Expression Of ROCK1 Research Articles

SiRNA Mediated Downregulation of RhoA Expression Reduces Oxidative Induced Apoptosis in Retinal Ganglion Cells.

Glaucoma is the second leading cause of blindness. Apoptosis of retinal ganglion cells (RGCs) is an important mechanism of glaucomatous optic injury. Rho kinase expression is significantly increased in apoptotic RGCs. This study aimed to investigate the role of RhoA, a Rho GTPase, on the survival of RGCs and further to explore its potential therapeutic applications. RGCs were treated with siRhoA for 24 hours in vitro. Knockdown of RhoA was confirmed with quantitative RT-PCR. Oxidative stress was induced by treating the RGCs with 200 μM of H2O2 for 1 hour, and apoptosis of RGCs was quantified with TUNEL assay in situ, and with flow cytometry. The mRNA expression levels of RhoA, Nogo receptor, caspase 3 and Bcl-2 were evaluated by quantitative RT-PCR, and the protein levels of RhoA, ROCK1, ROCK2, Nogo receptor, caspase 3 and Bcl-2 were evaluated by Western blot. We found siRhoA treatment efficiently downregulated the expression of RhoA in RGCs and protected against H2O2-induced injury in RGCs in vitro. Apoptosis of RGC cells under oxidative stress was quantified in situ using TUNEL assay and confirmed with flow cytometry (FCM). With the knockdown of RhoA, the expression of ROCK1, ROCK2, Nogo Receptor, Casepase-3 were decreased, while the expression of Bcl-2 was increased in both mRNA and protein level. Our data indicated that siRhoA prevented H2O2-induced apoptosis in RGC cells by modulating the RhoA/ROCK pathway. The results suggested that siRhoA may exert potentially effective neuroprotection for RGCs by reducing injury.

Myofilament calcium sensitization in non-human primate lower esophageal sphincter and esophagus

Background/Objectives: Peristalsis in the esophagus (ESO) moves ingested contents into the stomach. The lower esophageal sphincter (LES) is located at the distal end of the ESO and generates tone to prevent reflux of gastric contents. Previous studies suggest that the expression and/or phosphorylation of proteins involved in myofilament Ca2+ sensitization differs between phasic and tonic smooth muscles (Brozovich et al., 2016; PMID: 27037223). We hypothesize that differences in expression and/or phosphorylation of these proteins contributes to differences in contractile activity between LES and ESO. The aims were to 1) characterize contractile activity in Cynomolgus monkey LES and ESO, 2) evaluate myofilament Ca2+ sensitization protein expression and phosphorylation, 3) examine changes in phosphorylation when contractile activity is enhanced or inhibited pharmacologically. Methods: Contraction was assessed in strips of LES and ESO by isometric tension recordings. Carbachol (CCh, 1 μM) was used to enhance contractions. ROCK2 (SR3677; 2 μM) or CavL (nifedipine; 1 μM) antagonists were used to inhibit contractions. Tissues were snap frozen in liquid nitrogen after peak response and prepared for protein expression analysis using Jess immunoelectrophoresis. Results: Under basal conditions, the LES exhibited tone whereas the ESO had phasic activity. CCh significantly increased contractions. Nifedipine and SR3677 significantly decreased basal and CCh-induced contractions with nifedipine having the greater effect. MLCK and MYPT1 expression was significantly greater in LES than ESO while LC20 and CPI-17 expression was greater in ESO. ROCK2 expression was similar in LES and ESO. Under basal conditions MYPT1 and LC20 phosphorylation was greater in LES. MYPT1 and LC20 phosphorylation was decreased by SR3677 but not nifedipine in both tissues. CCh increased MYPT1 and LC20 phosphorylation in both tissues though was greater in LES. SR3677 inhibited phosphorylation of both proteins in LES and ESO. Discussion/Conclusions: The differences in expression and phosphorylation of key proteins involved in myofilament Ca2+ sensitization likely contribute to differences in contractile activity. Notably, there is greater LC20 phosphorylation in LES than ESO under basal conditions and with CCh. Furthermore, there is an important role for ROCK2 in regulating basal contractile activity in LES and ESO as both contraction and phosphorylation of MYPT1 and LC20 are reduced by SR3677. While nifedipine significantly reduced contractile activity in LES and ESO, it did not reduce phosphorylation of MYPT1 indicating that the ROCK2 pathway is independent of Ca2+ influx. These findings may improve understanding of the pathophysiology associated with esophageal dysmotility. NIH DK129528 to CAC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Caspase-resistant ROCK1 expression prolongs survival of Eµ-Myc B cell lymphoma mice.

