Expression Of ROCK1 Research Articles

An Agonistic Monoclonal Antibody Targeting cMet Attenuates Inflammation and Up-Regulates Collagen Synthesis and Angiogenesis in Type 2 Diabetic Mice Wounds.

Diabetic wounds account for 25 to 50 percent of total diabetic health care costs annually, and present overall healing rates of less than 50 percent. Because delayed diabetic wound healing is associated with impaired fibroblast function, the authors hypothesize that tyrosine kinase Met (cMet) agonistic monoclonal antibody will promote diabetic wound healing by means of stable activation of hepatocyte growth factor/cMet signaling. Two 6-mm dorsal wounds were created in each mouse (6-week-old, male BKS.Cg-Dock7 m +/+Lepr db /J; n = 5). After subcutaneous injections of agonist (20 mg/kg) at 0 and 72 hours, the wound sizes were measured at days 0, 1, 3, 6, and 10. Histologic and immunohistochemical analyses were performed at day 10 (cMet, α-smooth muscle actin, CD68, and transforming growth factor-β). In vitro cytotoxicity and migration tests with diabetic fibroblasts were performed with or without agonist treatment (1 or 10 nM). cMet pathway activation of fibroblasts was confirmed through p-p44/42 mitogen-activated protein kinase, p-mTOR, p-cMet, and ROCK-1 expression. The cMet agonistic monoclonal antibody-treated group showed 1.60-fold lower wound area ( p = 0.027), 1.54-fold higher collagen synthesis ( p = 0.001), and 1.79-fold lower inflammatory cell infiltration ( p = 0.032) than the saline-treated control. The agonist increased cMet (1.86-fold; p = 0.029), α-smooth muscle actin (1.20-fold; p = 0.018), and vascular endothelial growth factor (1.68-fold, p = 0.029) expression but suppressed CD68 (1.25-fold; p = 0.043), transforming growth factor-β (1.25-fold; p = 0.022), and matrix metalloproteinase-2 (2.59-fold; p = 0.029) expression. In vitro agonist treatment (10 nM) of diabetic fibroblasts increased their migration by 8.98-fold ( p = 0.029) and activated the hepatocyte growth factor/cMet pathway. Tyrosine kinase Met agonistic monoclonal antibody treatment improved diabetic wound healing in mice and reduced wound-site inflammatory cell infiltration. These results need to be validated in large animals before piloting human trials. Although further clinical studies are necessary to evaluate its therapeutic efficacy, our study suggested that cMet agonistic monoclonal antibody can be the alternative modality in order to improve wound healing cascade in diabetic foot patients.

Ferulic acid alleviates AFB1-induced duodenal barrier damage in rats via up-regulating tight junction proteins, down-regulating ROCK, competing CYP450 enzyme and activating GST

Previous studies reported that Aflatoxin B1 (AFB1) causes cell damage through its metabolite aflatoxin B1–8, 9-epoxide (AFBO), which is catalyzed by CYP450 enzymes. AFBO can be detoxified by glutathione S transferase (GST). Ferulic acid (FA) is known for its antioxidant capacity and intestinal protective function. However, the mechanism of AFB1 causing duodenal injury and the role of FA in AFB1-induced intestinal damage remains unclear. In this study, rats were exposed to AFB1 and treated with FA for 30 days. The results showed that I) FA alleviated the histopathological changes of duodenum and the ultrastructural changes of tight junctions between duodenal epithelial cells induced by AFB1. II) FA reduced the content of AFB1-ALB adduct in blood. III) The low expression of tight junction proteins (Claudin-1 and ZO-1) and the high expression of ROCK1 and ROCK2 induced by AFB1 were significantly reversed by FA. IV) The high expression of CYP2A6 and CYP3A4 were significantly down-regulated by FA, and the activity of GST was promoted by FA. V) The binding affinity of FA to CYP2A6 is very similar to the binding affinity of AFB1 to CYP2A6, which meaning that there is a competitive relationship between FA and AFB1 when conjugating to CYP2A6. These results suggested that FA proved effective in alleviating AFB1-induced duodenal barrier damage via up-regulating tight junction proteins, down-regulating ROCK, competing CYP450 enzyme, and activating GST in duodenal epithelial cells of rats.

