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Myocardin-related transcription factors (MRTFs) play a central role in the regulation of actin expression and cytoskeletal dynamics that are controlled by Rho GTPases. SRF is a ubiquitous transcription factor strongly expressed in muscular tissues. The depletion of SRF in the adult mouse heart leads to severe dilated cardiomyopathy associated with the down-regulation of target genes encoding sarcomeric proteins including α-cardiac actin. The regulatory triad, composed of SRF, its cofactor MRTFA and actin, plays a major role in the coordination of the nuclear transcriptional response to adapt actin filament dynamics associated with changes in cell shape, and contractile and migratory activities. Most of the knowledge on the regulation of the SRF–MRTF–Actin axis has been obtained in non-muscle cells with α-actin and smooth muscle cells with α-smooth actin. Here, we visualized for the first time by a time-lapse video, the nucleocytoplasmic shuttling of MRTFA induced by serum or pro-hypertrophic agonists such as angiotensin II, phenylephrine and endothelin-1, using an MRTFA-GFP adenovirus in cultures of neonatal rat cardiomyocytes. We showed that an inhibitor of the RhoA/ROCK signaling pathway leads to an α-cardiac actin polymerization disruption and inhibition of MRTFA nucleocytoplasmic shuttling. Moreover, inhibition of the PI3K/Akt signaling pathway also prevents the entry of MRTFA into the nuclei. Our findings point out a central role of the SRF–MRTFA–actin axis in cardiac remodeling.

With increasing demands on long-term storage of cells, cryopreservation of cells is gaining more importance in cell-based research and applications. Dimethyl sulfoxide (DMSO) is a commonly used chemical cryoprotectant, providing increased cell survival during the freezing process. However, its use is limited in clinical applications due to its low biocompatibility above cryogenic temperatures. Herein, we present a new approach for reducing the use of DMSO in cryopreservation by using biodegradable hyaluronic acids (HAs). By adding HAs into cryoprotectant media containing a low concentration of DMSO, higher cell viability and cell proliferation rate were observed upon thawing after cryopreservation. The HA-supplemented cryopreservation media did not reduce the size of the ice crystal, which significantly influenced cell viability during cell freezing, but decreased the Ras homolog family member A (RhoA)/Rho-associated protein kinase (ROCK) signaling pathway related to apoptosis. The cell-interactive cryoprotectants containing HA can be applied to the development of a new cryoprotectant that reduces the adverse effect of DMSO.

The destruction of the blood-brain barrier (BBB) contributes to a spectrum of neurological diseases such as stroke, and the hyperpermeability of endothelial cells is one of the characters of stroke, which is possibly exacerbated after reperfusion. However, the underlying mechanisms involving hyperpermeability after reperfusion between the endothelial cells remain poorly understood. Therefore, in the present study, the human microvascular endothelial cells (HBMECs) were exposed to oxygen-glucose deprivation/reperfusion (OGD/R) to mimic ischemic stroke condition in vitro with the aim to investigate the potential mechanisms induced by OGD/R. The permeability of cultured HBMECs was measured using FITC-labeled dextran in a Transwell system and transendothelial electrical resistance (TEER), while the RhoA activity was detected by pull-down assay. In addition, the phosphorylation of MYPT1, which reflects the activation of ROCK and the internalization of VE-cadherin, was detected by Western blot. It showed that OGD/R treatment significantly increased the permeability of HBMEC monolayers and facilitated the internalization of VE-cadherin in HBMEC monolayers. Pull-down assay showed that RhoA activation was obviously enhanced after OGD/R treatment, while RhoA and ROCK inhibitor significantly reversed OGD/R-induced HBMEC monolayers hyperpermeability and the internalization of VE-cadherin. Meanwhile, the knockdown assay showed that RhoA small interfering RNA (siRNA) led to similar effects. The inactivation of the downstream effector protein ROCK was also examined. Intriguingly, ROCK2 rather than ROCK1 exerted its adverse effects on HBMEC monolayer integrity, since ROCK2 knockdown markedly reverses the injury of OGD/R in HBMEC monolayers. In conclusion, the present study provides evidence that OGD/R may induce HBMEC monolayer hyperpermeability via RhoA/ROCK2-mediated VE-cadherin internalization, which may provide an impetus for the development of therapeutics targeting BBB damage in ischemic stroke.

