Levels Of RhoA Research Articles

P75NTR prevents the onset of cerebellar granule cell migration via RhoA activation.

Neuronal migration is one of the fundamental processes during brain development. Several neurodevelopmental disorders can be traced back to dysregulated migration. Although substantial efforts have been placed in identifying molecular signals that stimulate migration, little is known about potential mechanisms that restrict migration. These restrictive mechanisms are essential for proper development since it helps coordinate the timing for each neuronal population to arrive and establish proper connections. Moreover, preventing migration away from a proliferative niche is necessary in maintaining a pool of proliferating cells until the proper number of neuronal progenitors is attained. Here, using mice and rats, we identify an anti-migratory role for the p75 neurotrophin receptor (p75NTR) in cerebellar development. Our results show that granule cell precursors (GCPs) robustly express p75NTR in the external granule layer (EGL) when they are proliferating during postnatal development, however, they do not express p75NTR when they migrate either from the rhombic lip during embryonic development or from the EGL during postnatal development. We show that p75NTR prevented GCP migration by maintaining elevated levels of active RhoA. The expression of p75NTR was sufficient to prevent the migration of the granule cells even in the presence of BDNF (brain-derived neurotrophic factor), a well-established chemotactic signal for this cell population. Our findings suggest that the expression of p75NTR might be a critical signal that stops and maintains the GCPs in the proliferative niche of the EGL, by promoting the clonal expansion of cerebellar granule neurons.

Ferulic acid alleviates sciatica by inhibiting peripheral sensitization through the RhoA/p38MAPK signalling pathway

BackgroundNonsteroidal anti-inflammatory drugs are used to relieve sciatica, but their effects are not satisfactory. PurposeThis study aimed to explore the therapeutic effects of ferulic acid on sciatica. MethodsThirty-two SD rats were randomly divided into 4 groups, i.e., sham operation group, chronic constriction injury (CCI) group, mecobalamin group, and ferulic acid group. We conducted behavioural tests, ELISA, PCR, Western blots, and immunofluorescence analysis. Specific inhibitors were used in cell experiments to explore the related mechanisms. ResultsThermal hyperalgesia was induced after CCI operation, and ferulic acid relieved thermal hyperalgesia. In addition, ferulic acid decreased the IL1β, IL6, TNF-α, and CRP mRNA levels; the IBA-1, iNOS, IL1β, RhoA, RhoA-GTP, COX2, Rock1, TRPV1, TRPA1, and p-p38MAPK levels in dorsal root ganglion (DRG) neurons; and the LPS, CRP, substance P (SP), and prostaglandin E2 (PGE2) levels in serum, and these levels were higher in the CCI group. In the cell experiments, LPS induced M1 polarization of GMI-R1 cells via the RhoA/Rock pathway. Ferulic acid attenuated LPS-induced M1 polarization by decreasing the levels of M1 polarization markers, including IL1β, IL6, TNF-α, iNOS, and CD32, and increased M2 polarization by increasing the levels of M2 polarization markers, including CD206 and Arg-1. LPS treatment clearly increased the iNOS, IL1β, RhoA, Rock1, Rock2 and p-p38 MAPK levels and reduced Arg-1 expression, and ferulic acid reversed these changes. ConclusionFerulic acid can inhibit peripheral sensitization by reducing the levels of inflammatory factors, TRPA1 and TRPV1 through the RhoA/p38 MAPK pathway to alleviate sciatica.

Exercise-induced signaling pathways to counteracting cardiac apoptotic processes.

Cardiovascular diseases are the most common cause of death in the world. One of the major causes of cardiac death is excessive apoptosis. However, multiple pathways through moderate exercise can reduce myocardial apoptosis. After moderate exercise, the expression of anti-apoptotic proteins such as IGF-1, IGF-1R, p-PI3K, p-Akt, ERK-1/2, SIRT3, PGC-1α, and Bcl-2 increases in the heart. While apoptotic proteins such as PTEN, PHLPP-1, GSK-3, JNK, P38MAPK, and FOXO are reduced in the heart. Exercise-induced mechanical stress activates the β and α5 integrins and subsequently, focal adhesion kinase phosphorylation activates the Akt/mTORC1 and ERK-1/2 pathways, leading to an anti-apoptotic response. One of the reasons for the decrease in exercise-induced apoptosis is the decrease in Fas-ligand protein, Fas-death receptor, TNF-α receptor, Fas-associated death domain (FADD), caspase-8, and caspase-3. In addition, after exercise mitochondrial-dependent apoptotic factors such as Bid, t-Bid, Bad, p-Bad, Bak, cytochrome c, and caspase-9 are reduced. These changes lead to a reduction in oxidative damage, a reduction in infarct size, a reduction in cardiac apoptosis, and an increase in myocardial function. After exercising in the heart, the levels of RhoA, ROCK1, Rac1, and ROCK2 decrease, while the levels of PKCε, PKCδ, and PKCɑ are activated to regulate calcium and prevent mPTP perforation. Exercise has an anti-apoptotic effect on heart failure by increasing the PKA-Akt-eNOS and FSTL1-USP10-Notch1 pathways, reducing the negative effects of CaMKIIδ, and increasing the calcineurin/NFAT pathway. Exercise plays a protective role in the heart by increasing HSP20, HSP27, HSP40, HSP70, HSP72, and HSP90 along with increasing JAK2 and STAT3 phosphorylation. However, research on exercise and factors such as Pim-1, Notch, and FAK in cardiac apoptosis is scarce, so further research is needed. Future research is recommended to discover more anti-apoptotic pathways. It is also recommended to study the synergistic effect of exercise with gene therapy, dietary supplements, and cell therapy for future research.

