Expression Of RhoA Research Articles

Astragalus (Astragalus mongholicus) Improves Ventricular Remodeling via ESR1 Downregulation RhoA/ROCK Pathway.

Astragalus (Astragalus mongholicus) alleviates myocardial remodeling caused by hypertension. However, the detailed molecular mechanism is unclear. This study aims to investigate the effect of Astragalus on ventricular remodeling in ovariectomized spontaneous hypertensive rats (OVX-SHR).Female SHR/NCrl rats were subjected to bilateral ovariectomy to establish the OVX-SHR model and treated with Astragalus extract by gavage. The hemodynamics and cardiac function parameters were measured. HE and Masson staining were used to detect the pathological structure of myocardial remodeling and observe the hyperplasia of myocardial collagen fibers. The immunohistochemistry tested the level of α-SMA. The expression levels of inflammatory cytokines, IκB, p65, Cleaved-Caspase3, RhoA, and ROCK1/2 were detected using Western blot. The method of qRT-PCR measured the expression of matrix metalloproteinase (MMP-2 and MMP-9).Hemodynamic and cardiac function parameters were significantly improved after a high dose of Astragalus extract and Valsartan treatment. The myocardial integrity of the model group was significantly reduced, arranged loosely, and disordered, while the expression of α-SMA was increased. However, Astragalus extract and Valsartan treatments significantly reduced the pathological damage and α-SMA. The levels of TNF-α, IL-1β, IL-6, TGF-β, MMP-2, and MMP-9 in the model group were increased but decreased after Astragalus extract treatment. Adding an ESR1 inhibitor attenuated the improvement effect of Astragalus extract on myocardial remodeling and restored the expression of RhoA and ROCK1/2.Astragalus extract attenuates the cardiac damage in OVX-SHR by downregulating the RhoA/ROCK pathway through ESR1.

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.

The combination of phosphoinositol with inositol suppresses colorectal cancer growth and metastasis by regulating CLDN23/ RhoA /ROCK1 pathway

Colorectalcancerisa prevalentmalignanttumor, with liver metastases being the primary cause of death. The combination of inositol hexaphosphate (IP6) and inositol (INS) has been shown to inhibit colorectal cancer metastasis.We identified a correlation between CLDN23 expression and the occurrence of liver metastases in colorectal cancer patients. Following the transfection with the lentiviral vector (LV-CLDN23-RNAi), proteomic analysis of SW620 revealed significant alterations in the occludin, ZO-1, RhoA, and ROCK-1 genes expression by western blotting. We found that both SW620 and SW480 cells showed inhibited proliferation, invasion, and migration ability after the treatment with IP6 and INS. Additionally, this combination upregulated occludin and ZO-1, while downregulated CLDN23, RhoA, ROCK-1, and p-MLC2. In conclusion, the combination of IP6 and INS suppressed the proliferation, invasion, and migration of colorectal cancer cells and regulated CLDN23 expression by enhancing tightjunction proteins, such as occludin and ZO1, via the RhoA /ROCK1 pathway.

Pinocembrin attenuates susceptibility to atrial fibrillation in rats with pulmonary arterial hypertension

BackgroundPulmonary arterial hypertension (PAH) is a disease characterized by pulmonary vascular remodeling that triggers fibrosis and excessive myocardium apoptosis, ultimately facilitating atrial fibrillation (AF). In various rat models, Pinocembrin has anti-fibrotic and anti-apoptotic effects, reducing arrhythmia vulnerability. However, whether pinocembrin alleviates to AF in a PAH model remains unclear. The experiment aims to investigate how pinocembrin affects AF susceptibility in PAH rats and the possible mechanisms involved. MethodsThe PAH model was induced by monocrotaline (MCT; i. p. 60 mg/kg). Concurrently, rats received pinocembrin (i.p.50 mg/kg) or saline. Hemodynamics parameters, electrocardiogram parameters, lung H.E. staining, atrial electrophysiological parameters, histology, Western blot, and TUNEL assay were detected. ResultsCompared to the control rats, MCT-induced PAH rats possessed prominently enhancive mPAP (mean pulmonary artery pressure), pulmonary vascular remodeling, AF inducibility, HRV, right atrial myocardial fibrosis, apoptosis, atrial ERP, APD, and P-wave duration. Additionally, there were lowered protein levels of Cav1.2, Kv4.2, Kv4.3, and connexin 40 (CX40) in the MCT group in right atrial tissue. However, pinocembrin reversed the above pathologies and alleviated the activity of the Rho A/ROCKs signaling pathway, including the expression of Rho A, ROCK1, ROCK2, and its downstream MYPT-1, LIMK2, BCL-2, BAX, cleaved-caspase3 in right atrial and HL-1 cells. ConclusionPresent data exhibited pinocembrin attenuated atrial electrical, ion-channel, and autonomic remodeling, diminished myocardial fibrosis and apoptosis levels, thereby reducing susceptibility to AF in the MCT-induced PAH rats. Furthermore, we found that pinocembrin exerted inhibitory action on the Rho A/ROCK signaling pathway, which may be potentially associated with its anti-AF effects.

