Expression Of ROCK1 Research Articles

Inhibition of ROCK2 alleviates renal fibrosis and the metabolic disorders in the proximal tubular epithelial cells.

Non-specific inhibition of Rho-associated kinases (ROCKs) alleviated renal fibrosis in the unilateral ureteral obstruction (UUO) model, while genetic deletion of ROCK1 did not affect renal pathology in mice. Thus, whether ROCK2 plays a role in renal tubulointerstitial fibrosis needs to be clarified. In the present study, a selective inhibitor against ROCK2 or genetic approach was used to investigate the role of ROCK2 in renal tubulointerstitial fibrosis. In the fibrotic kidneys of chronic kidney diseases (CKDs) patients, we observed an enhanced expression of ROCK2 with a positive correlation with interstitial fibrosis. In mice, the ROCK2 protein level was time-dependently increased in the UUO model. By treating CKD animals with KD025 at the dosage of 50 mg/kg/day via intraperitoneal injection, the renal fibrosis shown by Masson's trichrome staining was significantly alleviated along with the reduced expression of fibrotic genes. In vitro, inhibiting ROCK2 by KD025 or ROCK2 knockdown/knockout significantly blunted the pro-fibrotic response in transforming growth factor-β1 (TGF-β1)-stimulated mouse renal proximal tubular epithelial cells (mPTCs). Moreover, impaired cellular metabolism was reported as a crucial pathogenic factor in CKD. By metabolomics analysis, we found that KD025 restored the metabolic disturbance, including the impaired glutathione metabolism in TGF-β1-stimulated tubular epithelial cells. Consistently, KD025 increased antioxidative stress enzymes and nuclear erythroid 2-related factor 2 (Nrf2) in fibrotic models. In addition, KD025 decreased the infiltration of macrophages and inflammatory response in fibrotic kidneys and blunted the activation of macrophages in vitro. In conclusion, inhibition of ROCK2 may serve as a potential novel therapy for renal tubulointerstitial fibrosis in CKD.

Whole-blood dysregulation of actin-cytoskeleton pathway in adult spinal muscular atrophy patients.

ObjectiveRecent advances in therapeutics have improved prognosis for severely affected spinal muscular atrophy (SMA) type 1 and 2 patients, while the best method of treatment for SMA type 3 patients with later onset of disease is unknown. To better characterize the SMA type 3 population and provide potential therapeutic targets, we aimed to understand gene expression differences in whole blood of SMA type 3 patients (n = 31) and age‐ and gender‐matched controls (n = 34).MethodsWe performed the first large‐scale whole blood transcriptomic screen with L1000, a rapid, high‐throughput gene expression profiling technology that uses 978 landmark genes to capture a representation of the transcriptome and predict expression of 9196 additional genes.ResultsThe primary downregulated KEGG pathway in adult SMA type 3 patients was "Regulation of Actin Cytoskeleton,” and downregulated expression of key genes in this pathway, including ROCK1, RHOA, and ACTB, was confirmed in the same whole blood samples using RT‐qPCR. SMA type 3 patient‐derived fibroblasts had lower expression of these genes compared to control fibroblasts from unaffected first‐degree relatives. Overexpression of SMN levels using an AAV vector in fibroblasts did not normalize ROCK1, RHOA, and ACTB mRNA expression, indicating the involvement of additional genes in cytoskeleton dynamic regulation.InterpretationOur findings from whole blood and patient‐derived fibroblasts suggest SMA type 3 patients have decreased expression of actin cytoskeleton regulators. These observations provide new insights and potential therapeutic targets for SMA patients with longstanding denervation and secondary musculoskeletal pathophysiology.