Shenqihuatan formula reduces inflammation by inhibiting transforming growth factor-beta-stimulated signaling pathway in airway smooth muscle cells.

To study the effects and mechanism of Shenqihuatan formula (, SQHT) of the transforming growth factor-beta (TGF-β)-stimulated cell processes in airway remodeling. The current study examined cell viability using a Cell Counting Kit-8 assay. Furthermore, a Transwell assay was conducted to detect the ability of cell migration, and apoptosis was detected via flowcytometry. Western Blot and quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine the expression levels of apoptosis or inflammation-related factors, such as TGF-β, Interleukin-1β (IL-1β), B cell lymphoma 2 (Bcl-2), Bcl-2-Associated X (Bax), Ras homolog gene family, member A (RhoA), recombinant rho associated coiled coil containing protein kinase 1/2 (ROCK1/2), extracellular regulated protein kinases 1/2 (ERK1/2), Snail, and Slug. Finally, the expression levels of matrix metalloproteinase-9 (MMP-9) and Tissue inhibitor of metalloproteinase (TIMP-1) were admeasured by enzyme-linked immuno sorbent assay. The results demonstrated that SQHT inhibited the viability and migration, as well as the the F-actin formation and cytoskeletal reorganization of airway smooth muscle cells (ASMCs) stimulated by TGF-β. By monitoring the changes of critical regulators in the presence of the formula, it was observed that the expression levels of TGF-β, IL-1β, Bcl-2, RhoA, ROCK1/2, ERK1/2, Snail, and Slug were markedly suppressed, whereas Bax expression exhibited the opposite effect. Compared with a well-characterized RhoA pathway inhibitor, Fasudil, SQHT generated equivalent or even higher inhibitory effects on these processes in ASMCs. Collectively, these suggested that SQHT can reduce airway inflammation by inhibiting TGF-β-stimulated signaling pathways in ASMCs. These findings may provide a novel remedy for treating ASMC inflammation, which causes thickening and obstruction of the airway in chronic obstructive pulmonary disease.

RhoA with Associated TRAb or FT3 in the Diagnosis and Prediction of Graves' Ophthalmopathy.

During Graves' disease (GD) treatment, Graves' ophthalmopathy (GO) is often ignored because only mild ocular symptoms are present in early GD. Therefore, we performed isobaric tags for relative and absolute quantification (iTRAQ) analysis and measured relevant endocrine hormones to identify predisposing factors of GO. Serum samples from 3 patients with mild GD and GO and 3 patients with GD but without GO were analyzed by iTRAQ. Based on their clinical data, 60 patients with GD were divided into the GO-free and GO groups. All patients were followed up for 7 months. Their eye conditions and changes in related biochemical indexes were recorded. The iTRAQ results showed that RhoA expression was upregulated and correlated significantly with the tight junction pathway and immunity. The changes in FT3 and RhoA from baseline to 7 months, the FT3 and RhoA baseline levels, and the TRAb titer levels in patients with GD significantly differed between the groups. ELISA and western blotting for RhoA, TRAb, and FT3 in the serum samples from GO patients showed significant upregulation, as well as elevated serum RhoA and TRAb levels in the mild stage of GO. At 7 months, the serum RhoA and FT3 levels were elevated. RhoA is a potential biomarker for mild GO. In GD patients, if an elevated serum RhoA level is accompanied by an elevated TRAb or FT3 level, GO is highly likely to occur, even when obvious ocular symptoms are absent.