Mechanism of RhoA regulating benign prostatic hyperplasia: RhoA-ROCK-β-catenin signaling axis and static & dynamic dual roles

BackgroundThe pathogenesis of benign prostatic hyperplasia (BPH) has not been fully elucidated. Ras homology family member A (RhoA) plays an important role in regulating cell cytoskeleton, growth and fibrosis. The role of RhoA in BPH remains unclear.MethodsThis study aimed to clarify the expression, functional activity and mechanism of RhoA in BPH. Human prostate tissues, human prostate cell lines, BPH rat model were used. Cell models of RhoA knockdown and overexpression were generated. Immunofluorescence staining, quantitative real time PCR (qRT-PCR), Western blotting, cell counting kit-8 (CCK-8), flow cytometry, phalloidine staining, organ bath study, gel contraction assay, protein stability analysis, isolation and extraction of nuclear protein and cytoplasmic protein were performed.ResultsIn this study we found that RhoA was localized in prostate stroma and epithelial compartments and was up-regulated in both BPH patients and BPH rats. Functionally, RhoA knockdown induced cell apoptosis and inhibited cell proliferation, fibrosis, epithelial-mesenchymal transformation (EMT) and contraction. Consistently, overexpression of RhoA reversed all aforementioned processes. More importantly, we found that β-catenin and the downstream of Wnt/β-catenin signaling, including C-MYC, Survivin and Snail were up-regulated in BPH rats. Downregulation of RhoA significantly reduced the expression of these proteins. Rho kinase inhibitor Y-27632 also down-regulated β-catenin protein in a concentration-dependent manner. However, overexpression of β-catenin did not affect RhoA-ROCK levels, suggesting that β-catenin was the downstream of RhoA-ROCK regulation. Further data suggested that RhoA increased nuclear translocation of β-catenin and up-regulated β-catenin expression by inhibiting its proteasomal degradation, thereby activating Wnt/β-catenin signaling. Overexpression of β-catenin partially reversed the changes in cell growth, fibrosis and EMT except cell contraction caused by RhoA downregulation. Finally, Y-27632 partially reversed prostatic hyperplasia in vivo, further suggesting the potential of RhoA-ROCK signaling in BPH treatment.ConclusionOur novel data demonstrated that RhoA regulated both static and dynamic factors of BPH, RhoA-ROCK-β-catenin signaling axis played an important role in the development of BPH and might provide more possibilities for the formulation of subsequent clinical treatment strategies.

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.

M4IDP stimulates ROS elevation through inhibition of mevalonate pathway and pentose phosphate pathway to inhibit colon cancer cells