LncRNA MAGI2-AS3 is downregulated in the distant recurrence of hepatocellular carcinoma after surgical resection and affects migration and invasion via ROCK2

Introduction and objectivesHepatocellular carcinoma (HCC) is the second most lethal cancer around the world, with poor survival rate and high metastasis rate in patients. Long noncoding RNAs (lncRNAs) have been reported to modulate the initiation and development of liver cancer. We aimed to investigate the role of lncRNA MAGI2-AS3 in HCC and underlying mechanisms. Materials and methodsThe expression levels of MAGI2-AS3 in plasma of HCC patients and the control participants were measured by qPCR. Hep3B and MHCC97-H cells were transfected with MAGI2-AS3 and ROCK2 expression vectors. Cell migration and invasion of HCC cells transfected with the vectors were investigated by transwell assay. In addition, flow cytometry and western blot were performed for apoptosis detection. ResultsWe found that MAGI2-AS3 was downregulated in plasma of early stage HCC patients compared to healthy controls. After surgical resection, the expression levels of MAGI2-AS3 were increased compared to pretreatment levels on the day of discharge. During the follow-up, MAGI2-AS3 was downregulated in patients developed distant recurrence, but not in other patients compared to the levels measured on the day of discharge. In HCC cells, overexpression of MAGI2-AS3 mediated the downregulation of ROCK2. Cell invasion and migration assay showed that overexpression of MAGI2-AS3 mediated the decreased cell invasion and migration rate, while ROCK2 played an opposite role and attenuated the effects of overexpression of MAGI2-AS3. ConclusionOur study indicated that MAGI2-AS3 was downregulated in the distant recurrence of HCC after surgical resection and affected the invasion and migration of HCC cells via ROCK2.

Epimedium flavonoids improve cognitive impairment and white matter lesions induced by chronic cerebral hypoperfusion through inhibiting the Lingo-1/Fyn/ROCK pathway and activating the BDNF/NRG1/PI3K pathway in rats

Chronic cerebral hypoperfusion is a common cause of cerebral small vascular disease (CSVD). White matter (WM) lesions are the typical pathological manifestation of CSVD and contribute to cognitive decline. Epimedium flavonoids (EF) are the main component in Epimedium brevicornu Maxim., which is commonly used in traditional Chinese medicine. The purpose of this study was to investigate the effects of EF on cognitive impairment and the underlying mechanisms in a CSVD rat model induced with chronic cerebral hypoperfusion. The model was established by permanent bilateral common carotid artery occlusion (2VO) in rats. EF (50, 100, and 200 mg/kg) was intragastrically administered once a day for 12 weeks starting 2 weeks after 2VO surgery. The learning and memory capacity of the rats were measured using the Morris water maze and step-through tests. WM lesions were observed by MRI-diffusion tensor imaging, transmission electron microscopy, and LFB staining. Oligodendrocytes were detected by immunohistochemistry. Western blotting assay was used to determine the level of protein expression. The results showed that EF significantly improved learning and memory impairment, alleviated WM nerve fiber injuries and demyelination, and increased the number of mature oligodendrocytes in the corpus callosum, subcortical WM, and periventricular WM in 2VO rats. Mechanistically, EF reduced the expression of Lingo-1 and ROCK2 and increased the levels of phosphorylated (p-) Fyn, brain-derived neurotrophic factor (BDNF), TrkB, neuregulin-1 (NRG-1), p-ErbB4, PI3K p85 and p110α, p-Akt, and p-CREB in the corpus callosum of 2VO rats. These results suggest that EF may improve cognitive impairment and WM lesions induced by chronic cerebral hypoperfusion through inhibiting the Lingo-1/Fyn/ROCK pathway and activating the BDNF/TrkB, NRG-1/ErbB4, and the downstream PI3K/Akt/CREB pathways in WM. Thus, EF can be used as a potential neuroprotective agent in CSVD therapy.

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.

MiR-599 regulates LPS-mediated apoptosis and inflammatory responses through the JAK2/STAT3 signalling pathway via targeting ROCK1 in human umbilical vein endothelial cells.

