Exploration of the Mechanisms Underlying Yu's Enema Formula in Treating Ulcerative Colitis by Blocking the RhoA/ROCK Pathway based on Network Pharmacology, High-performance Liquid Chromatography Analysis, and Experimental Verification.

BACKGROUND: The traditional Chinese medicine formula, Yu's Enema Formula (YEF), has demonstrated potential in the treatment of Ulcerative Colitis (UC). OBJECTIVE: This study aimed to unveil the anti-UC mechanisms of YEF. METHODS: Utilizing public databases, we obtained YEF and UC-related targets. GO and KEGG analyses were conducted via clusterProfiler and Reactome. The STRING database facilitated the construction of the PPI network, and hub targets were selected using cytoHubba. We used R software for differential expression and correlation analyses, and molecular docking was performed with PyMOL and AutoDock. HPLC analysis identified the compounds in YEF. For in vivo validation, a UC rat model was employed. RESULTS AND DISCUSSION: 495 YEF-UC overlapping targets were identified. GO and KEGG analyses indicated enrichment in exogenous stimuli response, peptide response, positive MAPK cascade regulation, interleukin- related signaling, and the TLR4 cascade. Hub targets included CTNNB1, JUN, MAPK1, MAPK3, SRC, STAT3, TLR4, TP53, and RELA, which were often interconnected. Molecular docking revealed quercetin's strong binding affinity with CTNNB1, MAPK1, MAPK3, SRC, STAT3, TLR4, and TP53, consistent with HPLC analysis. In vivo experiments suggested that YEF has the potential to alleviate UC symptoms and protect the intestinal mucosal barrier by inhibiting the RhoA/ROCK pathway. CONCLUSION: YEF may safeguard the intestinal mucosal barrier in UC by targeting CTNNB1, MAPK1, MAPK3, SRC, STAT3, TLR4, and TP53, while blocking the RhoA/ROCK pathway.

Berberine promotes lacteal junction zippering and ameliorates diet-induced obesity through the RhoA/ROCK signaling pathway.

BACKGROUND: Obesity has emerged as a global epidemic. Recent research has indicated that diet-induced obesity can be prevented by promoting lacteal junction zippering. Berberine, which is derived from natural plants, is found to be promising in weight reduction, but the underlying mechanism remains unspecified. PURPOSE: To determine whether berberine protects against obesity by regulating the lacteal junction and to explore potential molecular mechanisms. METHODS: Following the induction of the diet-induced obese (DIO) model, mice were administered low and high doses of berberine for 4 weeks. Indicators associated with insulin resistance and lipid metabolism were examined. Various methods, such as Oil Red O staining, transmission electron microscopy imaging, confocal imaging and others were used to observe the effects of berberine on lipid absorption and the lacteal junction. In vitro, human dermal lymphatic endothelial cells (HDLECs) were used to investigate the effect of berberine on LEC junctions. Western Blot and immunostaining were applied to determine the expression levels of relevant molecules. RESULTS: Both low and high doses of berberine reduced body weight in DIO mice without appetite suppression and ameliorated glucolipid metabolism disorders. We also found that the weight loss effect of berberine might contribute to the inhibition of small intestinal lipid absorption. The possible mechanism was related to the promotion of lacteal junction zippering via suppressing the ras homolog gene family member A (RhoA)/Rho-associated kinase (ROCK) signaling pathway. In vitro, berberine also promoted the formation of stable mature junctions in HDLECs, involving the same signaling pathway. CONCLUSION: Berberine could promote lacteal junction zippering and ameliorate diet-induced obesity through the RhoA/ROCK signaling pathway.

Acupuncture Improves Synaptic Plasticity of SAMP8 Mice through the RhoA/ROCK Pathway.

BACKGROUND: Studies have found synaptic plasticity damage to be an early marker of Alzheimer's disease (AD). RhoA/ROCK pathway is involved in the regulation of synaptic plasticity. Acupuncture can significantly improve the cognitive state of AD. OBJECTIVE: We aimed to use modern biological technology to detect the changes in synaptic plasticity and RhoA/ROCK pathway in SAMP8 mice, as well as the intervention effect of acupuncture. METHODS: Morris water maze and electrophysiological techniques were used in vivo to detect the changes in spatial memory and LTP of mice. Golgi Cox staining and CASEVIEWER2.1 software were used to quantitatively analyze the changes in the morphology and number of dendritic spines in the hippocampus of mice. The activity of RhoA and ROCK2 in the hippocampus of mice was detected, respectively, by pull-down technique and ELISA. WB technique was used to detect the protein expression of ROCK2 and phosphorylation level of MLC2, LIMK2, and CRMP2 in the hippocampus of mice. RESULTS: The neurobehavior and synaptic plasticity of 8-month-old SAMP8 mice were found to be significantly impaired. Acupuncture could improve the spatial learning and memory ability of SAMP8 mice, and partially prevent the reduction in the number of spines on the secondary branches of the apical dendrites in the hippocampus and the attenuation of LTP. The RhoA/ROCK pathway was significantly activated in the hippocampus of 8-month-old SAMP8 mice, and acupuncture had an inhibitory effect on it. CONCLUSION: Acupuncture can improve synaptic plasticity by inhibiting the abnormal activation of the RhoA/ROCK pathway, and improve the spatial learning and memory ability of AD, so as to achieve the purpose of treating AD.