Apoptosis is characterized by membrane blebbing and apoptotic body formation. Caspase cleavage of ROCK1 generates an active fragment that promotes actin-myosin-mediated contraction and membrane blebbing during apoptosis. Expression of caspase-resistant non-cleavable ROCK1 (Rock1 NC) prolonged survival of mice that rapidly develop B cell lymphomas due to Eµ-Myc transgene expression. Eµ-Myc; Rock1 NC mice had significantly fewer bone marrow cells relative to those in Eµ-Myc mice expressing wild-type ROCK1 (Rock1 WT), which was associated with altered cell cycle profiles. Circulating macrophage numbers were lower in Eµ-Myc; Rock1 NC mice, but there were higher levels of bone marrow macrophages, consistent with spontaneous cell death in Eµ-Myc; Rock1 NC mouse bone marrows being more inflammatory. Rock1 WT recipient mice transplanted with pre-neoplastic Eµ-Myc; Rock1 NC bone marrow cells survived longer than mice transplanted with Eµ-Myc; Rock1 WT cells, indicating that the survival benefit was intrinsic to the Eµ-Myc; Rock1 NC bone marrow cells. The results suggest that the apoptotic death of Eµ-Myc; Rock1 NC cells generates a proliferation-suppressive microenvironment in bone marrows that reduces cell numbers and prolongs B cell lymphoma mouse survival.

RhoA/ROCK Pathway Is Upregulated in Experimental Autoimmune Myocarditis and Is Inhibited by Simvastatin at the Stage of Myosin Light Chain Phosphorylation.

Many studies have proven the involvement of the RhoA/ROCK pathway in autoimmune and cardiovascular diseases and the beneficial effects of its downregulation. Here, we examined whether the effect of simvastatin on experimental autoimmune myocarditis (EAM) may be through targeting the Ras homolog family member A/Rho-associated coiled-coil containing kinases (RhoA/ROCK) pathway and whether previously shown downregulation of metalloproteinase 9 (MMP-9) could be associated with MLC phosphorylation. Two doses of simvastatin were administered to experimental rats with autoimmune myocarditis by gastric gavage for 3 weeks, at the stage of development of the inflammatory process. Immunohistochemical staining for RhoA and ROCK1 was evaluated semi-quantitatively with H-score. The RhoA staining showed no significant differences in expression between the groups, but the ROCK1 expression was significantly upregulated in the hearts of the EAM group and was not downregulated by simvastatin. The Western blotting analysis of the last downstream product of the RhoA/ROCK axis, phosphorylated myosin light chain (phospho-MYL9), revealed that protein content increased in EAM hearts and it was prevented by the highest dose of simvastatin. Our findings suggest that the RhoA/ROCK pathway is upregulated in EAM, and simvastatin in EAM settings inhibits the RhoA/ROCK pathway at the stage of phosphorylation of myosin light chains and provides a new insight into the molecular pathology of autoimmune myocarditis.

Zinc as a Mediator Through the ROCK1 Pathway of Cognitive Impairment in Aluminum-Exposed Workers: A Clinical and Animal Study.