MicroRNA plays an integral role in the development of atherosclerosis. Our study aimed to investigate the roles of miR-599 in lipopolysaccharide (LPS)-induced endothelial damage in human umbilical vein endothelial cells (HUVECs). HUVECs were transfected with a miR-599 mimic and negative control, and then exposed to LPS. The expression of miR-599 was detected by quantitative real time-polymerase chain reaction (RT-qPCR). Cell viability was analyzed by CCK-8 assay and trypan blue exclusion assay; the formation of DNA fragments was tested by Cell Death Detection ELISA Plus kit; the incidence of apoptosis was detected by flow cytometry; the expression of p53 and cleaved-caspase 3 (c-caspase 3) was evaluated by western blot. Moreover, the mRNA levels and concentrations of tumour necrosis factor (TNF)-α, interleukin (IL)-6, ICAM-1 and VCAM-1 were assayed by RT-qPCR and ELISA. The results showed that overexpression of miR-599 increased cell viability, reduced DNA fragments, the incidence of apoptosis, as well as the protein levels of p53 and c-caspase 3 in the presence of LPS. TNF-α, IL-6, ICAM-1 and VCAM-1 mRNA levels and concentrations were also decreased upon miR-599 upregulation. In addition, the dual luciferase reporter assay demonstrated that ROCK1 is a direct target of miR-599. MiR-599 overexpression inhibited ROCK1 expression. Induced expression of ROCK1 reversed the roles of miR-599 in apoptosis and inflammation. The gain function of miR-599 function inhibited activation of the JAK2/STAT3 signalling pathway, which was abrogated by overexpression of ROCK1. Taken together, our results indicate that miR-599 attenuates LPS-caused cell apoptosis and inflammatory responses through the JAK2/STAT3 signalling pathway via targeting ROCK1.

Ripasudil alleviated the inflammation of RPE cells by targeting the miR-136-5p/ROCK/NLRP3 pathway

BackgroundInflammation of RPE cells led to different kinds of eye diseases and affected the normal function of the retina. Furthermore, higher levels of ROCK1 and ROCK2 induced injury of endothelial cells and many inflammatory diseases of the eyes. Ripasudil, which was used for the treatment of glaucoma, was one kind of the inhibitor of ROCK1 and ROCK2, but whether ripasudil could relieve the LPS-induced inflammation and damage of RPE cells was not clear.MethodsWe used LPS to stimulate ARPE-19 cells, the RPE cell line. After that, we detected the levels of ROCK1 and ROCK2 by western-blotting after the stimulation of LPS and treatment of ripasudil. Then luciferase reporter assays were used to confirm the targeting effect of miR-136-5p on ROCK1 and ROCK2. At last, the levels of NLRP3, ASC, caspase1, IL-1β and IL-18 were detected with the western-blotting after the knockdown of miR-136-5p.ResultsThe levels of ROCK1, ROCK2 and miR-136-5p in ARPE-19 cells were promoted after the stimulation of LPS. After the treatment of ripasudil, the expression levels of ROCK1, ROCK2 and miR-136-5p were suppressed. The expression of ROCK1 and ROCK2 was targeted and inhibited by the miR-136-5p. The levels of inflammation related proteins NLRP3, ASC, caspase1, IL-1β and IL-18 was also inhibited after the treatment of ripasudil. However, the expression of these proteins was rescued after the knockdown of miR-136-5p.ConclusionRipasudil relieved the inflammatory injury of RPE cells by upregulating miR-136-5p, therefore inhibiting the expression of ROCK1, ROCK2, NLRP3, ASC, caspase1, IL-1β and IL-18.

Kangxian‐Yixin Granule alleviates myocardial fibrosis in rats with dilated cardiomyopathy through RhoA/ROCK1 pathway

ObjectiveKangxian‐Yixin granule (KXYX) is an effective traditional Chinese medicine prescription for the clinical treatment of dilated cardiomyopathy (DCM). The purpose of this study was to study the role of RhoA/ROCK1 signaling pathway in DCM myocardial fibrosis and the intervention mechanism of KXYX.MethodsDCM rats were randomly divided into model group (n = 8), KXYX group (n = 8), captopril group (n = 8) and control group (n = 8). After 4 weeks of drug intervention, the cardiac function of rats was detected by echocardiography, and the degree of myocardial fibrosis was observed by Masson staining. The expressions of RhoA, ROCK1, p‐MLC and fibrosis related factors CTGF and Col‐1 in RhoA/ROCK1 signaling pathway were detected by PCR and Western blot. At the same time, the myocardial fibroblast model was induced by angiotensin II, and treated with KXYX and inhibitor fasudil. RhoA/ROCK1 signaling pathway and the expression of fibrosis factors CTGF and Col‐1 were detected by Western blot and PCR.ResultsCompared with the control group, the expression of RhoA/ROCK1 signaling pathway in the model group was up‐regulated, the expression of p‐MLC was overactivated, and the expression of fibrosis‐related factors CTGF and Col‐1 was increased in the model group (P &lt; 0. 05). KXYX could inhibit myocardial fibrosis, inhibit the transformation of fibroblasts into myofibroblasts, and down‐regulate the expression of RhoA/ROCK1 signaling pathway and fibrosis factors CTGF and Col‐1 in DCM rats.ConclusionRhoA/ROCK1 signaling pathway plays an important role in DCM myocardial fibrosis, and KXYX can inhibit the up‐regulation of RhoA/ROCK1 signaling pathway, thus inhibiting the development of myocardial fibrosis in DCM rats. The correspondence to Lihua Han; hlhaa@sina.com.Support or Funding InformationThis work was financially supported by grants from the National Natural Science Foundation of China (Grant No. 81573920)