Effect of electroacupuncture on colonic motility and RhoA and ROCK protein expression in IBS-D rats

To observe the effect of electroacupuncture (EA) of "Tianshu"(ST25) and "Shangjuxu"(ST37)on gastrointestinal motility, psychological abnormality and expression of RhoA and ROCK protein in rats with diarrhea predominant irritable bowel syndrome (IBS-D), so as to explore its underlying mechanism in improving IBS-D. Thirty-six male rats were randomly and equally divided into control, model, EA and medication groups (n=9 in each group). The IBS-D model with psychological abnormality was established by gavage of dinitrobenzene sulfonic acid (DNBS) + chronic restraint stress. EA (2 Hz/100 Hz, 0.3 mA) was applied to bilateral ST25 and ST37 for 20 min, once a day for 7 days. Rats of the medication group received gavage of pinaverium bromide solution (15 mg/kg), once a day for 7 days. The rats' food intake in 24 h, body mass and colonic contraction waves were recorded. The anhedonia-related behavior was measured using the sucrose consumption test. The elevated plus maze test (the open-arm residence time ratio) was used to assess the anxiety-like behavior. The small intestinal propulsion rate test was used to assess the intestinal motility. The expression levels of RhoA and ROCK proteins in the colonic tissue were measured by Western blot. After modeling, the body mass, food consumption, sucrose preference index, the open-arm residence time ratio andlatency of colonic contraction waves were significantly decreased (P<0.05), and the number of contraction waves, intestinal propulsive rate, and the expression levels of RhoA and ROCK proteins considerably increased (P<0.01，P<0.05) in the model group relevant to the control group. Following the interventions, the decrease of body mass, food consumption, sucrose preference index, open-arm residence time ratio and latency of contraction waves, and the increase of the contraction waves, intestinal propulsive rate, and the expression levels of RhoA and ROCK proteins were all reversed by both EA and medication (P<0.05，P<0.01). The effect of EA was significantly superior to that of medication in increasing the sucrose pre-ference index (P<0.05). EA can improve both colonic motility and psychological disorders in IBS-D rats with psychological disorder, which may be related to its function in down-regulating the expression of colonic RhoA and ROCK proteins.

Low Molecular Weight Sericin Enhances the In Vitro of Immunological Modulation and Cell Migration.

Sericin, a waste product of the silk textile industry, has favorable physicochemical and biological properties. In this study, we extracted a low molecular weight (MW) sericin (LMW-sericin; below 10 kDa) by a performing high-temperature and high-pressure method and confirmed the MW using matrix-assisted laser desorption ionization-time of flight and liquid chromatography–mass spectrometry. Furthermore, we determined its biological effects on macrophages and human adipose stem cells (hASCs) as cell models to investigate the biocompatibility, immunomodulation behavior, and potential signaling pathway-related wound healing via analyses of gene expression of focal adhesion and human cytokines and chemokines using quantitative real-time polymerase chain reaction and cytokine assay. LMW-sericin showed good biocompatibility both in macrophages and hASCs. Macrophages cultured with 0.1 mg/ml LMW-sericin displayed an improved inflammatory response shown by the upregulation of CXCL9, IL12A, BMP7, and IL10, which developed Th1 and Th2 balance. LMW-sericin also improved the differentiation of macrophages toward the M2 phenotype by significantly enhancing the expression of Arg-1, which is conducive to the repair of the inflammatory environment. Moreover, the gene expression of hASCs showed that LMW-sericin promoted the secretion of beneficial adhesion molecules that potentially activate the gene transcription of differentiation and migration in hASCs, as well as significantly enhanced the levels of PKCβ1, RhoA, and RasGFR1 as fruitful molecules in wound healing. These findings provide insights into LMW-sericin application as a potential biomaterial for wound management.

RHOA protein expression correlates with clinical features in gastric cancer: a systematic review and meta-analysis

BackgroundGastric cancer (GC) is one of the most fatal cancers worldwide and is generally only detected after it has progressed to an advanced stage. Since there is a lack of comprehensive data on RHOA protein expression of patients with GC, this study utilized a systematic review and meta-analysis to address the limitation. The objective of this meta-analysis was to link GC clinical features with RHOA protein high- vs. low-expressing patients with GC.MethodsThe PubMed and Web of Science were used for a systematic literature review of GC related to RHOA. The included studies were obtained from two literature databases from past to Aug 31, 2021, by searching keywords. This meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. The odds ratios (ORs) and 95% confidential intervals (CIs) for clinical features were estimated according to the high and low protein expression levels of RhoA. The mean effect sizes of ORs were obtained using the random-effects and fixed-effects models of meta-analysis. Heterogeneity of the studies was assesed by using statistics: τ2, I2; and Q values. The symmetry of funnel plots were inspected for publication bias.ResultsFinally, 10 studies including 1,389 patients with GC (735 RHOA-positive and 654 RHOA-negative) were eligible for our meta-analysis to estimate associations between the protein expression and clinical features (e.g., Union for International Cancer Control [UICC] stage progression, differentiation, Lauren histological classification, and vascular invasion). In our meta-analysis, RHOA positive expression was determined to have a statistically significant association with UICC stage progression (P = 0.02) and poorly differentiated status (P = 0.02). The association between RHOA positivity and Lauren subtypes was not statistically significant (P = 0.07).ConclusionsThis meta-analysis suggested that RhoA protein expression in patients with GC was associated with clinical features: UICC stage progression and poorly differentiated status. Our findings are inconclusive but indicate that high RHOA protein expressing patients with GC could predict advanced UICC stages. A large prospective cohort study is required for validation in future.