Retinol binding protein 4 promotes the phenotypic transformation of vascular smooth muscle cells under high glucose condition via modulating RhoA/ROCK1 pathway.

Phenotypic switch of vascular smooth muscle cells (VSMCs) contributes to the pathogenesis of atherosclerosis (AS). High level of retinol binding protein 4 (RBP4) is regarded as a risk factor in cardiac-cerebral vascular disease. This study is performed to clarify the biological function of RBP4 in modulating the phenotypic switch of VSMCs induced via RhoA/ROCK1 signaling pathway. In vivo experiment, all the rats were divided into control group (NC), diabetic group (DM) and diabetic atherosclerosis group (DAS). The expressions of biochemical indicators, RhoA and Rho associated coiled-coil containing protein kinase 1 (ROCK1) were detected. In vitro experiment, VSMCs were cultured under high glucose condition, and ectogenic RBP4, HA-1100, rapamycin, or 3-methyladenine (3-MA) were supplemented to treat the VSMCs, respectively. The proliferation and migration of VSMCs were evaluated. The regulatory relationship between RBP4 and ROCK1 was predicted by bioinformatics analysis, and validated by qRT-PCR and Western blot. The regulatory effects of RBP4 on contractile phenotypic markers such as calponin, MYH11, α-SMA and autophagy markers including LC3II, LC3I, and Beclin-1 as well as mTOR were also detected. Moreover, VSMCs were cultured exposed to ROCK1 overexpressed plasmid or short hairpin RNA (shRNA), the proliferation and migration of VSMCs were evaluated and the regulatory effects of RhoA/ROCK1 signaling pathway on contractile phenotypic markers and autophagy markers were also detected. In vivo, RhoA, ROCK1, and mTOR were highly expressed in the rats intraperitoneally injected with RBP4. In vitro, the expressions of calponin, MYH11, α-SMA, LC3II, LC3I, and Beclin-1 were decreased in VSMCs treated with ROCK1-OA under high glucose condition, conversely, the expressions were increased in VSMCs exposed to ROCK1-shRNA. After incubated with rapamycin additionally, the expressions of calponin, MYH11, α-SMA, LC3II/I and Beclin-1 were up-regulated and the expression of p-mTOR was decreaed in VSMCs of HG+ROCK1-OA. Conversely, after incubated with 3-MA additionally, the expressions of calponin, MYH11, α-SMA, LC3II/I and Beclin-1 were down-regulated and the expression of p-mTOR was elevated in VSMCs of HG+ROCK1-shRNA. Ectogenic RBP4 facilitated high glucose-induced proliferation and migration of VSMCs, and it repressed the expression of calponin, MYH11, α-SMA, LC3II/I, and Beclin-1 in VSMCs. As expected, ROCK1 inhibit or counteracted the biological effects of RBP4 on VSMCs. In addition, the expressions of contractile phenotypic markers, LC3II/I, and Beclin-1 were promoted and mTOR were decreased after the VSMCs treated with autophagy agonist, whereas no significant difference was observed in the expressions of ROCK1, RhoA. RBP4 is an injurious factor in the pathogenesis of diabetic AS, and it promotes the phenotypic switch of VSMCs via activating RhoA/ROCK1 pathway and inhibiting autophagy.

Identification of a small RhoA GTPase inhibitor effective in fission yeast and human cells.

The Rho GTPase family proteins are key regulators of cytoskeletal dynamics. Deregulated activity of Rho GTPases is associated with cancers and neurodegenerative diseases, and their potential as drug targets has long been recognized. Using an economically effective drug screening workflow in fission yeast and human cells, we have identified a Rho GTPase inhibitor, O1. By a suppressor mutant screen in fission yeast, we find a point mutation in the rho1 gene that confers resistance to O1. Consistent with the idea that O1 is the direct inhibitor of Rho1, O1 reduced the cellular amount of activated, GTP-bound Rho1 in wild-type cells, but not in the O1-resistant mutant cells, in which the evolutionarily conserved Ala62 residue is mutated to Thr. Similarly, O1 inhibits activity of the human orthologue RhoA GTPase in tissue culture cells. Our studies illustrate the power of yeast phenotypic screens in the identification and characterization of drugs relevant to human cells and have identified a novel GTPase inhibitor for fission yeast and human cells.