Effects of RhoA on depression-like behavior in prenatally stressed offspring rats

Depression is a common mental disease that can lead to suicide when severe. Exposure to prenatal stress (PS) can lead to depression-like behavior in offspring, but the mechanism is unclear. RhoA (Ras homology family member A) plays an important role in stress-induced changes in synaptic plasticity, participating in the development of depression by activating the downstream effector ROCK (Rho-associated protein kinase). This study explored the influence in the expression of RhoA and downstream molecules ROCK1/2 in prenatally stressed rats, and the effect of RhoA inhibitor simvastatin on depression-like behavior induced by PS. Depression-like behavior in offspring was detected by sucrose preference test, forced swimming test, and open-field test. The mRNA and protein expression of RhoA and ROCK1/2 in the hippocampus and prefrontal cortex of offspring rats were detected by qRT-PCR and western blotting, respectively. Our results showed that PS causes depression-like behavior in offspring rats, associated with elevated expression of RhoA, ROCK1/2 in the hippocampus and prefrontal cortex. After administration of simvastatin to PS rats, the expression of RhoA and ROCK2 was significantly reduced, alleviating depression-like behavior. Our study demonstrated that RhoA participates in the depression-like behavior in prenatally stressed offspring rats, which may be a potential target for antidepressant therapy.

RHO KINASE BLOCKADE ALLEVIATE BLOOD PRESSURE ELEVATION AND INHIBIT VASCULAR REMODELING IN APATINIB-INDUCED HYPERTENSION TO BE CONFERMED

Objective: Hypertension is one of the major adverse effects of tyrosine kinase inhibitors (TKIs) targeting vascular endothelial growth factors. However, the mechanism underlying TKIs-induced hypertension remains unclear. Here, we evaluated BP responses at different doses of apatinib, a TKI used for anti-tumor therapy, in normotensive Wistar–Kyoto rats. Furthermore, we explored the role of the RhoA/Rho kinase (ROCK) emodellin pathway in elevation of blood pressure (BP) induced by apatinib, a selective TKI approved in China for treatment of advanced or metastatic gastric cancer. A non-specific ROCK inhibitor, Y27632, was then combined with apatinib and its efficacy in alleviating apatinib-induced hypertension was evaluated. Design and method: Normotensive female Wistar–Kyoto rats were exposed to two different doses of apatinib, or apatinib combined with Y27632, or vehicle for two weeks. BP was monitored by a tail-cuff plethysmography system. The mRNA levels and protein expression in the RhoA/ROCK pathway were determined, and vascular emodelling assessed. Results: Administration of either a high or low dose of apatinib was associated with a rapid rise in BP, reaching a plateau after 12 days. Apatinib treatment mediated upregulation of RhoA and ROCK 2 in the mid-aorta, more significant in the high dose group. However, ROCK1 expression showed no statistically significant differences. Furthermore, the mRNA level of GRAF3 decreased dose-dependently. Apatinib administration was also associated with decreased levels of MLCP, and elevated endothelin-1 (ET-1) and collagen 1, which was accompanied with increased mid-aortic media. However, treatment with Y27632 attenuated the above changes. Conclusions: Results of the present study indicated that apatinib treatment increased BP and promoted vascular emodelling by activating the RhoA/ROCK emodellin pathway. However, Y27632, a non-specific ROCK inhibitor, reversed apatinib-induced increase in BP and vascular emodelling. Therefore, RhoA/ROCK activation could be the underlying mechanism of apatinib-induced hypertension, while ROCK inhibitors could be potential therapeutic drugs.

M2 macrophage facilitated angiogenesis in cutaneous squamous cell carcinoma via circ_TNFRSF21/miR-3619-5p/ROCK axis.

In recent years, the role of circular RNA in cancer cells has been studied broadly; however, the functional significance of circular RNA in the regulation of the tumor microenvironment (TME) is not fully understood. In this study, we aimed to reveal the role of circ_TNFRSF21 in M2 macrophage-induced cutaneous squamous cell carcinoma (cSCC) angiogenesis. Quantitative polymerase chain reaction and Western blotting were performed to determine the levels of the indicated genes. Direct binding between circ_TNFRSF21 and miR-3619-5p, miR-3619-5p, and ROCK2 was verified by dual-luciferase activity. The migration and invasion of human umbilical vein endothelial cells were evaluated by wound healing and transwell assays. Tube formation was performed to detect in vitro angiogenesis. Circ_TNFRSF21 and ROCK2 were upregulated in cSCC tissue, while miR-3619-5p was downregulated. Circ_TNFRSF21 negatively regulated the expression of miR-3619-5p, while miR-3619-5p negatively regulated the expression of ROCK2. miR-3619-5p suppressed tube formation by inhibiting ROCK signaling. M2 macrophages facilitated tube formation via the circ_TNFRSF21/miR-3619-5p/ROCK2 axis. Our present study revealed that circ_TNFRSF21 was elevated in M2 macrophages and mediated M2 macrophage-induced tube formation in vitro.