Selenium Deficiency Leads to Inflammation, Autophagy, Endoplasmic Reticulum Stress, Apoptosis and Contraction Abnormalities via Affecting Intestinal Flora in Intestinal Smooth Muscle of Mice.

Selenium (Se) is a micronutrient that plays a predominant role in various physiological processes in humans and animals. Long-term lack of Se will lead to many metabolic diseases. Studies have found that chronic Se deficiency can cause chronic diarrhea. The gut flora is closely related to the health of the body. Changes in environmental factors can cause changes in the intestinal flora. Our study found that Se deficiency can disrupt intestinal flora. Through 16s high-throughput sequencing analysis of small intestinal contents of mice, we found that compared with CSe group, the abundance of Lactobacillus, Bifidobacterium, and Ileibacterium in the low selenium group was significantly increased, while Romboutsia abundance was significantly decreased. Histological analysis showed that compared with CSe group, the small intestine tissues of the LSe group had obvious pathological changes. We examined mRNA expression levels in the small intestine associated with inflammation, autophagy, endoplasmic reticulum stress, apoptosis, tight junctions, and smooth muscle contraction. The mRNA levels of NF-κB, IκB, p38, IL-1β, TNF-α, Beclin, ATG7, ATG5, LC3α, BaK, Pum, Caspase-3, RIP1, RIPK3, PERK, IRE1, elF2α, GRP78, CHOP2, ZO-1, ZO-2, Occludin, E-cadherin, CaM, MLC, MLCK, Rho, and RhoA in the LSe group were significantly increased. The mRNA levels of IL-10, p62 BcL-2 and BcL-w were significantly decreased in the LSe group compared with the CSe group. These results suggest that changes in the abundance of Lactobacillus, bifidobacterium, ileum, and Romboutsia may be associated with cellular inflammation, autophagy, endoplasmic reticulum stress, apoptosis, tight junction, and abnormal smooth muscle contraction. Intestinal flora may play an important role in chronic diarrhea caused by selenium deficiency.

Porcine circovirus-like virus P1 activates pancreatic secretion pathway by interacting with CHRM3 protein

The porcine circovirus-like virus P1, a member of the circovirus family, causes post-weaning multisystemic wasting syndrome (PMWS) in weaned piglets with progressive wasting as the main clinical symptom. The pancreatic secretion pathway induces pancreatic acinar cells to secrete various digestive enzymes and as such is an important signaling pathway for the digestive system and somatic growth. This study examined the effects and mechanism of P1 virus infection on the pancreatic secretion pathway. The experiment was conducted by transfecting double-copy plasmid P1 into PK-15 and 3D4 cells and by infecting cells with the P1 virus. Samples were collected at various times after transfection or infection. The pathway's transcription and translation levels of CHRM3, Gq, PLC-β2, PRKCA, Rab3D, RhoA, Rac1, and amyA proteins were detected by real-time PCR and Western blots; these analyses confirmed that the P1 virus infection could upregulate the expression level of key pancreatic secretion signaling molecules. Then, we confirmed that the VP1 protein of the P1 virus could interact with the pathway initiation protein CHRM3 using Co-IP, pull-downs, and confocal fluorescence microscopy. Finally, we demonstrated that the VP1 protein activates the pancreatic secretory pathway through the CHRM3 protein. In conclusion, this study demonstrated that the P1 virus can interact with the CHRM3 receptor protein to activate the pancreatic secretion pathway and promote the secretion of various digestive enzymes downstream of the pathway, thereby providing a basis for P1 virus pathogenesis.

Targeting Ferroptosis Attenuates Inflammation, Fibrosis, and Mast Cell Activation in Chronic Prostatitis.