Investigating the Anticancer Activity of G-Rh1 Using In Silico and In Vitro Studies (A549 Lung Cancer Cells).

Ginsenoside Rh1 (G-Rh1), a possible bioactive substance isolated from the Korean Panax ginseng Meyer, has a wide range of pharmacological effects. In this study, we have investigated the anticancer efficacy of G-Rh1 via in silico and in vitro methodologies. This study mainly focuses on the two metastatic regulators, Rho-associated protein kinase 1 (ROCK1) and RhoA, along with other standard apoptosis regulators. The ROCK1 protein is a member of the active serine/threonine kinase family that is crucial for many biological processes, including cell division, differentiation, and death, as well as many cellular processes and muscle contraction. The abnormal activation of ROCK1 kinase causes several disorders, whereas numerous studies have also shown that RhoA is expressed highly in various cancers, including colon, lung, ovarian, gastric, and liver malignancies. Hence, inhibiting both ROCK1 and RhoA will be promising in preventing metastasis. Therefore, the molecular level interaction of G-Rh1 with the ROCK1 and RhoA active site residues from the preliminary screening clearly shows its inhibitory potential. Molecular dynamics simulation and principal component analysis give essential insights for comprehending the conformational changes that result from G-Rh1 binding to ROCK1 and RhoA. Further, MTT assay was employed to examine the potential cytotoxicity in vitro against human lung cancer cells (A549) and Raw 264.7 Murine macrophage cells. Thus, G-Rh1 showed significant cytotoxicity against human lung adenocarcinoma (A549) at 100 µg/mL. In addition, we observed an elevated level of reactive oxygen species (ROS) generation, perhaps promoting cancer cell toxicity. Additionally, G-Rh1 suppressed the mRNA expression of RhoA, ROCK1, MMP1, and MMP9 in cancer cell. Accordingly, G-Rh1 upregulated the p53, Bax, Caspase 3, caspase 9 while Bcl2 is downregulated intrinsic pathway. The findings from our study propose that the anticancer activity of G-Rh1 may be related to the induction of apoptosis by the RhoA/ROCK1 signaling pathway. As a result, this study evaluated the functional drug-like compound G-Rh1 from Panax ginseng in preventing and treating lung cancer adenocarcinoma via regulating metastasis and apoptosis.

Tetrandrine Attenuates Podocyte Injury by Inhibiting TRPC6-Mediated RhoA/ROCK1 Pathway.

Tetrandrine (Tet), a compound found in a traditional Chinese medicine, presents the protective effect for kidney function. Our study is aimed at clarifying the efficacy and underlying mechanism of Tet on podocyte injury. In this study, podocyte injury was induced in rats with adriamycin (ADR), and MPC5 podocytes were constructed with TRPC6 overexpression. We found that Tet treatment reduced the levels of proteinuria, serum creatinine, and blood urea nitrogen and increased plasma albumin levels in ADR-induced rats. Tet reduced intracellular Ca2+ influx and apoptosis in MPC5 podocytes overexpressing TRPC6. Tet downregulated the expression of renal TRPC6, RhoA, and ROCK1 and upregulated the expression of synaptopodin; meanwhile, it reduced calcineurin activity in vivo and in vitro. In conclusion, Tet protects against podocyte by affecting TRPC6 and its downstream RhoA/ROCK1 signaling pathway.

Parthenolide modulates cerebral ischemia-induced microglial polarization and alleviates neuroinflammatory injury via the RhoA/ROCK pathway.