The mechanism of the immediate adverse drug reactions (ADRs) induced by ShenMai injection (SMI) has not been completely elucidated. Within 30 minutes, the ears and lungs of mice injected with SMI for the first time showed edema and exudation reactions. These reactions were different from the IV hypersensitivity. The theory of pharmacological interaction with immune receptor (p-i) offered a new insight into the mechanisms of immediate ADRs induced by SMI. In this study, we determined that the ADRs were mediated by thymus-derived T cells through the different reactions of BALB/c mice (thymus-derived T cell normal) and BALB/c nude mice (thymus-derived T cell deficient) after injecting SMI. The flow cytometric analysis, cytokine bead array (CBA) assay and untargeted metabolomics were used to explain the mechanisms of the immediate ADRs. Moreover, the activation of the RhoA/ROCK signaling pathway was detected by western blot analysis. In BALB/c mice, the vascular leakage and histopathology results showed the occurrence of the immediate ADRs induced by SMI. The flow cytometric analysis revealed that CD4 T cell subsets (Th1/Th2, Th17/Treg) were imbalanced. And the levels of cytokines such as IL-2, IL-4, IL12P70 and INF-γ increased significantly. However, in BALB/c nude mice, all the indicators mentioned above have not changed significantly. The metabolic profile of both BALB/c mice and BALB/c nude mice was significantly changed after injecting SMI, and the notable increase in lysolecithin level might have a greater association with the immediate ADRs induced by SMI. The Spearman correlation analysis revealed that LysoPC (18:3(6Z,9Z,12Z)/0:0) showed a significant positive correlation with cytokines. After injecting SMI, the levels of RhoA/ROCK signaling pathway-related protein increased significantly in BALB/c mice. Protein-protein interaction (PPI) showed that the increased lysolecithin levels might be related to the activation of the RhoA/ROCK signaling pathway. Together, the results of our study revealed that the immediate ADRs induced by SMI were mediated by thymus-derived T cells, and elucidated the mechanisms of such ADRs. This study provided new insights into the underlying mechanism of immediate ADRs induced by SMI.

Metabolites/impurities (MIs) of penicillin are normally considered to be the main substances inducing immediate hypersensitivity reactions in penicillin treatment. Our previous research found that penicillin can cause non-allergic hypersensitivity reactions (NAHRs) by directly triggering vascular hyperpermeability and exudative inflammation. However, the chief culprits and underlying mechanisms involved in penicillin-induced NAHRs have not yet been fully elucidated. In this study, we used a combination of approaches including a mouse non-allergic hypersensitivity reaction model, UPLC-MS/MS analyses of arachidonic acid metabolites (AAMs), immunoblotting technique, and molecular docking, etc to investigate the culprits involved in penicillin-induced hypersensitivity reactions. We found penilloic acid, one of the main MIs of penicillin, could trigger NAHRs via inducing increased vascular permeability, while the other MIs did no exhibit similar effect. Penilloic acid-induced reactions were not IgE-dependent. Significantly increased arachidonic acids and cascade metabolites in lungs, and activation of RhoA/ROCK signaling pathway in the ears and lungs of mice were noticed after once administration of penilloic acid. This study revealed that penilloic acid was the chief culprit involved in penicillin-induced immediate NAHRs in mice, which mainly associated with direct stimulation of vascular hyperpermeability and exudative inflammation. The activations of AAMs and RhoA/ROCK signaling pathway played important roles in these reactions.