Purpose Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a common urological disorder. Although ferroptosis is closely associated with inflammation, oxidative stress, and neuropathic pain, its role in CP/CPPS has not yet been elucidated. Therefore, we sought to explore the role and mechanism of ferroptosis in the prostatitis development. Methods The experimental autoimmune prostatitis (EAP) was established through intradermal immunization of prostate extract. Iron chelator deferoxamine (DFO) and free radical scavenger edaravone (EDA) were applied to evaluate the effects of ferroptosis inhibition on oxidative stress, ferroptosis, inflammation, fibrosis, and mast cell activation in the context of CP/CPPS. Results Increased generation of lipid peroxidation products (ROS and MDA) and decreased activities of antioxidant enzymes (SOD and CAT) suggested an aberrant oxidative stress status in EAP model. Elevated iron concentration was observed in the EAP model. Meanwhile, we discovered significant biological performances associated with ferroptosis in CP/CPPS, including the downregulation of the system Xc−/GPX4 axis and the upregulation of the ACSL4/LPCAT3 axis. EAP rats performed serious leukocyte infiltration, advanced inflammatory grade, and abnormal expression of inflammatory mediators. Abundant collagen deposition, enhanced RhoA, ROCK1, and α-SMA protein levels indicated that EAP rats were prone to suffer from stromal fibrosis compared with control group. An elevated number of degranulated mast cells and corresponding marker TPSB2 represented that mast cell-sensitized pain was amplified in the EAP model. Furthermore, reduction of NRF2/HO-1 indicated a vulnerability of EAP towards ferroptosis response. However, application of DFO and EDA had partially reversed the adverse influences mentioned above. Conclusion We first demonstrated that ferroptosis might be a crucial factor of chronic prostatitis progression. Inhibition of ferroptosis using DFO and EDA represented a promising approach for treating prostatitis by ameliorating inflammation, fibrosis, and mast cell activation.

Rho GTPase-activating protein 35 suppresses gastric cancer metastasis by regulating cytoskeleton reorganization and epithelial-to-mesenchymal transition

ABSTRACT Cytoskeletal reorganization and epithelial-to-mesenchymal transition (EMT) are key processes and typical characteristics of metastatic cancer cells. Rho GTPase‑activating protein 35 (ARHGAP35) is a GTPase-activating protein, which has a significant effect on cell motility. However, the particular function of ARHGAP35 in gastric cancer (GC) remains unknown. In the present study, the role of ARHGAP35 in GC was investigated by in vitro loss-of-function and gain-of-function experiments. Cytoskeletal reorganization in GC cells was evaluated using immunofluorescence staining and the protein expression levels of key molecules and active RhoA were detected by western blot analysis. Additionally, the clinical evaluation of proteins in human GC tissues was assessed by immunohistochemistry. The results showed that ARHGAP35, a tumor suppressor, was downregulated in GC tissues and its decreased expression was associated with the metastatic status of GC. Additionally, Transwell and wound healing assays demonstrated that ARHGAP35 knockdown promoted cell motility in vitro. However, the above effects were abrogated following ectopic ARHGAP35 expression. Furthermore, ARHGAP35 could affect cytoskeletal reorganization via directly regulating RhoA activation. In addition, ARHGAP35 upregulated E-cadherin and attenuated EMT in GC cells. Both ARHGAP35 and E-cadherin were associated with overall survival in patients with GC, while their combination allowed for an even greater capacity for distinguishing GC patients with different prognosis. Overall, the results of the current study suggested that ARHGAP35 could directly regulate cell morphology and motility via affecting cytoskeletal reorganization and EMT via targeting RhoA and E-cadherin, respectively. Targeting the ARHGAP35/RhoA/E-cadherin pathway could be a potential approach for treating GC.

Simvastatin alleviates epithelial-mesenchymal transition and oxidative stress of high glucose-induced lens epithelial cells in vitro by inhibiting RhoA/ROCK signaling.

Diabetic cataracts (DC) is one of the main causes of blindness among patients with diabetes mellitus. The aim of the present study was to examine the effect of simvastatin on lens epithelial cells in DC and the underlying mechanism. The viability of SRA01/04 cells treated with different concentrations of simvastatin was detected using a Cell Counting Kit-8 assay before and after high glucose (HG) treatment. The expression levels of E-cadherin, N-cadherin, Vimentin and α-smooth muscle actin (α-SMA), proteins associated with epithelial-mesenchymal transition (EMT), in addition to RhoA, Rho-associated kinases (ROCK)1 and ROCK2, proteins related to RhoA/ROCK signaling, were also measured in SRA01/04 cells treated with HG and simvastatin, with or without U46619, using western blot analysis. DCFH-DA dyes, superoxide dismutase (SOD) and glutathione (GSH)/glutathione disulfide (GSSG) kits were used to measure the levels of oxidative stress parameters in SRA01/04 cells treated with HG and simvastatin with or without U46619. The cell viability of SRA01/04 cells treated with simvastatin was found to be significantly elevated after HG treatment. The protein expression levels of E-cadherin were increased but those of N-cadherin, Vimentin and α-SMA decreased after HG and simvastatin treatment, and this was reversed by U46619. The levels of SOD and GSH-GSSG were found to be increased whereas reactive oxygen species levels were decreased, effects that were reversed by U46619. Additionally, the protein expression levels of RhoA, ROCK1 and ROCK2 were markedly decreased. These findings provided evidence that simvastatin increased HG-induced SRA01/04 cell viability and exerted inhibitory effects on EMT and oxidative stress that occurs during DC.