LncRNA HOXA11-AS promotes vascular endothelial cell injury in atherosclerosis by regulating the miR-515-5p/ROCK1 axis.

AimsLong non‐coding RNA HOXA11‐AS participated in heart disease. In this study, we aim to evaluate the potential roles of HOXA11‐AS in atherosclerosis and its underlying mechanisms.Methods and resultsThe expression levels of HOXA11‐AS in ox‐LDL‐treated HUVECs and arch tissues of high‐fat diet‐fed ApoE−/− mice (n = 10) were assessed by qRT‐PCR. The effects of HOXA11‐AS knockdown on the development of atherosclerosis were evaluated using in vitro and in vivo models. Luciferase reporter and RNA immunoprecipitation (RIP) assays verified the potential relationships between HOXA11‐AS or ROCK1 and miR‐515‐5p. The interactive roles between HOXA11‐AS and miR‐515‐5p and between miR‐515‐5p and ROCK1 were further characterized in ox‐LDL‐treated HUVECs. Our data showed that HOXA11‐AS was significantly up‐regulated (P < 0.001), whereas miR‐515‐5p was dramatically down‐regulated in AS mice tissues (P < 0.001) and ox‐LDL‐treated HUVECs (P < 0.01). Ox‐LDL could induce endothelial injuries by inhibiting cell proliferation (P < 0.001) and SOD synthesis (P < 0.001), promoting apoptosis (P < 0.01), ROS (P < 0.001), and MDA production (P < 0.001), increasing Bax (P < 0.001) and cleaved Caspase‐3 (P < 0.001), and decreasing Bcl‐2 (P < 0.001) and phosphorylated eNOS (P < 0.01). HOXA11‐AS knockdown attenuated endothelial injuries via increasing eNOS phosphorylation. Luciferase assay and RIP results confirmed that miR‐515‐5p is directly bound to HOXA11‐AS and ROCK1. HOXA11‐AS promoted ox‐LDL‐induced HUVECs injury by directly inhibiting miR‐515‐5p from increasing ROCK1 expression and subsequently decreasing the expression and phosphorylation of eNOS. MiR‐515‐5p mimics could partially reverse the effects of HOXA11‐AS knockdown.ConclusionsHOXA11‐AS contributed to atherosclerotic injuries by directly regulating the miR‐515‐5p/ROCK1 axis. This study provided new evidence that HOXA11‐AS might be a candidate for atherosclerosis therapy.

MiR-665 Participates in the Protective Effect of Dexmedetomidine in Ischemic Stroke by ROCK2/NF-κB Axis.

