serum T, ICPmax/MAP and NO levels in penile corpus cavernosum in the castration groups were significantly lower than those in other groups (p<.01). The protein expression of P2X1, P2X2, P2X3, ROCK1 and ROCK2 in the castration groups was significantly higher than those in other groups (p<.01). P-eNOS/eNOS of the castration groups were significantly lower than those of other groups (p<.01). The serum T level was negatively correlated with the expression of P2X1, P2X2 and P2X3 in the corpus cavernosum. Low androgen level inhibits erectile function by up-regulating the expression of P2X1, P2X2, P2X3 and RhoA/Rho-kinase resulting in reducing the ratio of p-eNOS/eNOS and the level of NO in corpus cavernosum of rats.

P6289Neuregulin-1 improves cardiac function through protecting the loss of endothelial barrier in septic rats via RhoA/ROCK pathway

Abstract Background The main path of sepsis-induced cardiomyopathy is the loss of endothelial barrier function. Neuregulin-1 (NRG-1) has beneficial effects on endothelial function. Purpose To investigate the effect of NRG-1 treatment on the protection of cardiac endothelial cells and the changes of RhoA/ROCK signaling in sepsis. Methods Rats were randomly divided into three groups: sham, LPS, and NRG-1. After successful induction of sepsis by lipopolysaccharide (LPS, 10mg/kg), rats were administered either a vehicle or recombinant human neuregulin-1 (rhNRG-1, 10μg/kg/d) for one or two days. We recorded their survival rate at 48h after sepsis. Hemodynamic methods were performed to assess cardiac function. We used immunofluorescence assay to detect von Willebrand Factor (vWF). Intercellular adhesion molecule-1 (ICAM-1) and vascular endothelial growth factor (VEGF)levels in serum were measured by enzyme-linked immunosorbent assay (ELISA), and serum nitric oxide (NO) was detected by reductase method. We used transmission electron microscopy to observe changes in myocardial ultrastructure and western blot to assess expression of RhoA and ROCK1 protein. Results Sepsis impaired endothelial function manifested by increased ICAM-1, NO and VEGF levels in serum, NRG-1 treatment could significantly alleviate these increase (P&lt;0.05). Compared with the vehicle, NRG-1 significantly decreased cardiac vascular permeability through increasing the expression of vWF on endothelial lining (P&lt;0.05). Moreover, NRG-1 alleviated damages of ultrastructure of myocardial cells and suppressed the expression of RhoA and ROCK1 protein (P&lt;0.05). Ultimately, NRG-1 improved the survival,and prevented hemodynamic derangement. Levels of endothelial biomarker in serum ICAM-1 (pg/ml) VEGF (pg/ml) NO (pg/ml) Sham 24h 1564.74±94.41 0.84±0.28 203.27±1.81 Sham 48h 322.92±7.92 1.78±0.61 6.57±0.38 LPS 24h 5139.85±284.15† 3.11±0.04† 679.05±78.59† LPS 48h 950.41±25.23†‡ 4.65±0.30† 180.40±30.08†‡ NRG 24h 2772.18±164.45§ 0.30±0.04§ 355.14±23.41§ NRG 48h 578.57±28.97§‡ 1.02±0.45§ 104.67±2.13§‡ †p&lt;0.05 vs. the sham group, §p&lt;0.05 vs. the LPS group and ‡p&lt;0.05 vs. the 24h group. Effect of NRG-1 on endothelial cells Conclusions NRG-1 could alleviate endothelial injury in sepsis by strengthening the barrier function of vascular, reducing the secretion of endothelial-related biomarkers and inhibiting oxidative stress, thus improving cardiac function and survival rate, these effects may base on RhoA/ROCK signaling pathway. These may contribute to reverse the impaired endothelial cells in sepsis in the future. Acknowledgement/Funding The national natural science foundation of China (81772044)

Long noncoding RNA PANDAR promotes progression and predicts poor prognosis via upregulating ROCK1 in prostate cancer.

Recent researches have proved that long noncoding RNAs (lncRNAs) play an important role in tumorigenesis. In this research, lncRNA PANDAR was explored to identify the role it played in the development of prostate cancer and how it achieves. Real-time quantitative polymerase chain reaction (RT-qPCR) was utilized to detect PANDAR expression in both prostate cancer tissue samples and cells. Moreover, the associations between expression level of PANDAR and patients' disease-free survival rate were studied respectively. Then wound healing assay and transwell assay were conducted. Furthermore, RT-qPCR and Western blot assay were used to explore the underlying mechanism. By comparison with PANDAR expression in adjacent tissues, PANDAR expression level was significantly higher in prostate cancer samples, which was closely associated with patients' disease-free survival time. Moreover, after PANDAR was upregulated, cell migration and cell invasion capacities of prostate cancer cells were enhanced in vitro. In addition, after overexpression of PANDAR, the mRNA and protein expression of ROCK1 was upregulated, respectively. Furthermore, it was found that ROCK1 expression was positively correlated to PANDAR expression in prostate cancer tissues. Results above suggest that PANDAR could enhance cell migration and invasion of prostate cancer by upregulating ROCK1, which may offer a potential therapeutic target in prostate cancer.

MicroRNA-340-5p suppresses osteosarcoma development by down-regulating the Wnt/β-catenin signaling pathway via targeting the STAT3 gene.

To study the effects of miR-340-5p on the proliferation of osteosarcoma (OS) U2OS cells. miR-340-5p expression was measured by quantitative Real-time Polymerase Chain Reaction (qRT-PCR) in cancer tissues and paracancerous tissues from OS patients, and in OS cell line U2OS and normal osteoblast cell line hFOB1.19. The dual luciferase reporter vector was constructed to verify whether Signal transducers and activators of transcription 3 (STAT3) were a potential target gene of miR-340-5p. MiR-340-5p mimics/inhibitor was transfected into U2OS cells using liposomes, and transfection results were verified by qRT-PCR. Cell Counting Kit-8 (CCK-8) and Annexin V/PI were used to analyze the proliferation and apoptosis of transfected cells, respectively. Western Blot was used to detect STAT3 protein expression and Wnt/β-catenin pathway-related proteins after transfection. U2OS cells were injected into nude mice to observe the effects of over expression of miR-340-5p on tumor growth in vivo. miR-340-5p gene expression in OS tissues and U2OS cells was significantly lower than that in paracancerous tissues and hFOB1.19 cells. miR-340-5p directly interacted with the 3'-untranslated region (3'-UTR) of STAT3 gene and negatively regulated its expression. The increase of miR-340-5p expression could significantly inhibit U2OS proliferation in vitro and induce apoptosis, and vice versa. STAT3, β-catenin, c-Myc, TCF-4, CyclinD1, and ROCK1 protein expression in U2OS cells was significantly decreased after miR-340-5p over-expression, and vice versa. MiR-340-5p over-expression in nude mice significantly decreased tumor size and weight. Low expression of miR-340-5p in OS and U2OS cells could inhibit the course of OS by negatively regulating Wnt/β-catenin signaling pathway through targeting STAT3 gene. Therefore, miR-340-5p might be a potential biomarker and target for the diagnosis and treatment of OS.