BACKGROUND: Microglia can be activated as proinflammatory (M1) phenotypes and anti-inflammatory (M2) phenotypes after stroke. Parthenolide (PTL) has anti-inflammatory and protective effects on neurological diseases, but until now, the exact mechanisms of these processes after stroke have been unclear. The purpose of this study was to determine the effect of PTL on microglial polarization after stroke and its target for inducing microglial polarization. METHODS: Triphenyltetrazolium chloride (TTC) staining, hematoxylin-eosin (HE) staining, and neurological evaluation were performed in a focal transient cerebral ischemia rat model. The human microglia exposed to lipopolysaccharide (LPS) was used for in vitro experiments. Microglial polarization was assessed by RT-PCR and immunostaining. Inflammatory cytokine assays and western blotting were used to investigate the molecular mechanisms underlying PTL-mediated microglial polarization in vivo and in vitro. RESULTS: PTL significantly reduced cerebral infarction and neuronal apoptosis in rats with cerebral ischemia, reduced the level of inflammatory factors and alleviated neurological deficits. PTL treatment decreased the expression of microglia/macrophage markers in M1 macrophages and increased the expression of microglia/macrophage markers in M2 macrophages after stroke, which induced the transformation of microglia cells from the M1 phenotype to the M2 phenotype. Furthermore, PTL significantly reduced RhoA/ROCK-NF-κB pathway activity and downregulated the effects of pentanoic acid (ROCK agonist). CONCLUSIONS: PTL has been shown to mediate neuroinflammation and protect against ischemic brain injury by regulating microglial polarization via the RhoA/ROCK pathway.

RhoA as a Signaling Hub Controlling Glucagon Secretion From Pancreatic α-Cells.

Glucagon hypersecretion from pancreatic islet α-cells exacerbates hyperglycemia in type 1 diabetes (T1D) and type 2 diabetes. Still, the underlying mechanistic pathways that regulate glucagon secretion remain controversial. Among the three complementary main mechanisms (intrinsic, paracrine, and juxtacrine) proposed to regulate glucagon release from α-cells, juxtacrine interactions are the least studied. It is known that tonic stimulation of α-cell EphA receptors by ephrin-A ligands (EphA forward signaling) inhibits glucagon secretion in mouse and human islets and restores glucose inhibition of glucagon secretion in sorted mouse α-cells, and these effects correlate with increased F-actin density. Here, we elucidate the downstream target of EphA signaling in α-cells. We demonstrate that RhoA, a Rho family GTPase, plays a key role in this pathway. Pharmacological inhibition of RhoA disrupts glucose inhibition of glucagon secretion in islets and decreases cortical F-actin density in dispersed α-cells and α-cells in intact islets. Quantitative FRET biosensor imaging shows that increased RhoA activity follows directly from EphA stimulation. We show that in addition to modulating F-actin density, EphA forward signaling and RhoA activity affect α-cell Ca2+ activity in a novel mechanistic pathway. Finally, we show that stimulating EphA forward signaling restores glucose inhibition of glucagon secretion from human T1D donor islets.

Regulation of cellular communication network factor 1 by Ras homolog family member A in bovine steroidogenic luteal cells.

Development of the corpus luteum (CL) requires the growth of a new capillary network from preexisting vasculature, a process known as angiogenesis. Successful building of this capillary network occurs through a sequence of cellular events-differentiation, proliferation, migration, and adhesion-which are regulated by a suite of angiogenic proteins that includes cellular communication network factor 1 (CCN1). We previously reported that the expression of CCN1 was highest in luteal tissue obtained from the early-cycle, 4-d-old bovine CL (i.e., corpus hemorrhagicum) compared to the mid- and late-cycle CL. In the present study, we treated steroidogenic bovine luteal cells from early-cycle CL with luteinizing hormone (LH), but it had no effect on CCN1 expression. Direct stimulation of the canonical LH pathway with forskolin and dibutyryl-cyclic adenosine monophosphate (cAMP), however, inhibited CCN1 mRNA expression. In endothelial cells, stimulation of Ras homolog family member A (RhoA) induces CCN1 expression, whereas RhoA inactivation inhibits it. Yet, it is unknown if regulation of CCN1 in steroidogenic luteal cells works likewise. We hypothesized that a similar mechanism of CCN1 regulation exists in bovine luteal cells and that thrombin, a known RhoA activator, may be a physiologic trigger for this mechanism in the early-cycle CL. To test this hypothesis, ovaries were collected from lactating dairy cows on days 3 or 4 of the estrous cycle, and corpora lutea were dissected and dissociated. Steroidogenic luteal cells were suspended in defined Ham's F12 medium, supplemented with insulin/transferrin/selenium and gentamicin, and seeded into 6-well plates. After 24 h, spent medium was replaced with fresh Ham's F12, and the cells were cultured for 24 to 48 h. Cells were treated for 2 h with defined medium, 10% fetal bovine serum (FBS), thrombin (1, 5, 10 U/mL), or Rho Activator II (0.25, 1, 2 µg/mL). Cells were then lysed for RNA extraction, followed by cDNA generation, and quantitative polymerase chain reaction (qPCR). Thrombin (1, 5, 10 U/mL; n = 3) and Rho Activator II (0.25, 1, 2 µg/mL; n = 6) increased (P < 0.05) CCN1 mRNA expression. In summary, CCN1 in bovine steroidogenic luteal cells was induced by thrombin and appeared to be regulated in a Rho-dependent manner. Future work will elucidate the signaling partners downstream of Rho which leads to CCN1 gene expression.