Aluminum (Al) exposure was implicated in neurodegenerative diseases and cognitive impairment, yet the involvement of zinc (Zn) and its mechanism in Al-induced mild cognitive impairment (MCI) remains poorly understood. The objective is to explore the role of Zn in Al-induced cognitive impairment and its potential mechanisms. Montreal cognitive assessment (MoCA) test scores and serum Al, Zn from Al industry workers were collected. A mediation analysis was performed to evaluate the role of serum Zn among serum Al and MoCA test scores. Subsequently, an Al-exposure study was conducted on a rat model categorized into control, low-, medium-, and high-dose groups. After a Morris Water Maze test and detection of Al, Zn content in the hippocampus, integrated transcriptomic and proteomic analyses between the control group and the high-dose group were performed to identify the differentially expressed genes (DEPs), proteins (DEPs), and pathways. To corroborate these findings, quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting (WB) were selected to identify the gene and protein results. Zn overall mediates the relationship between serum Al and cognitive function (mediation effect 17.82%, effect value = - 0.0351). In the Al-exposed rat model, 734 DEGs, 18 miRNAs, 35 lncRNAs, 64 circRNAs, and 113 DEPs were identified between the high-dose group and the control group. Among them, ROCK1, DMD, and other four DEPs were identified as related to zinc finger proteins (ZNF). Co-enrichment analyses of the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) linked these changes to the RHOA/ROCK1 signaling axis. ZNF-related proteins Rock1, DMD, and DHX57 in the high-dose group were downregulated (p = 0.006, 0.003, 0.04), and the expression of Myl9, Rhoa, miR431, and miR182 was also downregulated (p = 0.003, 0.032, 0.032, and 0.046). These findings also show correlations between Al, Zn levels in the hippocampus, water maze performance, and expressions of Myl9, Rhoa, miR431, miR182, DMD, ROCK1, and DHX57, with both negative and positive associations. Based on the results, we determined that Zn was involved in Al-induced MCI in Al workers and Al-exposed rat models. Al exposure and interaction with Zn could trigger the downregulation of ZNF of ROCK1, DMD, and DHX57. miR431, miR182 regulate RHOA/ROCK1 was one of the Zn-involved pathways in Al-induced cognitive impairment.

Rho-Kinase Is Differentially Expressed in the Adipose Tissue of Rodent and Human in Response to Aging, Sex, and Acute Exercise.

White adipose tissue (WAT) controls energy storage, expenditure, and endocrine function. Rho-kinase (ROCK) is related to impaired thermogenesis, downregulation of preadipocyte differentiation, and adipokine production. Furthermore, WAT ROCK responds to metabolic stress from high-fat diets or diabetes. However, ROCK distribution in adipose depots and its response to aging and sex remain unclear. Thus, we aim to investigate ROCK function in adipose tissue of rodent and human in response to aging and sex. We observed specific differences in the ROCK1/2 distribution in inguinal WAT (ingWAT), perigonadal WAT (pgWAT), and brown adipose tissue of male and female rodents. However, ROCK2 expression was lower in female ingWAT compared with males, a fact that was not observed in the other depots. In the pgWAT and ingWAT of male and female rodents, ROCK activity increased during development. Moreover, middle-aged female rodents and humans showed downregulation in ROCK activity after acute physical exercise. Interestingly, ROCK levels were associated with several inflammatory markers both in rats and humans WAT (Nfkb1, Tnf, Il1b, Il6, and Mcp1). Induction of cell senescence by etoposide elevates ROCK activity in human preadipocytes; however, silencing ROCK1/2 demonstrates improvement in the inflammatory and cell senescence state. Using public databases, several pathways were strongly associated with ROCK modulation in WAT. In summary, WAT ROCK increases with development in association with inflammatory markers. Further, ROCK activity was attenuated by acute physical exercise, implicating it as a possible therapeutic target for metabolism improvement mediated by adipose tissue inflammatory state changes.

Simvastatin-Loaded Nanoniosome Protects H9c2 Cells from Oxygen-Glucose Deprivation/ Reperfusion Injury by Downregulating Inflammation.

Simvastatin (SIM) has anti-inflammatory and antioxidant properties against cardiac ischemia/reperfusion injury (I/RI). However, it suffers from low bioavailability and a short half-life. Nanoniosomes are novel drug delivery systems that may increase SIM effectiveness. The present research evaluates the impact of SIM-loaded nanoniosomes on the oxygen-glucose deprivation/reperfusion (OGD/R) injury model of H9c2 cells. Cells were seeded based on five groups: (1) control; (2) OGD/R; (3) OGD/R receiving SIM; (4) OGD/R receiving nanoniosomes; and (5) OGD/R receiving SIM loaded nanoniosomes. OGD/R injury of the H9c2 cells was treated with SIM or SIM loaded nanoniosomes. Cell viability, two inflammatory factors, necroptosis factors, along with HMGB1 and Nrf2 gene expressions were assessed. The cells treated with SIM loaded nanoniosomes showed a significant elevation in the cell viability and a reduction in HMGB1, Nrf2, TNF-α, IL-1β, RIPK1, and ROCK1 expression levels compared to the OGD/R and SIM groups. Based on our findings, nanoniosomes could safely serve as a drug delivery system to counterbalance the disadvantages of SIM, resulting in improved aqueous solubility and stability.

The Distant Molecular Effects on the Brain by Cancer Treatment.