Maintaining redox homeostasis is an essential feature of cancer cells, and disrupting this homeostasis to cause oxidative stress and induce cell death is an important strategy in cancer therapy. M4IDP, a zoledronic acid derivative, can cause the death of human colorectal cancer cells by increasing the level of intracellular reactive oxygen species (ROS). However, its potential molecular mechanism is unclear. Our in vitro studies showed that treatment with M4IDP promoted oxidative stress in HCT116 cells, as measured by the decreased ratios of GSH/GSSG and NADPH/NADP+ and increased level of MDA. M4IDP could cause the decrease of GSH content, the increase of GSSG content, the decrease of NADPH content and pentose phosphate pathway flux, the downregulation of G6PD expression, the upregulation of unprenylated Rap1A and total expression of RhoA and CDC42. The increase of ROS and cytotoxicity induced by M4IDP could be reversed by the supplementation of NADPH, the overexpression of G6PD and the supplementation of GGOH. In vivo studies showed that M4IDP inhibited tumor growth in the human colorectal cancer xenograft mouse model, which was accompanied with a decreased [18F]FDG uptake. Collectively, these results provide evidence that M4IDP can promote oxidation in colon cancer cells by inhibiting mevalonate pathway and pentose phosphate pathway and produce therapeutic effect. This study revealed for the first time a possible mechanism of bisphosphonate-induced increase of ROS in malignant tumor cells. This is helpful for the development of new molecular therapeutic targets and can provide new ideas for the combined therapy of bisphosphonates in tumors.

PAR1 regulates sepsis-induced vascular endothelial barrier dysfunction by mediating ERM phosphorylation via the RhoA/ROCK signaling pathway

Sepsis begins with vascular endothelial barrier breakdown and causes widespread organ failure. Protease-activated receptor 1 (PAR1) is an important target for modulating vascular endothelial permeability; however, little research has been undertaken in sepsis, and its putative molecular mechanism remains unknown. The vascular endothelial permeability was examined by detecting FITC-dextran flux. F-actin was examined by immunofluorescence (IF). PAR1, ERM phosphorylation, and RhoA/ROCK signaling pathway expression in lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs) line were examined by IF and Western blot. To develop the sepsis model, cecal ligation and puncture (CLP) were conducted. The PAR1 inhibitor SCH79797 was utilized to inhibit PAR1 expression in vivo. Vascular permeability in main organs weres measured by Evans blue dye extravasation. The pathological changes in main organs were examined by HE staining. The expression of PAR1, ERM phosphorylation, and the RhoA/ROCK signaling pathway was examined using IF, immunohistochemical and WB in CLP mice. In vitro, in response to LPS stimulation of HUVECs, PAR1 mediated the phosphorylation of ERM, promoted F-actin rearrangement, and increased endothelial hyperpermeability, all of which were prevented by inhibiting PAR1 or RhoA. Additionally, inhibiting PAR1 expression reduced RhoA and ROCK expression. In vivo, we showed that inhibiting PAR1 expression will reduce ezrin/radixin/moesin (ERM) phosphorylation to relieve vascular endothelial barrier dysfunction and thereby ameliorate multiorgan dysfunction syndrome (MODS) in CLP-induced septic mice. This study revealed that PAR1-mediated phosphorylation of ERM induced endothelial barrier dysfunction, which in turn led to MODS in sepsis, and that the RhoA/ROCK signaling pathway underlay these effects.

Forebrain excitatory neuron-specific loss of Brpf1 attenuates excitatory synaptic transmission and impairs spatial and fear memory.

Bromodomain and plant homeodomain (PHD) finger containing protein 1 (Brpf1) is an activator and scaffold protein of a multiunit complex that includes other components involving lysine acetyltransferase (KAT) 6A/6B/7. Brpf1, KAT6A, and KAT6B mutations were identified as the causal genes of neurodevelopmental disorders leading to intellectual disability. Our previous work revealed strong and specific expression of Brpf1 in both the postnatal and adult forebrain, especially the hippocampus, which has essential roles in learning and memory. Here, we hypothesized that Brpf1 plays critical roles in the function of forebrain excitatory neurons, and that its deficiency leads to learning and memory deficits. To test this, we knocked out Brpf1 in forebrain excitatory neurons using CaMKIIa-Cre. We found that Brpf1 deficiency reduced the frequency of miniature excitatory postsynaptic currents and downregulated the expression of genes Pcdhgb1, Slc16a7, Robo3, and Rho, which are related to neural development, synapse function, and memory, thereby damaging spatial and fear memory in mice. These findings help explain the mechanisms of intellectual impairment in patients with BRPF1 mutation.

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.