The corpus luteum (CL) is a transient ovarian endocrine gland that secretes progesterone, the hormone of pregnancy. Development of an optimally functioning CL requires the creation of a dense capillary bed through growth of new blood vessels, which is an intricate process called angiogenesis. A myriad of factors regulates angiogenesis, including the angiogenic inducer protein, cellular communication network factor 1 (CCN1). Although it is highly expressed in the early-cycle bovine CL, the mechanisms of CCN1 regulation have not been fully elucidated. In the present study, we showed that CCN1 expression in steroidogenic luteal cells from the early-cycle bovine CL was induced by Ras homolog family member A (RhoA) and by thrombin, but not by luteinizing hormone (LH). To the best of our knowledge, the involvement of thrombin and its signaling partner, RhoA, in regulating CCN1 in bovine steroidogenic luteal cells has not been previously reported. These findings will inform our future work to determine how RhoA activation by thrombin leads to increased expression of CCN1.

Xueshuantong injection combined with Salvianolate lyophilized injection improves the synaptic plasticity against focal cerebral ischemia/reperfusion injury in rats through PI3K/AKT/mTOR and RhoA/ROCK pathways.

The combined use of two or more different drugs can better promote nerve recovery and its prognosis for treatment of stroke. Salvianolate lyophilized injection (SLI) made from the aqueous extraction of salvia miltiorrhiza and Xueshuantong injection (lyophilized) (XST) made from the Panax Notoginseng extraction are two herbal standardized preparations that have been widely used in China for the treatment of ischemic stroke. In this study, we investigated the neuroprotective effects of XST combined with SLI in the recovery stage of middle cerebral artery occlusion / reperfusion (MCAO/R) injury rat. Wistar rats were subjects to MCAO/R, then were treated with SLI or XST alone, or with their combination (1X1S) via tail injection daily for 14 days. The pathological status of the brain was detected by neurological deficit scores, TTC, regional cerebral blood flow and Nissl staining. Golgi-Cox staining was used to assess dendritic, axonal and synaptic remodeling. The expression of MAP-2, ß-Tubulin, PSD95, SYN, BDNF and VEGF were analyzed by western blotting and immunofluorescence. The results showed that administration of 1X1S not only significantly decreased neurological scores and infarct volumes, but also increased regional cerebral blood flow, strengthened dendritic and synaptic remodeling compared with XST, SLI used alone. And the mechanism of combined of 1X1S to exert neuroprotection may be associated with PI3K/ AKT/ mTOR and RhoA/ROCK2 pathways. Overall, these findings suggest that combination of XST and SLI promotes dendritic spine density and synaptic plasticity via upregulation of the PI3K/ AKT/ mTOR pathways and inhabitation the RhoA/ROCK2 signaling pathway in rat with MCAO/R, showing its multiple-action-multiple-target efficacy and suggest a potential new strategy for ischemia.

Hesperetin Inhibits TGF-ß1-Induced Migration and Invasion of Triple Negative Breast Cancer MDA-MB-231 Cells via Suppressing Fyn/Paxillin/RhoA Pathway.

Triple-negative breast cancer is an aggressive subtype of breast cancer with poor clinical outcomes and poor prognosis. Hesperetin is an active component extracted from Citrus fruits and Traditional Chinese Medicine has a wide range of pharmacological effects. Here, we assessed the anti-migration and anti-invasive effects and explored inhibitory mechanisms of hesperetin on metastasis of human triple negative breast cancer MDA-MB-231 cells. Cell viability experiments revealed that 200 µM hesperetin has a clear inhibitory effect on MDA-MB-231 cells. TGF-ß1 treatment induces apparent tumor progression in MDA-MB-231 cells including aberrant wound-healing and invasion ability, which is effectively suppressed by hesperetin co-treatment. Additionally, hesperetin inhibited the TGF-ß1-mediated actin stress fiber formation. Western blot results showed that hesperetin suppressed the TGF-ß1-mediated (i) activation of Fyn, (ii) phosphorylation of paxillin at Y31, Y88, and Y118 sites, (iii) the increased expression of RhoA, and (iv) activation of Rho-kinase. We demonstrated the increased interaction of Fyn with paxillin and RhoA protein in the TGF-ß1-induced metastasis of MDA-MB-231 cells. Small interfering RNA Fyn inhibited phosphorylation of paxillin (Y31) and activation of Rho-kinase induced by TGF-ß1. In conclusion, hesperetin has a significant inhibitory effect on migration and invasion of MDA-MB-231 cells induced by TGF-ß1, which might be attributed to inhibiting the Fyn/paxillin/RhoA pathway.