Cancer survivors experience cancer-related cognitive impairment (CRCI) secondary to treatment. Chemotherapy and radiation therapy independently contribute to cognitive dysfunction; however, the underlying mechanisms leading to dysfunction remain unclear. We characterized brain gene expression changes in a mouse model of CRCI to identify the mechanistic underpinnings. Eleven-to-twelve-week-old SKH1 mice were treated with doxorubicin (DOX), hindlimb radiation (RT), concurrent hindlimb radiation and doxorubicin (DOX-RT), or no treatment (control). Sixteen days following treatment, gene expression was measured from murine brains using the NanoString nCounter® glial profiling panel. Gene expression was normalized and compared between groups. No two groups shared the same expression pattern, and only Gnb1 and Srpr were upregulated in multiple treatment groups. Brains from DOX-treated mice had upregulated Atf2, Atp5b, Gnb1, Rad23b, and Srpr and downregulated Sirt5 expression compared to control brains. Brains from RT-treated mice demonstrated increased Abcg2 and Fgf2 and decreased C1qa and C1qb expression compared to control brains. Brains from DOX-RT-treated mice had upregulated Adar, E2f3, Erlec1, Gnb1, Srpr, Vim, and Pdgfra expression and downregulated Rock2 and Inpp5f expression compared to control brains. The gene expression changes demonstrated here highlight roles for neuronal transmission and oxidative stress in the pathogenesis of doxorubicin-related CRCI and inflammation in RT-related CRCI.

THE ROLE OF N6-METHYLADENOSINE METHYLTRANSFERASE RBM15 IN NONALCOHOLIC FATTY LIVER DISEASE.

Nonalcoholic fatty liver disease (NAFLD) is a prevalent liver disorder with significant health implications. N6-methyladenosine (m6A) methyltransferase is known to exert regulatory functions in liver-related diseases. This study investigates the intricate role of RNA binding motif protein 15 (RBM15) in modulating inflammation and oxidative stress in NAFLD. An NAFLD model was induced in mice (male, C57BL/6J, 72 mice in the sham group) through a high-fat diet for 9 weeks, and hepatocytes were exposed to long chain-free fatty acids. The expression levels of RBM15, ring finger protein 5 (RNF5), and rho-kinase 1 (ROCK1) were assessed. RBM15 expression was intervened (injection of AAV9 virus at week 9 and detection at week 11). Liver damage was evaluated using staining assays, along with assessments of weight changes and lipid levels. Notably, RBM15 (decreased approximately 40%/60%) and RNF5 (decreased approximately 60%/75%) were poorly expressed while ROCK1 (increased approximately 2.5-fold) was highly expressed in liver tissues and cells. RBM15 overexpression mitigated liver damage, inflammation, and oxidative stress in NAFLD mice, resulting in reduced liver-to-body weight ratio (20%) and decreased levels of alanine aminotransferase (54%), aspartate aminotransferase (36%), total cholesterol (30%), and triglycerides (30%), and inhibited inflammation and oxidative stress levels. Mechanistically, RBM15 upregulated RNF5 expression through m6A methylation modification, and RNF5 suppressed ROCK1 protein levels through ubiquitination modification. RNF5 knockdown or ROCK1 overexpression accelerated inflammation and oxidative stress in NAFLD. Taken together, RBM15 upregulated RNF5 expression through m6A methylation modification. RNF5 inhibited ROCK1 expression through ubiquitination modification to mitigate NAFLD.

Inhibition of microRNA-34c reduces detrusor ROCK2 expression and urinary bladder inflammation in experimental cystitis

Interstitial cystitis (IC), also called painful bladder syndrome (PBS), is 2 to 5 times more common in women than in men, yet its cause and pathogenesis remain unclear. In our study using the cyclophosphamide (CYP)-induced mouse model of cystitis, histological evaluation of the urinary bladder (UB) lamina propria (LP) showed immune cell infiltrations, indicating moderate to severe inflammation. In this study, we noticed a differential expression of a subset of microRNAs (miRs) in the UB cells (UBs) of CYP-induced cystitis as compared to the control. UB inflammatory scores and inflammatory signaling were also elevated in CYP-induced cystitis as compared to control. We identified eight UBs miRs that exhibited altered expression after CYP induction and are predicted to have a role in inflammation and smooth muscle function (miRs-34c-5p, −34b-3p, −212-3p, −449a-5p, −21a-3p, −376b-3p, −376b-5p and − 409-5p). Further analysis using ELISA for inflammatory markers and real-time PCR (RT-PCR) for differentially enriched miRs identified miR-34c as a potential target for the suppression of UB inflammation in cystitis. Blocking miR-34c by antagomir ex vivo reduced STAT3, TGF-β1, and VEGF expression in the UBs, which was induced during cystitis as compared to control. Interestingly, miR-34c inhibition also downregulated ROCK2 but elevated ROCK1 expression in bladder and detrusor cells. Thus, the present study shows that targeting miR-34c can mitigate the STAT3, TGF-β, and VEGF, inflammatory signaling in UB, and suppress ROCK2 expression in UBs to effectively suppress the inflammatory response in cystitis. This study highlights miR-34c as a potential biomarker and/or serves as the basis for new therapies for the treatment of cystitis.