Crosstalk between integrin/FAK and Crk/Vps25 governs invasion of bovine mammary epithelial cells by S. agalactiae

Streptococcus agalactiae (S.agalactiae) is a contagious obligate parasite of the udder in dairy cows. Here, we examined S.agalactiae-host interactions in bovine mammary epithelial cells (BMECs) invitro. We found that S.agalactiae infected BMECs through laminin β2 and integrin. Crk, Vps25, and RhoA were differentially expressed in S.agalactiae-infected cells. S.agalactiae infection activated FAK and Crk. FAK deficiency decreased the number of intracellular S.agalactiae and Crk activation. Knockdown of Crk or Vps25 increased the level of intracellular S.agalactiae, whereas its overexpression had the opposite effect. RhoA expression and actin cytoskeleton were altered in S.agalactiae-infected BMECs. Crk and Vps25 interact in cells, and invaded S.agalactiae also activates Crk, allowing it to cooperate with Vps25 to defend against intracellular infection by S.agalactiae. This study provides insights into the mechanism by which intracellular infection by S.agalactiae is regulated in BMECs.

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.

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.

MiR-155 enhances phagocytosis of alveolar macrophages through the mTORC2/RhoA pathway.

Alveolar macrophage phagocytosis is significantly reduced in Chronic obstructive pulmonary disease, and cigarette smoke extract is one of the chief reasons for this decrease. Nevertheless, the specific underlying mechanism remains elusive. In this study, the role and possible mechanism of miR-155-5p/mTORC2/RhoA in the phagocytosis of mouse alveolar macrophages (MH-S) were explored. Our results revealed that cigarette smoke extract intervention reduced MH-S cell phagocytosis and miR-155-5p expression. Meanwhile, the dual-luciferase reporter assay validated that Rictor is a target of miR-155-5p. On the one hand, transfecting miR-155-5p mimic, mimic NC, miR-155-5p inhibitor, or inhibitor NC in MH-S cells overexpressing miR-155-5p increased the Alveolar macrophage phagocytotic rate, up-regulated the expression level of RhoA and p-RhoA, and down-regulated that of mTOR and Rictor mRNA and protein. On the other hand, inhibiting the expression of miR-155-5p lowered the phagocytotic rate, up-regulated the expression of mTOR, Rictor mRNA, and protein, and down-regulated the expression of RhoA and p-RhoA, which taken together, authenticated that miR-155-5p participates in macrophage phagocytosis via the mTORC2/RhoA pathway. Finally, confocal microscopy demonstrated that cells overexpressing miR-155-5p underwent cytoskeletal rearrangement during phagocytosis, and the phagocytic function of cells was enhanced, signaling that miR-155-5p participated in macrophage skeletal rearrangement and enhanced alveolar macrophage phagocytosis by targeting the expression of Rictor in the mTORC2/RhoA pathway.

Role of short-wave-sensitive 1 (sws1) in cone development and first feeding in larval zebrafish.

Color vision is mediated by the expression of different major visual pigment proteins (opsins) on retinal photoreceptors. Vertebrates have four classes of cone opsins that are most sensitive to different wavelengths of light: short wavelength sensitive 1 (SWS1), short wavelength sensitive 2 (SWS2), medium wavelength sensitive (RH2), and long wavelength sensitive (LWS). UV wavelengths play important roles in foraging and communication. However, direct evidence provide links between sws1 and first feeding is lacking. Here, CRISPR/Cas9 technology was performed to generate mutant zebrafish lines with sws1 deletion. sws1 mutant zebrafish larvae exhibited decreased sws1, rh2-2, and lws1 expression, and increased rod gene (rho and gnat1) expression. Furthermore, the sws1-deficient larvae exhibited significantly reduced food intake, and the orexigenic genes npy and agrp signaling were upregulated at 6days postfertilization (dpf). The transcription expression of sws1 and rh2-3 genes decreased in sws1-/- adults compared to wild type. Surprisingly, the results of feeding at the adult stage were not the same with larvae. sws1 deficiency did not affect food intake and appetite gene expression at adult stages. These results reveal a role for sws1 in normal cone development and first feeding in larval zebrafish.