Dietary patterns in association with the expression of pro-metastatic genes in primary breast cancer.

Metastasis is a major leading cause of mortality in female breast cancer (BrCa). Cellular motility is a pathological process of metastasis remarked by the overexpression of cortactin (CTTN), Ras homolog family member-A (RhoA), and Rho-associated kinase (ROCK) genes.Their balanceisresponsible forupholding the integrity of healthy epithelial cell junctions. This study aimed to explore the associations between a posteriori dietary patterns and theexpression levels of pro-metastatic genes in primary BrCa. In this consecutive case series, 215 eligible women, newly diagnosed with histologically confirmednon-metastatic BrCa (stage I-IIIA), were recruited from Hospitals in Tabriz, NorthwesternIran (2015-2017). The tumoral expression levels of genes were quantified using real-time reverse transcription-polymerase chain reaction. Dietary data assessment was carried out using a validated food frequency questionnaire. Three dietary patterns were identified using principal component analysis (KMO = 0.699). Adherence to the "vegan" pattern (vegetables, fruits, legumes, nuts, seeds, and whole grains) was inversely associated with the expression levels of RhoA (ORAdj.T3vs.T1 = 0.24, 95%CI 0.07-0.79) and ROCK (ORAdj.T3vs.T1 = 0.26, 95%CI 0.08-0.87). In addition, the highest adherence to the "prudent" pattern (spices, seafood, dairy, and vegetable oils) decreased the odds of overexpressions at RhoA (ORAdj.T3vs.T1 = 0.26, 95%CI 0.08-0.84) and ROCK genes (ORAdj.T3vs.T1 = 0.29, 95%CI 0.09-0.95). The highest adherence to "Western" pattern (meat, processed meat, hydrogenated fat, fast food, refined cereals, sweets, and soft drinks) was a risk factor associated with the overexpression of RhoA (ORAdj.T3vs.T1 = 3.15, 95%CI 1.12-8.85). Adherence to healthy dietary patterns was significantly associated with the downregulation of pro-metastatic genes. Findings provided new implications to advance the nutrigenomic knowledge to prevent the odds ofover-regulations in pro-metastatic genes of theprimary BrCa.

Potential effect of amniotic fluid-derived stem cells on hyperoxia-induced pulmonary alveolar injury

BackgroundWith the widespread of Coronavirus Disease 2019 pandemic, in spite of the newly emerging vaccines, mutated strains remain a great obstacle to supportive and preventive measures. Coronavirus 19 survivors continue to face great danger of contacting the disease again. As long as no specific treatment has yet to be approved, a great percentage of patients experience real complications, including among others, lung fibrosis. High oxygen inhalation especially for prolonged periods is per se destructive to the lungs. Nevertheless, oxygen remains the first line support for such patients. In the present study we aimed at investigating the role of amniotic fluid-mesenchymal stem cells in preventing versus treating the hyperoxia-induced lung fibrosis in rats.MethodsThe study was conducted on adult albino rats; 5 pregnant female rats were used as amniotic fluid donors, and 64 male rats were randomly divided into two groups: Control group; where 10 rats were kept in normal atmospheric air then sacrificed after 2 months, and hyperoxia-induced lung fibrosis group, where 54 rats were exposed to hyperoxia (100% oxygen for 6 h/day) in air-tight glass chambers for 1 month, then randomly divided into the following 5 subgroups: Hyperoxia group, cell-free media-treated group, stem cells-prophylactic group, stem cells-treated group and untreated group. Isolation, culture and proliferation of stem cells were done till passage 3. Pulmonary function tests, histological examination of lung tissue under light and electron microscopes, biochemical assessment of oxidative stress, IL-6 and Rho-A levels, and statistical analysis of data were performed. F-test (ANOVA) was used for normally distributed quantitative variables, to compare between more than two groups, and Post Hoc test (Tukey) for pairwise comparisons.ResultsLabelled amniotic fluid-mesenchymal stem cells homed to lung tissue. Stem cells administration in the stem cells-prophylactic group succeeded to maintain pulmonary functions near the normal values with no significant difference between their values and those of the control group. Moreover, histological examination of lung tissues showed that stem cells-prophylactic group were completely protected while stem cells-treated group still showed various degrees of tissue injury, namely; thickened interalveolar septa, atelectasis and interstitial pneumonia. Biochemical studies after stem cells injection also showed decreased levels of RhoA and IL-6 in the prophylactic group and to a lesser extent in the treated group, in addition to increased total antioxidant capacity and decreased malondialdehyde in the stem cells-injected groups.ConclusionsAmniotic fluid-mesenchymal stem cells showed promising protective and therapeutic results against hyperoxia-induced lung fibrosis as evaluated physiologically, histologically and biochemically.