Effects of combined electroacupuncture and medication therapy on the RhoA/ROCK-2 signaling pathway in the striatal region of rats afflicted by cerebral ischemia

ObjectiveTo investigate the effects of electroacupuncture(EA), gastrodin(Gas), and their combination on the signaling pathways involving Ras homologous gene family member A (RhoA) and Rho-associated frizzled helix protein kinase (ROCK-2) within the striatal region of rats subjected to cerebral ischemia. Additionally, we aim to elucidate the therapeutic effects and potential underlying mechanisms associated with the concurrent application of electroacupuncture and medication in the treatment of cerebral ischemia. MethodsRats were randomly assigned to one of five groups, namely, the sham operation (Sham) group, model group, EA group, Gas group, and the EA combined with Gas group (referred to as the "EA+Gas group"). Each group consisted of ten rats. Following the induction of cerebral ischemia, the EA group and EA+Gas group received EA stimulation at the Baihui(GV20) and Zusanli(ST36) acupoints for 30 min per session, administered once daily for 14 consecutive days. The Gas group and EA+Gas group were intraperitoneally injected with Gas at a dosage of 10 mg/kg, also administered once daily for 14 consecutive days. Nissl staining was employed to observe morphological alterations in the striatal nerve cells of rats in each group. Immunohistochemistry and western blot techniques were employed to evaluate the expression levels of striatal RhoA and ROCK-2 proteins. ResultsIn comparison to the Sham group, the model group exhibited a substantial reduction in the number of striatal nerve cells on the ischemic side, accompanied by notable changes in cell morphology, characterized by reduced cytoplasm, defective and atrophied cytosol, solidified nuclei, loosely arranged cells, and enlarged intercellular spaces. Additionally, there was a notable increase in the positive expression of RhoA and ROCK-2. In contrast, when compared to the model group, the EA, Gas, and EA+Gas groups demonstrated an elevated number of normal nerve cells within the ischemic striatal region, with a significant improvement in cell count and morphology. Furthermore, positive expression levels of RhoA and ROCK-2 were notably reduced in these groups. Compared with the EA group or the GAS group, the number of normal nerve cells in the striatum on the ischemic side of the EA+GAS group was further increased, and the positive expression level of RhoA and ROCK-2 were both further reduced. ConclusionThe protective mechanism underlying the therapeutic efficacy of EA combined with Gas against cerebral ischemic striatal injury in rats may be associated with the inhibition of the activation of the RhoA/ROCK-2 signaling pathway. Importantly, the therapeutic effects observed with the combination of electroacupuncture and medication were superior to those achieved with EA alone or the sole administration of Gas.

ECM1 promotes migration and invasion in endometriosis

Extracellular matrix protein 1 (ECM1) is a glycoprotein that may be a key player in tumorigenesis and tumor progression. However, knowledge regarding the role of ECM1 in endometriosis (EM) is still lacking. Microarray analyses were performed to compare the mRNA expression patterns between paired EU tissues and ectopic endometrial (EC) tissues (n = 4) from EM patients. ECM1 expression was significantly increased in the eutopic endometrial (EU) tissues than paired EC tissues of endometriotic patients and normal endometrial (NE) tissues of controls without EM. Blocking ECM1 with siRNA attenuated the migration and invasion of hEM15A cells and modified the distribution of the F-actin cytoskeleton. We conducted microarray analyses and bioinformatics analyses to investigate the differentially expressed genes (DEGs) and related pathways regulated by ECM1. A total of 161 DEGs between the siECM1 and the negative control (siNC) treatments were identified, consisting of 79 downregulated genes and 82 upregulated genes. Enriched DEGs were associated with 9 gene ontology (GO) terms. Moreover, a protein-protein interaction (PPI) network was constructed for the hub genes and modules. Radixin (RDX) was the second most downregulated gene in the siECM1 group compared with the siNC group. ECM1 knockdown significantly decreased the expression of RDX, RhoC, ROCK1, N-cadherin and β-catenin but not ROCK2. ECM1 showed high tissue-specific expression in EU tissues from EM patients, and may contribute to the migration, invasion and reorganization of the F-actin cytoskeleton in eutopic endometrial stromal cells via the RhoC/ROCK1 signaling pathway in EM.