TAGAP activates Th17 cell differentiation by promoting RhoA and NLRP3 to accelerate rheumatoid arthritis development.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder that can give rise to joint swelling and inflammation, potentially affecting the entire body, closely linked to the state of T cells. The T-cell activation Rho GTPase activating protein (TAGAP) is associated with many autoimmune diseases including RA and is directly linked to the differentiation of Th17 cells. The present study intends to investigate the influence of TAGAP on the RA progression and its mechanism to empower new treatments for RA. A collagen-induced-arthritis (CIA) rat model was constructed, as well as the extraction of CD4+ T cells. RT-qPCR, H&E staining and safranin O/fast green staining revealed that TAGAP interference reduced TAGAP production in the ankle joint of CIA rats, and joint inflammation and swelling were alleviated, which reveals that TAGAP interference reduces synovial inflammation and cartilage erosion in the rat ankle joint. Expression of inflammatory factors (TNF-α, IL-1β, and IL-17) revealed that TAGAP interference suppressed the inflammatory response. Expression of pro-inflammatory cytokines, matrix-degrading enzymes, and anti-inflammatory cytokines at the mRNA level was detected by RT-qPCR and revealed that TAGAP interference contributed to the remission of RA. Mechanistically, TAGAP interference caused a significant decrease in the levels of RhoA and NLRP3. Assessment of Th17/Treg levels by flow cytometry revealed that TAGAP promotes Th17 cells differentiation and inhibits Treg cells differentiation in vitro and in vivo. In conclusion, TAGAP interference may decrease the differentiation of Th17 cells by suppressing the expression of RhoA and NLRP3 to slow down the RA progression.

Expression of Ras signaling pathway proteins and developmental factors in peripheral nerve sheath tumors of patients with neurofibromatosis type 1.

To characterize expression of factors relevant for Ras signaling and developmental factors in a large series of peripheral nerve sheath tumors (PNST) obtained from patients with neurofibromatosis type 1 (NF1). Tissue micro-array technique was applied to study 520 PNST of 385 NF1 patients by immunohistochemistry for mTor, Rho, phosphorylated MEK, Pax7, Sox9, and periaxin expression. PNST comprised cutaneous neurofibroma (CNF) (n=114), diffuse neurofibroma (DNF) (n=109), diffuse plexiform neurofibroma (DPNF) (n=108), plexiform neurofibroma (PNF) (n=110), and malignant PNST (MPNST) (n=22). All proteins examined showed highest expression levels/highest frequency of expression in MPNST. Benign PNF with potential for malignant dedifferentiation expressed mTor, phosphorylated MEK, Sox9, and periaxin significantly higher/more frequently than other benign neurofibroma subtypes. In NF1-associated PNST, expression of proteins involved in Ras-signaling and development is upregulated not only in MPNST, but also in benign PNF with the potential for malignant dedifferentiation. The differences in protein expression may provide clues for understanding the therapeutic effects of substances applied for reduction of PNST in NF1.

Optimization of Neferine Purification Based on Response Surface Methodology and Its Anti-Metastasis Mechanism on HepG2 Cells.

Liver cancer continues to be a focus of scientific research due to its low five-year survival rate. One of its main core issues is the high metastasis of cells, for which there is no effective treatment. Neferine was originally isolated from Plumula nelumbinis and demonstrated to have a good antitumor effect. In order to extract high-purity Neferine in a more efficient and environmentally friendly manner, response surface methodology (RSM) was used to optimize the isolation and purification procedures in this study. The extract conditions of a 7:3 ratio for the eluent of dichloromethane: methanol, 1:60 for the mass ratio of the extract amount: silica gel, and 3 mL/min of the elution flow rate were shown to be the optimal conditions. These conditions resulted in the highest yield of 6.13 mg per 66.60 mg of starting material, with productivity of 8.76% and purity of 87.04%. Compared with the previous methods, this method can prepare Neferine in large quantities more quickly. We subsequently evaluated the antitumor activity of the purified Neferine against HepG2 hepatic cancer cells. The purified Neferine was found to inhibit the proliferation of HepG2 cells through the CCK-8 assay, with an IC50 of 33.80 μM in 24 h, 29.47 μM in 48 h, 24.35 μM in 72 h and 2.78 μM in 96 h of treatment. Neferine at a concentration of 3 μM could significantly inhibit the migration and invasion abilities of the HepG2 cells in vitro. We also explored the mechanism of action of Neferine via Western blot. We showed that Neferine could reduce RhoA expression by effectively inhibiting the phosphorylation of MYPT1, thereby effectively exerting anti-metastasis activity against HepG2 cells. Thus, we have optimized the isolation procedures for highly pure Neferine by response surface methodology (RSM) in this study, and purified Neferine is shown to play an essential role in the anti-metastasis process of liver cancer cells. The Neferine purification procedure may make a wide contribution to the follow-up development of other anti-metastasis lead compounds.