Ischemic stroke is a grievous intimidation to the healthiness of sufferers. Previous studies have reported that dexmedetomidine (DEX) has a protective effect on a variety of organs. This paper aimed to explore the regulatory mechanism of DEX in ischemic stroke through miR-665/ROCK2 axis. The mice model of ischemic stroke was constructed by middle cerebral artery occlusion (MCAO). The cell model of ischemic stroke was constructed by oxygen-glucose deprivation (OGD). Cell viability and apoptosis were assessed by CCK-8 assay and flow cytometry. The expression of cytokines was detected by ELISA. Lactate dehydrogenase (LDH) concentration was evaluated by LDH kit. The cerebral infarct volume of MCAO mice was detected by TTC staining, and the apoptosis of brain cells was detected by TUNEL staining. The target relationship between ROCK2 and miR-665 was analyzed by dual-luciferase reporter assay. DEX contributed cell viability from 42 to 66% (1μM) and restrained cell apoptosis from 26 to 18% in HT22 cells treated with OGD (P < 0.01). Meanwhile, DEX decreased the expression of cytokines and LDH concentration from 184 to 126% (P < 0.001). Moreover, the expression of miR-665 enhanced 2.9 times (P < 0.05) and the expression of ROCK2 (P < 0.05) and NF-κB p65 (P < 0.01) reduced 1.8 times and 2.2 times after DEX treatment in OGD induced HT22. And miR-665 knockdown attenuated the effect of DEX on inflammation damage (the levels of TNF-α, IL-1β and IL-6 increased 1.36 times, 1.31 times, 1.43 time, respectively, and IL-10 decreased 1.68 times) and apoptosis from 17 to 25% (P < 0.01). MiR-665 directly targeted ROCK2 and regulated ROCK2 and NF-κB p65 expression (P < 0.01). Furthermore, ROCK2 overexpression inhibited the protective effect of DEX in HT22 induced by OGD (P < 0.001), while miR-665 overexpression reversed the regulatory of ROCK2 (P < 0.01). In vivo, DEX decreased cerebral infarction volume and inhibited apoptosis of brain cell (P < 0.001). DEX has a protective effect in ischemic stroke by promoting miR-665 expression to downregulate ROCK2/NF-κB axis, suggesting DEX has a beneficial effect on ischemic stroke and miR-665 is a conceivable target for the therapeutics and diagnosis of ischemic stroke.

S1PR2/RhoA/ROCK1 pathway promotes inflammatory bowel disease by inducing intestinal vascular endothelial barrier damage and M1 macrophage polarization

Vascular and immune dysfunctions are thought to be related to the pathogenesis of inflammatory bowel disease (IBD), but behind this, the exact mechanism of mucosal vascular endothelial barrier dysfunction and macrophage phenotypic transition is not fully understood. Here, we explored the mechanistic role of sphingosine 1-phosphate receptor 2 (S1PR2) and its downstream G protein RhoA/Rho kinase 1 (ROCK1) signaling pathway in the intestinal endothelial barrier damage and M1 macrophage polarization in IBD. We found that the expression of S1PR2 in intestinal mucosal vascular endothelial cells and macrophages of IBD patients and DSS-induced colitis mice as well as vascular endothelial cells and macrophages treated with LPS in vitro was significantly increased. Knocking down or pharmacologically inhibiting S1PR2 significantly downregulated the expression of RhoA and ROCK1 in vascular endothelial cells and macrophages. Furthermore, inhibition of S1PR2 and ROCK1 reversed the impaired vascular barrier function and M1 macrophage polarization in vivo and in vitro, while reducing ER stress in vascular endothelial cells and glycolysis in macrophages. In addition, inhibition of ER stress or glycolysis reversed LPS-induced impairment of vascular endothelial cell barrier function and M1 macrophage polarization. Collectively, our results indicate that the S1PR2/RhoA/ROCK1 signaling pathway may participate in the pathogenesis of IBD by regulating vascular endothelial barrier function and M1 macrophage polarization.

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.

Neutrophil-derived extracellular vesicles induce endothelial inflammation and damage through the transfer of miRNAs

The critical role of neutrophils in pathological inflammation, notably in various autoimmune disorders, is currently the focus of renewed interest. Here, we demonstrate for the first time that activation of neutrophils with various inflammatory stimuli induces the release of extracellular vesicles (EVs) that are internalized by endothelial cells (ECs), thus leading to the transfer of miR-223, miR-142–3p and miR-451 and subsequent endothelial damage. Indeed, while miR-223 has little effect on EC responses, we show that the induced expression of miR-142–3p and miR-451 in ECs results in profound cell damage, especially in inflammatory conditions, characterized by a dramatic increase in cell apoptosis, impaired angiogenic repair responses, and the induction of IL-6, IL-8, CXCL10 and CXCL11 expression. We show that the strong deleterious effect of miR-142–3p may be due in part to its ability to block the activation of ERK1/2 and eNOS-mediated signals in ECs. miR-142–3p also inhibits the expression of RAC1, ROCK2 and CLIC4, three genes that are critical for EC migration and angiogenic responses. Importantly, miR-223, miR-142–3p and miR-451 are markedly increased in kidney biopsies from patients with active ANCA-associated vasculitis, a severe autoimmune disease that is prototypical of a neutrophil-induced microvascular damage. Taken together, our results suggest that miR-142–3p and miR-451 released in EVs by activated neutrophils can target EC to trigger an inflammatory cascade and induce direct vascular damage, and that therapeutic strategies based on the inhibition of these miRNAs in ECs will have implications for neutrophil-mediated inflammatory diseases.