Triptolide Reduces MDA-MB-231 Cell Metastasis by Attenuating Epithelial-Mesenchymal Transition through the ROCK/PTEN/Akt Axis.

Triple-negative breast cancer (TNBC) is a highly heterogeneous and invasive subtype of breast cancer. The prognosis of TNBC is poor because of its high distant metastasis rate. Triptolide is a type of diterpene trioxide natural compound with potential anti-tumor activities. This study explored the metastatic inhibitory effect of triptolide on MDA-MB-231 cells and its underlying mechanism. Triptolide suppressed cell proliferation and induced cell apoptosis in a time- and dose-dependent manner. Low doses of triptolide (0-8 nM) reduced the migration and invasion capabilities of MDA-MB-231 cells. Triptolide decreased ROCK1, p-Akt, N-cadherin, vimentin and MMP-9 expressions, but increased PTEN and E-cadherin expressions on protein and mRNA levels. Furthermore, the down-regulation of ROCK1 expression in MDA-MB-231 cells after being treated by triptolide could be rescued by ROCK1 specific inhibitor Y27632. Molecular docking showed that triptolide and Y27632 shared the same active center of ROCK1 protein. This article's findings taken together showed that ROCK1 is the primary target of triptolide, which can cause cell apoptosis and inhibit the epithelial-mesenchymal transition of MDA-MB-231 cells.

The role of KDM4A-mediated histone methylation on temozolomide resistance in glioma cells through the HUWE1/ROCK2 axis.

Temozolomide(TMZ) resistance presents a significant challenge in the treatment of gliomas. Although lysine demethylase 4A (KDM4A) has been implicated in various cancer-related processes, its role in TMZ resistance remains unclear. This study aims to elucidate the contribution of KDM4A to TMZ resistance in glioma cells and its potential implications for glioma prognosis. We assessed the expression of KDM4A in glioma cells (T98G and U251MG) using qRT-PCR and Western blot assays. To explore the role of KDM4A in TMZ resistance, we transfected siRNA targeting KDM4A into drug-resistant glioma cells. Cell viability was assessed using the CCK-8 assay and the TMZ IC50 value was determined. ChIP assays were conducted to investigate KDM4A, H3K9me3, and H3K36me3 enrichment on the promoters of ROCK2 and HUWE1. Co-immunoprecipitation confirmed the interaction between HUWE1 and ROCK2, and we examined the levels of ROCK2 ubiquitination following MG132 treatment. Notably, T98G cells exhibited greater resistance to TMZ than U251MG cells, and KDM4A displayed high expression in T98G cells. Inhibiting KDM4A resulted in decreased cell viability and a reduction in the TMZ IC50 value. Mechanistically, KDM4A promoted ROCK2 transcription by modulating H3K9me3 levels. Moreover, disruption of the interaction between HUWE1 and ROCK2 led to reduced ROCK2 ubiquitination. Inhibition of HUWE1 or overexpression of ROCK2 counteracted the sensitization effect of si-KDM4A on TMZ responsiveness in T98G cells. Our findings highlight KDM4A's role in enhancing TMZ resistance in glioma cells by modulating ROCK2 and HUWE1 transcription and expression through H3K9me3 and H3K36me3 removal.

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.