S1PR2/Wnt3a/RhoA/ROCK1/β-catenin signaling pathway promotes diabetic nephropathy by inducting endothelial mesenchymal transition and impairing endothelial barrier function

AimsHyperglycemia and hyperlipidemia are key factors in the pathogenesis of diabetic nephropathy (DN), and renal fibrosis is the most common pathway leading to the disease. Endothelial mesenchymal transition (EndMT) is a crucial mechanism for the production of myofibroblasts, and impaired endothelial barrier function is one of the mechanisms for the generation of microalbuminuria in DN. However, the specific mechanisms behind these are not yet clear. Main methodsProtein expression was detected by immunofluorescence, immunohistochemistry and Western blot. Knocking down or pharmacological inhibition of S1PR2 were used to inhibit Wnt3a, RhoA, ROCK1, β-catenin, and Snail signaling. Changes in cell function were analyzed by CCK-8 method, cell scratching assay, FITC-dextran permeability assay, and Evans blue staining. Key findingsConsistent with increased gene expression of S1PR2 in DN patients and mice with kidney fibrosis disease, S1PR2 expression was significantly increased in glomerular endothelial cells of DN mice and HUVEC cells treated with glucolipids. Knocking down or pharmacological inhibition of S1PR2 significantly decreased the expression of Wnt3a, RhoA, ROCK1, and β-catenin in endothelial cells. Furthermore, inhibition of S1PR2 in vivo reversed EndMT and endothelial barrier dysfunction in glomerular endothelial cells. Inhibition of S1PR2 and ROCK1 in vitro also reversed EndMT and endothelial barrier dysfunction in endothelial cells. SignificanceOur results suggest that the S1PR2/Wnt3a/RhoA/ROCK1/β-catenin signaling pathway is involved in the pathogenesis of DN by inducing EndMT and endothelial barrier dysfunction.

RhoA/ROCK1 regulates the mitochondrial dysfunction through Drp1 induced by Porphyromonas gingivalis in endothelial cells.

Porphyromonas gingivalis (P. gingivalis) is a pivotal pathogen of periodontitis. Our previous studies have confirmed that mitochondrial dysfunction in the endothelial cells caused by P. gingivalis was dependent on Drp1, which may be the mechanism of P. gingivalis causing endothelial dysfunction. Nevertheless, the signalling pathway induced the mitochondrial dysfunction remains unclear. The purpose of this study was to investigate the role of the RhoA/ROCK1 pathway in regulating mitochondrial dysfunction caused by P. gingivalis. P. gingivalis was used to infect EA.hy926 cells (endothelial cells). The expression and activation of RhoA and ROCK1 were assessed by western blotting and pull-down assay. The morphology of mitochondria was observed by mitochondrial staining and transmission electron microscopy. Mitochondrial function was measured by ATP content, mitochondrial DNA and mitochondrial permeability transition pore openness. The phosphorylation and translocation of Drp1 were evaluated using western blotting and immunofluorescence. The role of the RhoA/ROCK1 pathway in mitochondrial dysfunction was investigated using RhoA and ROCK1 inhibitors. The activation of RhoA/ROCK1 pathway and mitochondrial dysfunction were observed in P. gingivalis-infected endothelial cells. Furthermore, RhoA or ROCK1 inhibitors partly prevented mitochondrial dysfunction caused by P. gingivalis. The increased phosphorylation and mitochondrial translocation of Drp1 induced by P. gingivalis were both blocked by RhoA and ROCK1 inhibitors. In conclusion, we demonstrate that the RhoA/ROCK1 pathway was involved in mitochondrial dysfunction caused by P. gingivalis by regulating the phosphorylation and mitochondrial translocation of Drp1. Our research illuminated a possible new mechanism by which P. gingivalis promotes endothelial dysfunction.