Ex Vivo Hydrostatic Pressure Loading of Atrial Tissues Activates Profibrotic Transcription via TGF-β Signal Pathway.

Excessive mechanical stress causes fibrosis-related atrial arrhythmia. Herein, we tried to investigate the mechanism of atrial fibrogenesis in response to mechanical stress by ex vivo approach. We collected atrial tissues from mice and then cultured them as "explants" under atmospheric pressure (AP group) or 50 mmHg hydrostatic pressure loading (HP group) conditions. Pathway-specific PCR array analysis on the expression of fibrosis-related genes indicated that the loading of atrial tissues to 50 mmHg for 24 hours extensively upregulated a series of profibrotic genes. qRT-PCR data also showed that loading atrial tissues to 50 mmHg enhanced Rhoa, Rock2, and Thbs1 expression at different time points. Interestingly, the enhanced expression of Thbs1 at 1 hour declined at 6-24 hours and then increased again at 72 hours. In contrast, an enhanced expression of Tgfb1 was observed at 72 hours. In contrast, daily loading to 50 mmHg for 3 hours significantly accelerated the outgrowth of mesenchymal stem-like stromal cells from atrial tissues; however, we did not observe significant phenotypic changes in these outgrowing cells. Our ex vivo experimental data clearly show the induction of profibrotic transcription of atrial tissues by HP loading, which confirms the common pathological feature of atrial fibrosis following pressure overload.

Dual efficacy of Fasudil at improvement of survival and reinnervation of flap through RhoA/ROCK/PI3K/Akt pathway.

Fasudil is reported to be effective at protecting against ischaemic diseases, and at augmenting axon growth. In this study, we aim to evaluate its efficacy in promoting flap survival and reinnervation. Ninety-two Institute of Cancer Research (ICR) mice were used and divided into the control, Fasudil, LY294002, Fasudil+LY294002 groups, receiving a daily intraperitoneal injection of normal saline, Fasudil (10mg/kg), LY294002 (5mg/kg), and Fasudil (10mg/kg) + LY294002 (5mg/kg), respectively. On days 0 and 5, the blood perfusion and diameter of the iliolumbar artery in the pedicle of the flaps in the four groups were evaluated using laser speckling contrast imaging (LSCI). On day 5, the flaps were photographed and the necrosis rate of the flaps was calculated using Photoshop CS6. In addition, tissues were harvested from the flaps and divided into two parts. One part underwent routine cryosection and immunofluorescent staining using the antibody against CD31 for evaluation of the microvascular density in the four groups. In the other part, the expression of RhoA, ROCK1+2, p-CPI-17, p-MYPT, p-PTEN, p-PI3K, p-Akt, and vascular endothelial growth factor (VEGF) within the flaps were determined using western blotting. Moreover, at days 0, 7, 15, and 30 after flap surgery, the axons within the flaps were evaluated using immunofluorescent staining with the antibody against Neurofilament-200. It turned out that the necrosis rate was (24.4 ± 7.7)%, (5.2 ± 1.6)%, (29.8 ± 4.2)%, and (30.9 ± 7.1)%, respectively, in the control, Fasudil, LY294002, LY294002+Fasudil groups. There was a significant reduction in the necrosis rate of the flaps in the Fasudil group (P < .001). The LSCI and immunofluorescent staining demonstrated that Fasudil could significantly expand the diameter of the iliolumbar artery in the pedicle, boost the overall blood perfusion, and increase the microvascular density of the flaps in the Fasudil group (P < .05), which could all be abolished by PI3K inhibitor LY294002. On day 5, the expression of p-CPI-17, p-MYPT, and p-PTEN were downregulated, whereas pPI3K, p-Akt, and VEGF were upregulated in the Fasudil group (P < .001). As for reinnervation, Neurofilament-200 fluorescent staining revealed that at days 15 and 30 after flap harvest, only in the Fasudil group could new axons be observed. It can be concluded that Fasudil could simultaneously improve the survival and axon growth after flap harvest, a dual efficacy achieved by inhibition of the RhoA/ROCK pathway, which in turn activates /PI3K/AKT pathway.