ROCK1 and ROCK2 Are Down-regulated in Aggressive and Advanced Skin Melanomas - A Clinicopathological Perspective.

RhoA and its downstream effectors Rho-associated coiled-coil kinases (ROCK) 1 and 2 are central controllers of cytoskeleton dynamics, and therefore influence cell shape, adhesion and migration. Since modulation of these processes holds promise for an effective anticancer strategy, effects of ROCK inhibition have been evaluated in a number of malignancies. Using immunohistochemistry, ROCK1 and ROCK2 expression was semi-quantitatively assessed in 129 patient-derived primary melanomas. There was a striking predilection for low melanocytic expression of both kinases in thick, ulcerated and mitogenic tumors, as well as in nodular histological type. ROCK1 and -2 expression in tumor-infiltrating lymphocytes (TILs) was preferentially down-regulated in advanced and aggressive tumors. Moreover, diminished ROCK2 reactivity in melanoma cells and TILs was associated with shorter melanoma-specific and recurrence-free survival. This is the first analysis of ROCK1 and -2 protein expression in clinical melanoma samples and the results indicated the suppression of ROCK signaling in melanocytes of aggressive and late-stage tumors. Functional models that more accurately represent the clinical setting are necessary to dissect the role of ROCK1 and -2 in melanoma. Additionally, our study indicates that ROCK activity in TILs may be involved in the pathogenesis of cancer, and thus merits further investigations.

MRNA level of ROCK1, RHOA, and LIMK2 as genes associated with apoptosis in evaluation of effectiveness of adalimumab treatment

BackgroundPsoriasis is a multifactorial autoimmune disease, which underlies the abnormalities of the apoptotic process. In cases of psoriasis and psoriatic arthritis, biological treatment is used. This study aimed to determine any changes in the expression of the genes associated with apoptosis in patients with psoriatic arthritis treated with adalimumab and to assess any phenotypic modifications based on changes in dermatological indexes.MethodsThe study included 20 patients with psoriatic arthritis treated biologically and 20 healthy volunteers. The research material consisted of peripheral blood mononuclear cells (PBMCs) from which the total RNA was isolated. Changes in the gene expression were determined using oligonucleotide microarrays and RT-qPCR. The clinical condition was assessed based on selected indicators: PASI, BSA [%], DAS28, and DLQI, which were determined every 3 months.ResultsThere were changes in the expression of genes associated with apoptosis. Significant differences were found for ROCK1, RhoA, and LIMK2 expression profiles in PBMCs. At the initial stage of treatment, a decrease in the PASI and BSA rates was observed. At the later stages, the values of these indicators increased once again. There were correlations between the changes in these genes’ expression and the dermatological markers.ConclusionAdalimumab influences the expression of genes related to apoptosis and the values of dermatological indicators of patients. Changes in the expression level of genes associated with apoptosis suggest that ROCK1, RhoA, and LIMK2 may be genes that can potentially be indicators of treatment effectiveness and lack of response to biological treatment.

MicroRNA-152 Inhibits Cell Proliferation, Migration, and Invasion in Breast Cancer.