Effect of cadmium on Rho GTPases signal transduction during osteoclast differentiation.

Osteoclasts are the key target cells for cadmium (Cd)-induced bone metabolism diseases, while Rho GTPases play an important role in osteoclast differentiation and bone resorption. To identify new therapeutic targets of Cd-induced bone diseases; we evaluated signal transduction through Rho GTPases during osteoclast differentiation under the influence of Cd. In osteoclastic precursor cells, 10nM Cd induced pseudopodia stretching, promoted cell migration, upregulated the levels of Cdc42, and RhoQ mRNAs and downstream Rho-associated coiled-coil kinase 1 (ROCK1) and ROCK2 proteins, and downregulated the actin-related protein 2/3 (ARP2/3) levels. Cd at 2 and 5μM shortened the pseudopodia, inhibited cell migration, and decreased ROCK1, ROCK2, and ARP2/3 protein levels; Cd at 5μM also reduced the mRNA expression levels of Rac1, Rac2, and RhoU mRNAs and decreased the level of phosphorylated (p)-cofilin. In osteoclasts, 10nM Cd induced the formation of sealing zones, slightly upregulated Cdc42 mRNA levels and ROCK2 and ARP2/3 protein levels and significantly reduced p-cofilin levels. Cd at 2μM and 5μM Cd blocked the fusion of precursor cells; and 5μM Cd downregulated the expression levels of RhoB, Rac1, Rac3, and RhoU mRNAs, and ROCK1, p-cofilin and ARP2/3 protein levels, significantly. In vivo, Cd (at 5 or 25 mg/L) increased the levels of key proteins RhoA, Rac1/2/3, Cdc42, and RhoU and their mRNAs in bone marrow cells. In summary, the results suggested that Cd affected the differentiation process of osteoclast and altered the expression of several Rho GTPases, which might be crucial targets of Cd during the differentiation of osteoclasts.

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.

Oxycodone relieves permeability damage and apoptosis of oxygen-glucose deprivation/reoxygenation-induced brain microvascular endothelial cells through ras homolog family member A (RhoA)/ Rho-associated coiled-coil containing kinases (ROCK)/ myosin light chain 2 (MLC2) signal

ABSTRACT Cerebrovascular disease, an important cause of acute ischemic stroke, has attracted worldwide attention. Oxycodone has been widely used to treat various painful disorders. This study was designed to explore the mechanism of oxycodone in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced brain microvascular endothelial cell model. For the reliability of the results in the following experiments, the viability was firstly detected using CCK-8. With the application of LDH, TEER and TUNEL assays, the LDH expression, permeability and apoptosis of brain microvascular endothelial cells were detected, respectively. Besides, the mRNA and protein expressions of tight junction proteins and RhoA were measured using RT-qPCR and Western blot. Moreover, RT-qPCR was employed to evaluate the expressions of inflammatory cytokines. Western blot was adopted to measure the levels of RhoA, ROCK, MLC2 and apoptosis-related proteins. The results revealed that oxycodone attenuated permeability damage, inflammatory factor release and apoptosis of OGD/R-induced brain microvascular endothelial cells in a dose-dependent manner. It was also found that oxycodone could reduce the expressions of RhoA, ROCK and MLC2 in brain microvascular endothelial cells induced by OGD/R. More importantly, oxycodone exhibited desirable effects on OGD/R-induced brain microvascular endothelial cells through RhoA/ROCK/MLC2 signal. In conclusion, oxycodone relieved permeability damage and apoptosis of OGD/R-induced brain microvascular endothelial cells through RhoA/ROCK/MLC2 signal, suggesting that oxycodone might be an effective method for the improvement of cerebral ischemia-reperfusion injury.

Wnt4 regulates bone metabolism through IKK-NF-κB and ROCK signaling under occlusal traumatic periodontitis.