Moxibustion pretreatment inhibits RhoA/ROCK signaling to prevent lung inflammation in asthmatic rats: 艾灸预处理降低RhoA/ROCK信号表达预防哮喘大鼠肺炎

Objective: To determine whether pretreatment moxibustion prevents asthma by down-regulating the lung RhoA/ROCK pathway in rats with bronchial asthma and benignly mediating the lung inflammatory response. Methods: Twenty Sprague Dawley (SD) rats were randomly divided into normal control group (C), asthma model group (M), suspended moxibustion 40 min +asthma group (SM40), and suspended moxibustion 10 min +asthma group (SM10). Ovalbumin was used as a sensitizer. The two moxibustion groups completed moxibustion treatment lasted 40 min or 10 min respectively 30 min before modeling onset, and was repeated five times in each modeling cycle, for a total of 15 times. Samples were harvested on day 30. Results: Lung impairment was significant in the M group, whereas pretreatment with SM10 and SM40 dramatically attenuated the injury. After modeling, mRNA expression of RhoA and ROCK2 in the lung tissue was significantly higher than that in C group (both P < 0.001), resulting in significant increase in protein levels of IL-17A (P < 0.001). Significant decrease in RhoA and ROCK2 mRNA expression was seen in the SM10 (P<0.001, P<0.01) and SM40 (both P<0.001) groups compared to that with M rats. The differential trend in the SM40 group was more evident than that in the SM10 group. Regarding IL-10 or IL-17A protein concentration, an upregulation or down-regulation was observed in both SM10 (P<0.05, P < 0.01) and SM40 groups (both P < 0.001) compared to that with the M group. Conclusions: Moxibustion pretreatment significantly prevented pulmonary inflammation in asthmatic rats, potentially via inhibition of the RhoA/ROCK pathway. The efficacty of moxibustion appeared to be significantly associated with the duration of intervention with moxibustion.

Protective effects of tadalafil against cisplatin-induced spermatogenic dysfunction

BackgroundCisplatin (CDDP) is an effective anticancer drug for the treatment of malignant tumors, such as lung cancer, bladder cancer, and testicular cancer. However, oligozoospermia and azoospermia after administration of CDDP are clinical problems. One of the testicular toxicities of CDDP is known to cause oxidative stress. Tadalafil has been reported to exhibit antioxidant effects and is widely used in clinical practice to treat benign prostatic hyperplasia and erectile dysfunction. Rho-kinase α (ROCK2) regulates cell migration and apoptosis and has been reported to be involved in CDDP-induced nephrotoxicity. The excessive expression of ROCK2 is known to cause oxidative stress. ObjectiveThe objective of the current study was to test the effect of tadalafil on the testicular toxicity of CDDP. Material and methodsThirty-two rats were used and divided into the following four groups. (1) The control group (CONT), treated with saline on day 1 and saline and dimethyl sulfoxide (DMSO) on days 1–10 intraperitoneally (i.p.) (2) The Tadalafil Group (TAD), treated with saline on day 1, and 0.4 mg/kg tadalafil on days 1–10 i.p. (3) The CDDP group (CD), treated with 7 mg/kg CDDP, saline, and DMSO on days 1–10 i.p. and (4) The CDDP + TAD group (CDT) was treated with 7 mg/kg CDDP on day 1, and 0.4 mg/kg tadalafil on days 1–10 i.p. Testes and epididymides samples were collected on day 11. Biochemical and pathological analyses and quantitative polymerase chain reaction were performed on the excised specimens. ResultsCDDP treatment resulted in testicular atrophy, decreased sperm concentration, and atrophy of seminiferous tubules as observed from the testicular histology. Increased apoptosis of seminiferous tubules, oxidative stress, and ROCK2 mRNA expression were observed after CDDP treatment. Treatment with tadalafil improved these adverse effects. ConclusionTadalafil is a potential drug for reducing CDDP-induced spermatogenic dysfunction. The antioxidant effect of tadalafil may be partly responsible for this phenomenon. ROCK2 and oxidative stress markers may be involved in the possible antioxidant effects of tadalafil. Tadalafil may be considered as one of a treatment option for reducing spermatogenic dysfunction after administration of CDDP.

MiRNA-448 Regulates the Development of Glioblastoma (GBM) by Regulating Rho-Associated Protein Kinase 1.