The aim of the present study was to investigate the roles of microRNA-152 (miR-152) in the initiation and progression of breast cancer. The expression level of miR-152 was detected in human breast cancer tissue and a panel of human breast cancer cell lines using qRT-PCR. Results found that miR-152 expression was significantly downregulated in breast cancer tissue samples compared to adjacent noncancerous tissues as well as in breast cancer cell lines. Overexpression of miR-152 significantly suppressed breast cancer cell proliferation, migration, and invasion. Luciferase reporter assay results found that ROCK1 is a direct and functional target gene of miR-152 in breast cancer. In addition, downexpression of ROCK1 could inhibit breast cancer cell proliferation, migration, and invasion. These findings indicate that miR-152 inhibited breast cancer growth and metastasis through negative regulation of ROCK1 expression. These data suggest that miR-152/ROCK1 pathway may be a useful therapeutic target for breast cancer treatment.

Abstract TP350: Escin Attenuates the Impairment of Neurological Function by Ameliorating Systemic Inflammation in Intracerebral Hemorrhagic Mice

Background and Purpose: Escin could attenuate cerebral ischemia-induced brain injury. The present study aimed to investigate whether Escin may attenuate the impairment of neurological function by ameliorating systemic inflammation in ICH mice. Methods: The ICH model was prepared by intrastriatal injection of collagenase. At 0.5 h, 2 h, 6 h and 12 h after Escin injection, the concentration of Escin in the serum and brain were detected. The effects of Escin on the Garcia test were evaluated. Brain water content was observed via a wet/dry weight method. Evans blue extravasation in the cerebrums were also assayed. The serum IL-1β level was detected with the ELISA kit. The ICH mice were further treated with Escin plus IL-1β. At 24 h of the ICH, Garcia test, and brain water content, the Evans blue extravasation and serum IL-1β levels were evaluated. Additionally, the expression of RhoA, ROCK1, IκBα, nuclear NF-κB, cytosolic NF-κB, Occludin and Claudin-5 in mice brain tissue were investigated with the Western blot. Results: Escin was detected in the serum of the Control group and ICH group at 0.5 h, 2 h, 6 h and 12 h after Escin administration. Escin was not detected in brain tissues at any time points in the animals. At 24 h and 72 h of ICH, the Garcia test scores in the Escin groups were significantly increased ( P &lt; 0.05 or P &lt; 0.01). Brain water contents and Evans blue extravasation of the right basal ganglia in the Escin groups were significantly reduced ( P &lt; 0.05 or P &lt; 0.01). Escin abated the increase of IL-1β induced by ICH ( P &lt; 0.05 or P &lt; 0.01). IL-1β administration reversed the effect of Escin on Garcia test scores, the brain water contents, and the Evans blue extravasation ( P &lt; 0.01). In the ICH group, the levels of RhoA, ROCK1, nuclear NF-κB were increased while the expression of IκBα, cytosolic NF-κB, Occludin, Claudin-5 were reduced ( P &lt; 0.05 or P &lt; 0.01). However, Escin abated RhoA, ROCK1, nuclear NF-κB and augmented IκBα, cytosolic NF-κB, Occludin, and Claudin-5. IL-1β administration partially blocked the Escin-mediated regulation of IL-1β/RhoA/NF-κB signaling pathway. Conclusion: Escin cannot penetrate the blood brain barrier (BBB). Escin improves neurological function and by inhibiting systemic inflammation, and then regulating IL-1β/RhoA/NF-κB signaling pathway in BBB.

CircPSMC3 inhibits cell proliferation and induces cell apoptosis in nasopharyngeal carcinoma by downregulating ROCK1.

Currently, the importance of circular RNAs in malignant tumors has attracted much attention. However, the role of circPSMC3 in nasopharyngeal carcinoma (NPC) remains unclear. The aim of this study was to investigate the function of circPSMC3 in the proliferation and apoptosis of NPC and to explore its possible underlying mechanism. Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) was utilized to determine the level of circPSMC3 in NPC tissues and cell lines. The association between circPSMC3 expression and patients' prognosis was analyzed. CircPSMC3 lentivirus was constructed and transfected into NPC cells. Cell growth ability and apoptosis were detected through Cell Counting Kit-8 (CCK-8) assay, colony formation assay, and flow cytometry, respectively. Western blot was performed to analyze the target protein of circPSMC3. Furthermore, the function of circPSMC3 was explored in nude mice. CircPSMC3 was lowly expressed in NPC tissues compared with adjacent normal tissues. Low circPSMC3 expression was associated with poor prognosis of NPC patients. Meanwhile, the expression of circPSMC3 was significantly down-regulated in NPC cell lines as well. The growth ability of NPC cells was markedly inhibited after circPSMC3 was overexpressed. Overexpression of circPSMC3 significantly promoted the apoptosis of NPC cells in vitro. ROCK1 expression decreased markedly via overexpression of circPSMC3. Furthermore, tumor formation was inhibited after the up-regulation of circPSMC3 in vivo. CircPSMC3 could suppress cell growth and promote cell apoptosis in NPC by downregulating ROCK1.