Hyperoxia but not high tidal volume contributes to ventilator-induced lung injury in healthy mice

BackgroundMechanical ventilation is a supportive therapy used to maintain respiratory function in several clinical and surgical cases but is always accompanied by lung injury risk due to improper treatment. We investigated how tidal volume and oxygen delivery would contribute independently or synergistically to ventilator-induced lung injury (VILI).MethodsUnder general anesthesia and tracheal intubation, healthy female C57BL/6 N mice (9 weeks old) were randomly ventilated for 2 h by standard (7 ml/kg) or high (14 ml/kg) tidal volume at positive end-expiratory pressure (PEEP) of 2 cmH2O, with room air, 50% O2 (moderate hyperoxia), or 100% O2 (severe hyperoxia); respectively. Mice were sacrificed 4 h after mechanical ventilation, and lung tissues were collected for experimental assessments on lung injury.ResultsCompared with the healthy control, severe hyperoxia ventilation by either standard or high tidal volume resulted in significantly higher wet-to-dry lung weight ratio and higher levels of IL-1β and 8-OHdG in the lungs. However, moderate hyperoxia ventilation, even by high tidal volume did not significantly increase the levels of IL-1β and 8-OHdG in the lungs. Western blot analysis showed that the expression of RhoA, ROCK1, MLC2, and p-MLC2 was not significantly induced in the ventilated lungs, even by high tidal volume at 2 cmH2O PEEP.ConclusionSevere hyperoxia ventilation causes inflammatory response and oxidative damage in mechanically ventilated lungs, while high tidal volume ventilation at a reasonable PEEP possibly does not cause VILI.

CTNNAL1 deficiency suppresses CFTR expression in HDM-induced asthma mouse model through ROCK1-CAL signaling pathway.

The downregulation of adhesion molecule catenin alpha-like 1 (CTNNAL1) in airway epithelial cells of asthma patients and house dust mite (HDM)-induced asthma animal models was illustrated in our previous study. It is assumed to contribute to airway inflammation and mucus hypersecretion. In this work, we further explore the underlying mechanism of CTNNAL1 in asthma. CTNNAL1-silenced female mice exhibit a decreased level of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated and ATP-gated Cl - channel that correlates with mucus hypersecretion. Our previous study demonstrated that ROCK1 expression decreases but ROCK2 expression increases in the lungs of a CTNNAL1-silenced mouse model. Inhibition of ROCK1 leads to a reduction in CFTR expression in CTNNAL1-overexpressing and CTNNAL1-silenced human bronchial epithelial (HBE) cells. It has been reported that ROCK1 is a downstream target of RhoA and that activation of RhoA increases CFTR expression after CTNNAL1 deficiency in vitro and in vivo. The above results indicate that CTNNAL1 regulates CFTR expression through the ROCK1 pathway. In addition, the expression of CFTR-associated ligand (CAL) is increased after CTNNAL1 silencing, and immunoprecipitation results confirm the interaction between ROCK1 and CAL. Inhibition of CAL does not influence ROCK1 expression but increases CFTR expression in CTNNAL1-silenced HBE cells. These data suggest that CTNNAL1 deficiency decreases CFTR expression in the HDM-induced asthma mouse model through the ROCK1-CAL signaling pathway.

Transcriptome analysis of the effects of maternal fructose exposure during gestation and lactation on the cytoskeleton of offspring hippocampus

Excessive fructose consumption poses a public health concern. Maternal fructose overconsumption impairs offspring hippocampal function, indicating hippocampal sensitivity to fructose. The hippocampus is the vital for spatial learning and memory, and the cytoskeletal structure is crucial for hippocampal neuronal plasticity. The Rho GTPase is a key regulator of neuronal cytoskeleton formation, with RhoA protein governing neuronal growth and development. The RhoA/ROCK2 pathway affects myosin light chain phosphorylation, leading to cytoskeletal reorganization, actin cytoskeleton changes, and neurite retraction. However, the effects of maternal fructose exposure during gestation and lactation on the offspring's neural cytoskeleton remain unclear. We established maternal fructose exposure models during gestation and lactation, assessing fasting blood glucose levels in offspring confirm model effectiveness. Whole transcriptome sequencing examined offspring hippocampal developmental programming. Immunofluorescence and Western blot analyses assessed RhoA/ROCK2 pathway expression changes in the hippocampus, with PCR validating transcriptome sequencing results. Transcriptome sequencing identified 193 and 298 differentially expressed genes in the control, 13% fructose, and 40% fructose groups, respectively. GO and KEGG analyses revealed significant changes related to neural development, learning memory, and cytoskeleton in both fructose-exposed groups. GSEA indicated substantial cytoskeleton-related alterations in the offspring. Immunofluorescence demonstrated significant CRMP2 protein down-regulation in both fructose-exposed groups. Western blotting showed significantly higher expression of RhoA, ROCK2, p-ROCK2, and p-MLC2 in the fructose-exposed groups (P < 0.05). Maternal high-fructose exposure significantly impacts gene expression in offspring. These alterations may influence hippocampal neurodevelopment by up-regulating the RhoA/ROCK2 pathway, leading to cytoskeletal damage and axon growth inhibition.