Uncaria Rhynchophylla attenuates angiotensin â ¡-induced myocardial fibrosis via suppression of the RhoA/ROCK1 pathway.

Uncaria rhynchophylla (UR), a traditional Chinese medicine, has been proven effective in treating hypertensive patients in China. However, the mechanisms of action of UR in reducing hypertension and myocardial fibrosis are still unclear. The purpose of this study was to explore the role of UR in an angiotensin Ⅱ (Ang Ⅱ) induced mouse model. The mice were randomly divided into 5 groups and infused with Ang Ⅱ (500 ng/kg/min) or saline, then administered UR (0.78, 1.56 or 3.12 g/kg/d) or saline for 4 weeks. UR treatment significantly attenuated the elevation of blood pressure caused by Ang Ⅱ. It enhanced myocardial function and attenuated the increase in the heart weight index and the pathological changes in the Ang Ⅱ-induced hypertensive mice. Furthermore, UR treatment inhibited cardiac fibrosis and significantly down-regulated collagen I, collagen Ⅲ, and α-SMA protein expression in cardiac tissues. UR also attenuated the expression of RhoA, ROCK1, CTGF, and TGF-ß1. In cultured cardiac fibroblasts stimulated with Ang Ⅱ, UR significantly down-regulated the expression of Collagen I, Collagen III, RhoA, ROCK1, and α-SMA. In summary, UR can significantly attenuate Ang Ⅱ-induced hypertension and cardiac fibrosis, partly via suppression of the RhoA/ROCK1 signaling pathway.

Brusatol-Enriched Brucea javanica Oil Ameliorated Dextran Sulfate Sodium-Induced Colitis in Mice: Involvement of NF-κB and RhoA/ROCK Signaling Pathways.

Brucea javanica oil (BJO) is beneficial for the treatment of ulcerative colitis (UC), and that quassinoids in particular brusatol are bioactive components. However, it is still uncertain whether or not other components in BJO, such as oleic acid and fatty acids, have an anti-UC effect. The present study is aimed at comparing the anti-UC effects between brusatol-enriched BJO (BE-BJO) and brusatol-free BJO (BF-BJO) and at exploring the effects and mechanisms of BE-BJO on colon inflammation and intestinal epithelial barrier function. Balb/C mice received 3% (wt/vol) DSS for one week to establish the UC model. Different doses of BE-BJO, BF-BJO, or BJO were treated. The result illustrated that BE-BJO alleviated DSS-induced loss of body weight, an increase of disease activity index (DAI), and a shortening of colon, whereas BF-BJO did not have these protective effects. BE-BJO treatment improved the morphology of colon tissue, inhibited the production and release of TNF-α, IFN-γ, IL-6, and IL-1ß in the colon tissue, and reversed the decreased expressions of ZO-1, occludin, claudin-1, and E-cadherin induced by DSS but augmented claudin-2 expression. Mechanistically, BE-BJO repressed phosphorylation of NF-κB subunit p65, suppressed RhoA activation, downregulated ROCK, and prevented phosphorylation of myosin light chain (MLC) in DSS-treated mice, indicating that the protective effect of BE-BJO is attributed to suppression of NF-κB and RhoA/ROCK signaling pathways. These findings confirm that brusatol is an active component from BJO in the treatment of UC.

Panax Notoginseng Saponin Attenuates Gastric Mucosal Epithelial Cell Injury Induced by Dual Antiplatelet Drugs through COX and PI3K/Akt/ VEGF-GSK-3ß-RhoA Network Pathway.