Impact of RhoA overexpression on clinical outcomes in cervical squamous cell carcinoma treated with concurrent chemoradiotherapy.

The Rho-associated coiled-coil-containing protein kinase (ROCK) pathway is known to influence metastasis in several cancers; however, the impact of the pathway on clinical outcomes in patients undergoing radiotherapy remains unknown. In the present study, the expression of RhoA, RhoC, ROCK-1, ROCK-2 and p53 was immunohistochemically evaluated using biopsy specimens obtained from 49 patients with stage II–III cervical squamous cell carcinoma treated with concurrent chemoradiotherapy (CCRT). The relationship between the expression of these proteins and patient outcomes was investigated. RhoA overexpression was associated with significantly impaired disease-free survival and distant metastasis-free survival (P = 0.045 and P = 0.041, respectively) in stage III cancer patients. No differences in survival were observed based on the expression of the other proteins among stage III cancer patients. In stage II cancer patients, no differences in survival were noted based on the expression of any of the proteins. The expression of RhoA was able to successfully differentiate cervical cancer patients with distant metastasis after CCRT. This information may help stratify patients according to the risk of metastasis, thereby leading to the potential to provide individualized treatment.

Assessment of CIP2A and ROCK-I expression and their prognostic value in breast cancer.

Breast cancer is themost leading cause ofcancer death in females worldwide. Identification ofnovel biomarkers for prognosis is required. Imunohistochemical evaluation ofCIP2A and ROCK-1 expressions in 126 breast tissue specimens stratified as 21 ductal hyperplasias, 17 duct carcinoma in situ (DCIS) and 88 invasive carcinomas (56 invasive ductal carcinomas NST, 32 invasive lobular carcinomas) was studied. High CIP2A expression was detected in 48.9% ofinvasive carcinomas. CIP2A overexpression was significantly related to Nottingham prognostic index (NPI) (p=0.011), stage (p=0.01), ER negativity (p=0.031), PR negativity (p=0.048), and HER-2 positivity (p=0.02). CIP2A was significantly overexpressed in triple-negative breast cancer (TNBC) (p=0.004). ROCK-1 expression was detected in 54.5% ofinvasive carcinomas. Statistically significant associations were observed between ROCK-1 expression and NPI (p=0.032), stage (p=0.002), ER negativity (p=0.012), PR negativity (p=0.023), HER-2 positivity (p=0.016), and TNBC subtype (p=0.033). Apositive association between CIP2A and ROCK-1 expressions (p<0.0001) was documented. There was asignificant association between shorter overall survival and high CIP2A and positive ROCK-1 expressions (p<0.0001) and (p<0.0001). CIP2A and ROCK-1 expressions could be used as markers for thepoor prognosis ofbreast cancer.

YQFM Alleviates Side Effects Caused by Dasatinib through the ROCK/MLC Pathway in Mice.

Dasatinib, as a second-generation broad-spectrum tyrosine kinase inhibitor, presents an antitumor effect by inhibiting tyrosine kinases. However, dasatinib causes serious side effects, such as gastrointestinal bleeding and liver toxicity, possibly through the activation of ROCK kinase and MLC phosphorylation. At present, there is no effective prevention and treatment method. Previous research studies have shown that YQFM (YiQiFuMai powder injection) protects the blood-brain barrier by inhibiting the ROCK/MLC signaling pathway; whether YQFM can alleviate the side effects of dasatinib is unknown. In this study, dasatinib was injected (i.p. 70 mg/kg) and YQFM (i.p. 0.336 g/kg, 0.672 g/kg, 1.342 g/kg) was given in advance for 3 days to mice, to explore the effect of YQFM on side effects induced by Dasatinib. The results confirmed that YQFM significantly decreased Evans blue leakage in the small intestine and increased intestinal blood flow, increased the expression of ZO-1, Occludin, and VE-cadherin, and reduced the contents of D-lactic acid, s-VE-cadherin, Alanine aminotransferase (ALT), and Aspartate aminotransferase (AST) in serum. Finally, YQFM inhibited the expression of ROCK-1 and phosphorylation of MLC induced by Dasatinib. These findings suggested that YQFM could improve the side effects caused by Dasatinib linked with the ROCK/MLC signaling pathway, as shown in the graphical abstract.