Identification of ROCK1 as a novel biomarker for postmenopausal osteoporosis and pan-cancer analysis.

Postmenopausal osteoporosis (PMOP) is a prevalent bone disorder with significant global impact. The elevated risk of osteoporotic fracture in elderly women poses a substantial burden on individuals and society. Unfortunately, the current lack of dependable diagnostic markers and precise therapeutic targets for PMOP remains a major challenge. PMOP-related datasets GSE7429, GSE56814, GSE56815, and GSE147287, were downloaded from the GEO database. The DEGs were identified by "limma" packages. WGCNA and Machine Learning were used to choose key module genes highly related to PMOP. GSEA, DO, GO, and KEGG enrichment analysis was performed on all DEGs and the selected key hub genes. The PPI network was constructed through the GeneMANIA database. ROC curves and AUC values validated the diagnostic values of the hub genes in both training and validation datasets. xCell immune infiltration and single-cell analysis identified the hub genes' function on immune reaction in PMOP. Pan-cancer analysis revealed the role of the hub genes in cancers. A total of 1278 DEGs were identified between PMOP patients and the healthy controls. The purple module and cyan module were selected as the key modules and 112 common genes were selected after combining the DEGs and module genes. Five Machine Learning algorithms screened three hub genes (KCNJ2, HIPK1, and ROCK1), and a PPI network was constructed for the hub genes. ROC curves validate the diagnostic values of ROCK1 in both the training (AUC = 0.73) and validation datasets of PMOP (AUC = 0.81). GSEA was performed for the low-ROCK1 patients, and the top enriched field included protein binding and immune reaction. DCs and NKT cells were highly expressed in PMOP. Pan-cancer analysis showed a correlation between low ROCK1 expression and SKCM as well as renal tumors (KIRP, KICH, and KIRC). ROCK1 was significantly associated with the pathogenesis and immune infiltration of PMOP, and influenced cancer development, progression, and prognosis, which provided a potential therapy target for PMOP and tumors. However, further laboratory and clinical evidence is required before the clinical application of ROCK1 as a therapeutic target.

Fasudil and SR1001 synergistically protect against sepsis-associated pancreatic injury by inhibiting RhoA/ROCK pathway and Th17/IL-17 response

Sepsis is defined as a dysregulated host response to infection that can result in organ dysfunction and high mortality, which needs more effective treatment urgently. Pancreas is one of the most vulnerable organs in sepsis, resulting in sepsis-associated pancreatic injury, which is a fatal complication of sepsis. The aim of this study was to investigate the effect of combination of fasudil and SR1001 on sepsis-associated pancreatic injury and to explore the underlying mechanisms. The model of sepsis-associated pancreatic injury was induced by cecal ligation and puncture. Pancreatic injury was evaluated by HE staining, histopathological scores and amylase activity. The frequency of Th17 cells was analyzed by flow cytometry. Serum IL-17 level was determined by ELISA. Protein levels of RORγt, p-STAT3, GEF-H1, RhoA and ROCK1 were determined by Western blot. The apoptosis of pancreatic cells was examined by TUNEL analysis and Hoechst33342/PI staining. Compared to the sham group, the model group showed significant pathological injury including edema, hyperemia, vacuolization and necrosis. After treatment with fasudil, model mice showed an obvious reduction of Th17 cells and IL-17. SR1001 significantly reduced the expressions of GEF-H1, RhoA and ROCK1 in the model mice. The combination treatment with fasudil and SR1001 significantly inhibited the differentiation of Th17 cells, expressions of IL-17, GEF-H1, RhoA and ROCK1, which were more effective than each mono-treatment. In addition, our data revealed a remarkable decrease of apoptosis in pancreatic acinar cells culturing with fasudil or SR1001, which was further inhibited by their combination culture. Lipopolysaccharide remarkably upregulated the differentiation of Th17 cells in vitro, which could be significantly downregulated by fasudil or SR1001, and further downregulated by their combination treatment. Taken together, the combination of fasudil with SR1001 has a synergistic effect on protecting against sepsis-associated pancreatic injury in C57BL/6 mice.