Background Glioblastoma (GBM) is an aggressive adult brain tumor that poses a huge threat to people's health. Previous studies have shown that microRNAs (miRNAs) are important regulators in the progression of GBM. However, the role of miR-448 in GBM remains largely unknown. Therefore, the regulatory mechanism of miR-448 in the development of GBM is elucidated in this study. Methods The protein and mRNA expressions of miR-448 and ROCK1 were measured by Western blot analysis and RT-qPCR. Cell proliferation, migration, and invasion were detected by CCK-8 assay and Transwell assay. The relationship between miR-448 and ROCK1 was probed by luciferase reporter assay. Results miR-448 expression was downregulated in GBM tissues and cells. And poor clinical outcomes of GBM patients were related to miR-448 downregulation. Functionally, overexpression of miR-448 restrained cell viability, migration, and invasion in GBM. Additionally, miR-448 reduced ROCK1 expression by binding to its 3′-UTR. Moreover, knockdown of ROCK1 inhibited the progression of GBM. Furthermore, overexpression of ROCK1 abolished the antitumor effect of miR-448 in GBM. Conclusion miR-448 restrained cell viability, invasion, and migration in GBM by inhibiting ROCK1 expression.

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.

Human adipose mesenchymal stem cells modulate inflammation and angiogenesis through exosomes

Stem cell-derived exosomes are efficient and safe therapeutic tools for transferring endogenous biological cargo or functional biomolecules for regenerative medicine. The regulation of inflammation and angiogenesis plays a pivotal role in wound healing and tissue regeneration. The purpose of this study was to investigate the anti-inflammatory and pro-angiogenic roles of human adipose mesenchymal stem cell-derived exosomes, focusing on the underlying mechanisms. Exosomes inhibited LPS-induced inflammation by activating ROCK1 and PTEN expression. Moreover, microRNAs (miR-132 and miR-146a) released from exosomes upregulated the expression of pro-angiogenic genes and promoted proliferation activity and tube formation in human umbilical vein endothelial cells. Exosomal effects were verified using ROCK1/PTEN inhibitors for anti-inflammation and miR-132/miR-146a inhibitors for pro-angiogenesis. Our findings suggest that exosomes exert anti-inflammatory effects by targeting the ROCK1/PTEN pathway and exhibit pro-angiogenic effects via delivery of miR-132 and miR-146a. Taken together, these results suggest that exosomes may be promising therapeutic candidates for curing diseases involved in inflammation and angiogenesis.

ROCK2 Polymorphism and Expression Contribute to Increased Susceptibility and Poor Prognosis in Hepatocellular Carcinoma.

AimTo analyze the association between ROCK2 gene polymorphism/expression and susceptibility/prognosis of hepatocellular carcinoma (HCC).MethodsPeripheral venous blood from 204 hepatocellular carcinoma patients (HCC group), 242 chronic hepatitis B patients (CHB group), 215 liver cirrhosis patients (LC group) and 201 healthy volunteers (normal group) were collected. ROCK2 gene (including the rs9808232 locus) was amplified by PCR and the products were sequenced. Tumor tissues from patients with HCC and liver tissues from CHB, LC and normal groups were collected, and mRNA and protein expression of ROCK2 gene in liver tissues were tested by RT-qPCR and immunofluorescence, respectively.ResultsCompared with the normal group, the mRNA and protein expression levels of ROCK2 in the HCC group increased significantly (P < 0.05), but there are no obvious increases in the CHB and LC groups (P > 0.05). ROCK2 gene rs9808232 polymorphism was associated with an increased susceptibility of HCC, and genotypes AC and CC may be risk factors for HCC (P < 0.001). There was a significant association between the ROCK2 gene rs9808232 polymorphism and the route of metastasis in HCC patients (P < 0.05). According to the Kaplan–Meier analysis, the overall survival of patients with AC and CC genotypes of ROCK2 rs9808232 was much shorter than that of patients with AA genotype (median overall survival, 25.5 months vs 16.0 months; Log rank P = 0.006). The overall survival time of patients with high ROCK2 expression is much shorter than that of patients with low ROCK2 expression (median overall survival time, 15.0 months vs 20.5 months; Log rank P = 0.008). Compared with genotype AA, the ROCK2 protein levels of genotypes AC and CC were distinctly increased in the HCC group, especially genotype CC (P< 0.05).ConclusionROCK2 gene rs9808232 polymorphism may contribute to its expression in liver tissue, which may increase the susceptibility and poor prognosis of HCC.