OBJECTIVE: To elucidate the underlying mechanism of Panax notoginseng saponin (PNS) on gastric epithelial cell injury and barrier dysfunction induced by dual antiplatelet (DA). METHODS: Human gastric mucosal epithelial cell (GES-1) was cultured and divided into 4 groups: a control, a DA, a PNS+DA and a LY294002+PNS+DA group. GES-1 apoptosis was detected by flow cytometry, cell permeability were detected using Transwell, level of prostaglandins E2 (PGE2), 6-keto-prostaglandin F1α (6-keto-PGF1α) and vascular endothelial growth factor (VEGF) in supernatant were measured by enzyme linked immunosorbent assay (ELISA), expression of phosphatidylinositide 3-kinase (PI3K), phosphorylated-PI3K (p-PI3K), Akt, phosphorylated-Akt (p-Akt), cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), glycogen synthase kinase-3ß (GSK-3ß) and Ras homolog gene family member A (RhoA) were measured by Western-blot. RESULTS: DA induced apoptosis and hyper-permeability in GES-1, reduced supernatant level of PGE2, 6-keto-PGF1α and VEGF (P<0.05). Addition of PNS reduced the apoptosis of GES-1 caused by DA, restored the concentration of PGE2, 6-keto-PGF1α and VEGF (P<0.05). In addition, PNS attenuated the alteration of COX-1 and COX-2 expression induced by DA, up-regulated p-PI3K/p-Akt, down-regulated RhoA and GSK-3ß. LY294002 mitigated the effects of PNS on cell apoptosis, cell permeability, VEGF concentration, and expression of RhoA and GSK-3ß significantly. CONCLUSIONS: PNS attenuates the suppression on COX/PG pathway from DA, alleviates DA-induced GES-1 apoptosis and barrier dysfunction through PI3K/Akt/ VEGF-GSK-3ß-RhoA network pathway.

Lathyrus sativus diamine oxidase reduces Clostridium difficile toxin A-induced toxicity in Caco-2 cells by rescuing RhoA-GTPase and inhibiting pp38-MAPK/NF-κB/HIF-1α activation.

Clostridium difficile toxin A (TcdA) impairs the intestinal epithelial barrier, increasing the mucosa permeability and triggering a robust inflammatory response. Lathyrus sativus diamino oxidase (LSAO) is a nutraceutical compound successfully used in various gastrointestinal dysfunctions. Here, we evaluated the LSAO (0.004-0.4 µM) ability to counter TcdA-induced (30 ng/mL) toxicity and damage in Caco-2 cells, investigating its possible mechanism of action. LSAO has improved the transepithelial electrical resistance (TEER) score and increased cell viability in TcdA-treated cells, significantly rescuing the protein expression of Ras homolog family members, A-GTPase (RhoA-GTPase), occludin, and zonula occludens-1 (ZO-1). LSAO has also exhibited an anti-apoptotic effect by inhibiting the TcdA-induced expression of Bcl-2-associated X protein (Bax), p50 nuclear factor-kappa-B (p50), p65nuclear factor-kappa-B (p65), and hypoxia-inducible transcription factor-1 alpha (HIF-1α), and the release of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and vascular endothelial growth factor (VEGF) in the cell milieu. Our data showed that LSAO exerts a protective effect on TcdA-induced toxicity in Caco-2 cells, placing itself as an interesting nutraceutical to supplement the current treatment of the Clostridium difficile infections.

The ginsenoside metabolite compound K stimulates glucagon-like peptide-1 secretion in NCI-H716 cells by regulating the RhoA/ROCKs/YAP signaling pathway and cytoskeleton formation.

Ginsenoside Rb1 has been shown to have antidiabetic and anti-inflammatory effects. Its major metabolite, compound K (CK), can stimulate the secretion of glucagon-like peptide-1 (GLP1), a gastrointestinal hormone that plays a vital role in regulating glucose metabolism. However, the mechanism underlying the regulation of GLP1 secretion by compound K has not been fully explored. This study was designed to investigate whether CK ameliorates incretin impairment by regulating the RhoA/ROCKs/YAP signaling pathway and cytoskeleton formation in NCI-H716 cells. Using NCI-H716 cells as a model cell line for GLP1 secretion, we analyzed the effect of CK on the expression of RhoA/ROCK/YAP pathway components. Our results suggest that the effect of CK on GLP1 secretion depends on the anti-inflammatory effect of CK. We also demonstrated that CK can affect the RhoA/ROCK/YAP pathway, which is downstream of transforming growth factor ß1 (TGFß1), by maintaining the capacity of intestinal differentiation. In addition, this effect was mediated by regulating F/G-actin dynamics. These results provide not only the mechanistic insight for the effect of CK on intestinal L cells but also the molecular basis for the further development of CK as a potential therapeutic agent to treat type 2 diabetes mellitus (T2D).

Mapping the Endothelial Cell S-Sulfhydrome Highlights the Crucial Role of Integrin Sulfhydration in Vascular Function.