LncRNA OIP5-AS1 targets ROCK1 to promote cell proliferation and inhibit cell apoptosis through a mechanism involving miR-143-3p in cervical cancer.

Opa-interacting protein 5 antisense transcript 1 (OIP5-AS1) is one kind of cytoplasmic long non-coding RNA (lncRNA), which has been demonstrated to play a critical function in multiple cancers. However, the detailed mechanism of OIP5-AS1 in the regulation of cervical cancer progression is still obscure. Here, we demonstrated that lncRNA OIP5-AS1 was upregulated in cervical cancer and was correlated with poor prognosis by bioinformatics studies. OIP5-AS1 depletion inhibited cell proliferation and promoted cell apoptosis in cervical cancer cells. Furthermore, we clarified that ROCK1 was the downstream effector of OIP5-AS1 and OIP5-AS1 acted as a molecular sponge of miR-143-3p. Finally, we verified that OIP5-AS1 exerted its function in the regulation of cervical cancer progression via interacting with miR-143-3p to regulate ROCK1 expression. Our study revealed novel mechanisms about how lncRNA OIP5-AS1 executed its function in cervical cancer and thus provided potential therapeutic targets for the disease.

Tetrandrine Suppresses Transient Receptor Potential Cation Channel Protein 6 Overexpression- Induced Podocyte Damage via Blockage of RhoA/ROCK1 Signaling.

ObjectivePodocyte damage is common in many renal diseases characterized by proteinuria. Transient receptor potential cation channel protein 6 (TRPC6) plays an important role in renal function through its regulation of intracellular Ca2+ influx and RhoA/ROCK pathways. Chinese herb Stephania tetrandra, with the main active component being tetrandrine, has been used for the treatment of various kidney diseases for several years and has shown a positive effect. This study aimed at investigating the effect and mechanism of tetrandrine in podocyte damage induced by high expression of TRPC6.MethodsImmortalized, differentiated murine podocytes, MPC5 were treated with valsartan (0–800 μM) and tetrandrine (0–40 μM) for 48 h. The maximum safe concentrations of valsartan and tetrandrine were selected using a cell viability assay. MPC5 podocytes stably expressing TRPC6 were constructed using a lentivirus packaging system, followed by treatment with valsartan, tetrandrine, and Y-27632 for 48 h and U73122 (10 μM) for 10 min. The RhoA/ROCK pathway and podocyte-specific proteins (nephrin and synaptopodin) levels were quantified. Podocyte apoptosis and intracellular Ca2+ concentration were measured.ResultsMaximum safe concentrations of 100 μM valsartan and 10 μM tetrandrine showed no observable toxicity in podocytes. MPC5 podocytes stably expressing TRPC6 had higher intracellular Ca2+ influx, apoptotic percentages, and expression of RhoA/ROCK proteins, but lower expression of nephrin and synaptopodin proteins. U73122 treatment for 10 min did not inhibit TRPC6, but suppressed RhoA/ROCK protein. Y-27632 decreased ROCK1 expression, but did not influence the expression of TRPC6 protein. Both 100 μM valsartan and 10 μM tetrandrine for 48 h significantly inhibited intracellular Ca2+ influx, apoptosis, and RhoA/ROCK pathway, and increased nephrin and synaptopodin proteins in podocytes stably expressing TRPC6.ConclusionElevated TRPC6 expression can lead to podocyte injury by inducing intracellular Ca2+ influx and apoptosis of podocytes, and this effect may be mediated by activation of the RhoA/ROCK1 pathway. Tetrandrine can alleviate podocyte injury induced by TRPC6 expression through inhibition of the RhoA/ROCK pathway, suggesting a protective role in podocyte damage.

Hyperglycemia-induced transcriptional regulation of ROCK1 and TGM2 expression is involved in small artery remodeling in obese diabetic Göttingen Minipigs.