CTNNAL1 participates in the regulation of mucus overproduction in HDM-induced asthma mouse model throughthe YAP-ROCK2 pathway.

Our previous study indicated that adhesion molecule catenin alpha‐like 1(CTNNAL1) is downregulated in airway epithelial cells of asthma patients and asthma animal model but little is known about how the CTNNAL1 affects asthma pathogenesis. To reveal the direct relationship between asthma and CTNNAL1, CTNNAL1‐deficient mouse model in bronchopulmonary tissue was constructed by introducing CTNNAL1‐siRNA sequence using adeno‐associated virus (AAV) as vector. The mouse model of asthma was established by stimulation of house dust mite (HDM). After HDM‐challenged, there was marked airway inflammation, especially mucus hypersecretion in the CTNNAL1‐deficient mice. In addition, the CTNNAL1‐deficient mice exhibited an increase of lung IL‐4 and IL‐13 levels, as well as a significant increase of goblet cell hyperplasia and MUC5AC after HDM exposure. The expression of Yes‐associated protein (YAP), protein that interacted with α‐catenin, was downregulated after CTNNAL1 silencing and was upregulated due to its overexpression. In addition, the interaction between CTNNAL1 and YAP was confirmed by CO‐IP. Besides, inhibition of YAP could decrease the secretion of MUC5AC, IL‐4 and IL‐13 in CTNNAL1‐deficient 16HBE14o‐cells. Above results indicated us that CTNNAL1 regulated mucus hypersecretion through YAP pathway. In addition, the expression of ROCK2 increased when CTNNAL1 was silenced and decreased after YAP silencing, and inhibition of YAP decreased the expression of ROCK2 in CTNNAL1‐deficient HBE cells. Inhibition of ROCK2 decreased MUC5AC expression and IL‐13 secretion. In all, our study demonstrates that CTNNAL1 plays an important role in HDM‐induced asthma, mediating mucus secretion through the YAP‐ROCK2 pathway.

LncRNA ZFAS1 regulates the proliferation, oxidative stress, fibrosis, and inflammation of high glucose-induced human mesangial cells via the miR-588/ROCK1 axis

BackgroundDiabetic nephropathy (DN) is a critical and the most common microvascular complication and its pathogenesis is still faintly understood. Thus, this study was performed to examine the long non-coding RNA ZNFX1 Antisense Gene Protein 1 (lncRNA ZFAS1) biological function and mechanism of regulation in DN.MethodHuman glomerular mesangial cells (HGMC) were induced with high glucose (HG, 25 mM) to establish HG-induced cell viability, pro-inflammation observed in DN. After, target miRNA and mRNA were predicted through Lncbase and Targetscan. Subsequently, the expression of ZFAS1, miR-588, and ROCK1 in DN clinical samples and cell-model was examined through qRT-PCR and western blot analysis. We upheld the targeted interaction between miR-588 and ZFAS1 or ROCK1 through a dual-luciferase reporter assay. The proliferation of the cell was also examined through CCK-8 assay, while the level of HG-induced oxidative stress was established by measuring reactive oxygen species (ROS) level, and also the activities of antioxidant enzymes in the cell. Lastly, the level of accumulated extracellular matrix (ECM) protein-fibronectin and collagen type IV, and inflammatory cytokines produced by the cell was analyzed through western blot analysis and ELISA.ResultsZFAS1 was significantly upregulated in the DN blood samples and HG-induced HGMC. Prediction result revealed that the ZFAS1 endogenously targets the miR-588 seed sequence while miR-588 plays a role in post-transcriptional regulation of ROCK1 mRNA. Moreover, we found that miR-588 expression was significantly downregulated in DN blood samples and negatively correlates with ZFAS1 expression. Further results show that silencing ZFAS1 had a protective effect on HG-induced proliferation, oxidative stress, fibrosis, and inflammation in HGMC while miR-588 inhibition and ROCK1 overexpression reversed this effect.ConclusionsAltogether, our data suggest that ZFAS1 regulates the proliferation, oxidative stress, fibrosis, and inflammation of high glucose-induced diabetic nephropathy through the miR-588/ROCK1 axis.