BACKGROUND: In vascular endothelial cells, cysteine metabolism by the cystathionine γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (H2Sn), that exert their biological actions via cysteine S-sulfhydration of target proteins. This study set out to map the "S-sulfhydrome" (ie, the spectrum of proteins targeted by H2Sn) in human endothelial cells. METHODS: Liquid chromatography with tandem mass spectrometry was used to identify S-sulfhydrated cysteines in endothelial cell proteins and ß3 integrin intraprotein disulfide bond rearrangement. Functional studies included endothelial cell adhesion, shear stress-induced cell alignment, blood pressure measurements, and flow-induced vasodilatation in endothelial cell-specific CSE knockout mice and in a small collective of patients with endothelial dysfunction. RESULTS: Three paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low); (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression; and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing H2Sn donor, SG1002. The endothelial cell "S-sulfhydrome" consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on ß3 integrin in detail we found that S-sulfhydration affected intraprotein disulfide bond formation and was required for the maintenance of an extended-open conformation of the ß leg. ß3 integrin S-sulfhydration was required for endothelial cell mechanotransduction in vitro as well as flow-induced dilatation in murine mesenteric arteries. In cultured cells, the loss of S-sulfhydration impaired interactions between ß3 integrin and Gα13 (guanine nucleotide-binding protein subunit α 13), resulting in the constitutive activation of RhoA (ras homolog family member A) and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low H2Sn generation, impaired flow-induced dilatation, and failure to detect ß3 integrin S-sulfhydration, all of which were rescued after the administration of an H2Sn supplement. CONCLUSIONS: Vascular disease is associated with marked changes in the S-sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short-term H2Sn supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease.

RhoA/Rock2/Limk1/cofilin1 pathway is involved in attenuation of neuronal dendritic spine loss by paeonol in the frontal cortex of D-galactose and aluminum­induced Alzheimer's disease­like rat model.

Alzheimer's disease (AD) has become the most prevalent neurodegenerative disorder. Given the pathogenesis of AD is unclear, there is currently no drug approved to halt or delay the progression of AD. Therefore, it is pressing to explore new targets and drugs for AD. In China, polyphenolic Chinese herbal medicine has been used for thousands of years in clinical application, and no toxic effects have been reported. In the present study, using D­galactose and aluminum­induced rat model, the effects of paeonol on AD were validated via the Morris water maze test, open field test, and elevated plus maze test. Neuronal morphology in frontal cortex was assessed using ImageJ's Sholl plugin and RESCONSTRUCT software. RhoA/Rock2/Limk1/cofilin1 signaling pathway­related molecules were determined by Western blotting. Cofilin1 and p­cofilin1 were analyzed by immunofluorescence. Results showed that pre­treatment with paeonol attenuated D­galactose and aluminum­induced behavioral dysfunction and AD­like pathological alterations in the frontal cortex. Accompanied by these changes were the alterations in the dendrite and dendritic spine densities, especially the mushroom­type and filopodia­type spines in the apical dendrites, as well as actin filaments. In addition, the activity and intracellular distribution of cofilin1 and the molecules RhoA/Rock2/Limk1 that regulate the signaling pathway for cofilin1 phosphorylation have also changed. Our data suggests that paeonol may be through reducing Aß levels to alleviate the loss of fibrillar actin and dendrites and dendritic spines via the Rho/Rock2/Limk1/cofilin1 signaling pathway in the frontal cortex, and ultimately improving AD­like behavior.

Electroacupuncture promotes axonal regrowth by attenuating the myelin-associated inhibitors-induced RhoA/ROCK pathway in cerebral ischemia/reperfusion rats.

The limited capacity of central nerve regeneration after cerebral ischemia has been the focus of attention in the field. Electroacupuncture (EA) is an effective therapy for functional rehabilitation after cerebral stroke. However, the underlying mechanism is still unclear. This study explored whether EA can improve the inhibitory microenvironment, attenuate RhoA/ROCK-mediated neurite regrowth inhibitory pathways, andpromote the expression of neuroplasticity proteins, thus exerting a protective role in cerebral ischemia/reperfusion (I/R) injury. Rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h followed by 7 days of reperfusion, and they received EA or fasudil once daily for 7 days. The Garcia JH score, 2,3,5-triphenyltetrazolium chloride (TTC) staining, and transmission electron microscopy (TEM) were used to assess neural injury. The protein and mRNA levels of myelin-associated inhibitors (MAIs), RhoA/ROCK pathway-related molecules and neuroplasticity-related proteins were examined to explore the effect of EA on rats with cerebral I/R injury. We found that EA significantly decreased the infarct size and improved neurological function and hippocampal ultrastructure in the rats with cerebral ischemia/reperfusion (I/R) injury. EA ameliorates the inhibition of axonal regrowth and provides a protective role in functional rehabilitation after cerebral stroke by downregulating the MAI-induced RhoA/ROCK signaling pathway and by promoting the expression of GAP43 and BDNF to protect against cerebral I/R injury. Our findings provide a better understanding of the molecular mechanism underlying EA as an effective therapy for ischemic stroke.