Obesity and diabetes in humans are associated with hypertrophic remodeling and increased media:lumen ratio of small resistance arteries, which is an independent predictor of cardiovascular events. In order to minimize increases in media:lumen ratio, hypertrophic remodeling should be accompanied by outward remodeling. We aimed to investigate the mechanisms of structural remodeling in small pial arteries (PAs) and terminal mesenteric arteries (TMAs) from obese Göttingen Minipigs with or without diabetes. Göttingen Minipigs received either control diet (lean control (LC)), high fat/high fructose/high cholesterol diet (FFC), or FFC diet with streptozotocin (STZ)-induced diabetes (FFC/STZ) for 13 months. At the end of the study (20 months), we assessed body weight, fasting plasma biochemistry, passive vessel dimensions, mRNA expression (matrix metallopeptidases 2/9 (MMP2, MMP9), tissue inhibitor of metallopeptidase 1 (TIMP1), transglutaminase 2 (TGM2), Rho-kinase 1 (ROCK1), TGFβ-receptor 2 (TGFBR2), and IGF1-receptor (IGFR1) genes), and immunofluorescence in PAs and TMAs. We performed multiple linear correlation analyses using plasma values, structural data, and gene expression data. We detected outward hypertrophic remodeling in TMAs and hypertrophic remodeling in PAs from FFC/STZ animals. ROCK1 and TGM2 genes were up-regulated in PAs and TMAs from the FFC/STZ group. Passive lumen diameter (PLD) of TMAs was correlated with plasma values of glucose (GLU), fructosamine (FRA), total cholesterol (TC), and triglycerides (TGs). ROCK1 and TGM2 expressions in TMAs were correlated with PLD, plasma GLU, fructosamine, and TC. ROCK1 and TGM2 proteins were immunolocalized in the media of PAs and TMAs, and their fluorescence levels were increased in the FFC/STZ group. Hyperglycemia/hyperlipidemia is involved in regulation of ROCK1 and TGM2 expression leading to outward remodeling of small resistance arteries in obese diabetic Göttingen Minipigs.

Metformin Protects against Oxidative Stress Injury Induced by Ischemia/Reperfusion via Regulation of the lncRNA-H19/miR-148a-3p/Rock2 Axis.

Previous studies have shown that metformin not only is a hypoglycemic agent but also has neuroprotective effects. However, the mechanism of action of metformin in ischemic stroke is unclear. Oxidative stress is an important factor in the pathogenesis of cerebral ischemia-reperfusion injury. It has been reported that metformin is associated with stroke risk in the clinical population. This study is aimed at investigating the effect and mechanism of metformin in an experimental model of oxidative stress induced by ischemia/reperfusion (I/R) in vivo and oxygen glucose deprivation/reperfusion (OGD/R) in vitro. Metformin (100, 200, and 300 mg/kg) was administered intraperitoneally immediately after induction of cerebral ischemia. The indicators of oxidative stress selected were antioxidant enzyme activities of catalase, malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), and glutathione peroxidation enzyme (GSHPx). First, we demonstrated that metformin can significantly alleviate acute and chronic cerebral I/R injury and it has a strong regulatory effect on stroke-induced oxidative stress. It can reduce the elevated activities of MDA and NO and increase the levels of GSHPx and SOD in the cerebrum of mice and N2a cells exposed to I/R. Furthermore, real-time PCR and western blot were used to detect the expression of long noncoding RNA H19 (lncRNA-H19), microRNA-148a-3p (miR-148a-3p), and Rho-associated protein kinase 2 (Rock2). The direct interaction of lncRNA-H19, miR-148a-3p, and Rock2 was tested using a dual luciferase reporter assay. lncRNA-H19 altered OGD/R-induced oxidative stress by modulating miR-148a-3p to increase Rock2 expression. The expression of lncRNA-H19 and Rock2 could be downregulated with metformin in vivo and in vitro. In conclusion, our study confirmed that metformin exerts neuroprotective effects by regulating ischemic stroke-induced oxidative stress injury via the lncRNA-H19/miR-148a-3p/Rock2 axis. These results provide new evidence that metformin may represent a potential treatment for stroke-related brain injury.