Occlusal trauma is one of the most important local contributing factors of periodontitis. It has been reported that Wnt4, a noncanonical Wnt ligand, can inhibit osteoclast formation and inflammation and promote bone formation in vivo. However, the prospects of Wnt4 application in occlusal trauma and periodontitis have not yet been described. This study aimed to investigate the function and the corresponding mechanism of Wnt4 to regulate bone metabolism in occlusal trauma and periodontitis. Osteogenic-induced MC3T3-E1 cells were treated with or without Porphyromonas gingivalis lipopolysaccharide (Pg. LPS) under cyclic uniaxial compressive stress. After treatment with mouse recombinant protein Wnt4 (rWnt4), the expression of osteogenic markers and activation of the IKK-NF-κB signaling pathway were evaluated in vitro. To investigate whether Wnt4 can promote osteogenesis via the ROCK signaling pathway, the expression of RhoA was evaluated in vitro. Finally, we evaluated the change in bone quantity and the activation of the IKK-NF-κB and ROCK signaling in mice with occlusal trauma and periodontitis to demonstrate the therapeutic efficacy of rWnt4 injection. Stimulation of traumatic force and Pg. LPS stimulation suppressed the expression of osteoblast markers, but their expression was rescued after rWnt4 treatment in vitro. In addition, the inhibition of the ROCK signaling pathway induced by force loading was reversed when rWnt4 was applied in vitro. Micro-CT, H&E, and TRAP staining of the mandibles showed increased bone loss in the occlusal trauma-aggravated periodontitis group, whereas it was rescued after rWnt4 injection. The expression levels of IκBα and p65 were upregulated in occlusal trauma and periodontitis-bearing mice, whereas the expression levels of Runx2 and RhoA were downregulated. After rWnt4 injection, remarkably upregulation of Runx2 and RhoA expression was observed in occlusal trauma and periodontitis- bearing mice. Wnt4 not only inhibits IKK-NF-κB signaling but also activates ROCK signaling to inhibit osteoclast formation and promote bone regeneration in occlusal trauma and periodontitis-bearing mice.

Mechanism study of the protective effects of selective cyclooxygenase-2 enzyme inhibitors on the liver of rats with type 2 diabetes mellitus combined with nonalcoholic steatohepatitis via Rho/ROCK pathway

Objective: To investigate whether the selective cyclooxygenase-2 enzyme inhibitors celecoxib has protective effect on the liver of rats with type 2 diabetes mellitus (T2DM) combined with nonalcoholic steatohepatitis (NASH) via inhibiting the expression of Rho/ROCK pathway. Methods: Forty male SD rats were randomly divided into four groups: type 2 diabetes mellitus combined with nonalcoholic steatohepatitis (T2DM-NASH) group, T2DM-NASH + celecoxib group, control group, and control+celecoxib group. The T2DM-NASH and T2DM-NASH + celecoxib groups were fed with high-sugar and fat diet, and the control group and control + celecoxib group were fed with basal diet (25 kJ/kg). Four weeks later, streptozotocin (STZ, 30 mg/kg) was intraperitoneally injected into the NASH group and T2DM-NASH + celecoxib group to induce T2DM model, and the control group and control + celecoxib group were intraperitoneally injected with isovolumic citric acid-sodium citrate buffer. Four weeks after STZ injection, the T2DM-NASH + celecoxib group and the control + celecoxib group were gavaged with celecoxib (10 mg·kg·d) dissolved in normal saline for 4 weeks, and the remaining two groups of rats were gavaged with isovolumic normal saline for 4 weeks. Animals were sacrificed at the end of the 12- weeks, and the liver tissue was collected. Liver pathological changes were observed by HE staining. The expressions of RhoA, RhoA, ROCK1 and ROCK2 proteins in liver were detected by immunohistochemistry and western blot. The expressional condition of RhoA, ROCK1 and ROCK2 mRNA in liver were detected by real-time quantitative PCR. The differences were compared between protein and mRNA expression among the groups by analysis of variance and t-test. Results: Compared with the control group and the control + celecoxib group, the liver tissue of the T2DM-NASH group and the T2DM-NASH + celecoxib group had severe steatosis, and there was partial inflammatory cell infiltration under the light microscope. The expression levels of RhoA, ROCK1 and ROCK2 protein and mRNA were significantly increased (P < 0.05) in each liver tissue, while liver steatosis was reduced to certain extent in T2DM-NASH + celecoxib group than T2DM-NASH group, and the expression levels of RhoA, ROCK1 and ROCK2 protein and mRNA were decreased in each liver tissue of T2DM-NASH group (P < 0.05). Conclusion: The selective cyclooxygenase-2 enzyme inhibitors celecoxib has a protective effect on the liver of rats with T2DM-NASH, and its effect may be achieved by inhibiting the expression of Rho/ROCK pathway.