Objective: This study investigated the role of dexmedetomidine (DEX) in dextran sulfate sodium (DSS)-induced NCM460 cells. Material and Methods: The viability and apoptosis of NCM460 cells treated with DEX with or without DSS were detected by CCK-8 and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay. The level of inflammatory factors and expression of inflammation-related proteins, tight junction proteins and Ras homolog gene family, member A/Rho-associated coiled-coil containing protein kinase (RhoA/ROCK) signaling-related proteins in NCM460 cells treated with DEX and/or U46619 (RhoA/ROCK agonist) and/or DSS were detected by the respective enzyme-linked immunosorbent assay (ELISA) kits and Western blot analysis. The permeability of NCM460 monolayers was examined with transepithelial electrical resistance (TEER) assay. Results: DEX had no effect on NCM460 cell viability. However, DEX improved the viability and barrier damage and suppressed the apoptosis and inflammation of DSS-induced NCM460 cells. Correspondingly, the expression of inflammation-related proteins was reduced and the expression of tight junction proteins was increased in DSS-induced NCM460 cells after treatment with DEX. In addition, RhoA/ROCK signaling was activated in NCM460 cells induced by DSS, which was suppressed by DEX. The protective effects of DEX on DSS-indued NCM460 cells were reversed by U46619. Conclusion: DEX improved viability and barrier damage while suppressed apoptosis and inflammation in DSS-indued NCM460 cells by inhibiting RhoA/ROCK signaling pathway.

[This corrects the article DOI: 10.3389/fcvm.2022.873829.].[This corrects the article DOI: 10.3389/fcvm.2022.873829.].

Methamphetamine (METH) is a powerful addictive psychostimulant that gives rise to severe abusers worldwide. While many studies have reported on the neurotoxicity of METH, blood–brain barrier (BBB) dysfunction has recently attracted attention as an essential target in METH-induced pathological changes in the brain. However, its mechanism has not been fully understood. We found that METH increased paracellular permeability and decreased vascular integrity through FITC–dextran and trans-endothelial electrical resistance (TEER) assay in primary human brain endothelial cells (HBMECs). Also, redistribution of tight junction proteins (zonula occluden-1 and claudin-5) and reorganization of F-actin cytoskeleton were observed in METH-exposed HBMECs. To determine the mechanism of METH-induced BBB disruption, the RhoA/ROCK signaling pathway was examined in METH-treated HBMECs. METH-activated RhoA, followed by an increase in the phosphorylation of downstream effectors, myosin light chain (MLC) and cofilin, occurs in HBMECs. Pretreatment with ROCK inhibitors Y-27632 and fasudil reduced the METH-induced increase in phosphorylation of MLC and cofilin, preventing METH-induced redistribution of junction proteins and F-actin cytoskeletal reorganization. Moreover, METH-induced BBB leakage was alleviated by ROCK inhibitors in vitro and in vivo. Taken together, these results suggest that METH induces BBB dysfunction by activating the RhoA/ROCK signaling pathway, which results in the redistribution of junction proteins via F-actin cytoskeletal reorganization.

Shuanghuanglian injection (SHLI) is a famous Chinese medicine used as an intravenous preparation for the treatment of acute respiratory tract infections. In the recent years, the immediate hypersensitivity reactions induced by SHLI have attracted broad attention. However, the mechanism involved in these reactions has not yet been elucidated. The present study aims to explore the characteristics of the immediate hypersensitivity reactions induced by SHLI and deciphers the role of the RhoA/ROCK signaling pathway in these reactions. SHLI-immunized mice or naive mice were intravenously injected (i.v.) with SHLI (600 mg/kg) once, and vascular leakage in the ears was evaluated. Passive cutaneous anaphylaxis test was conducted using sera collected from SHLI-immunized mice. Naive mice were administered (i.v.) with a single dose of 150, 300, or 600 mg/kg of SHLI, and vascular leakage, histamine release, and histopathological alterations in the ears, lungs, and intestines were tested. , human umbilical vein endothelial cell (HUVEC) monolayer was incubated with SHLI (0.05, 0.1, or 0.15 mg/mL), and the changes in endothelial permeability and cytoskeleton were observed. Western blot analysis was performed and ROCK inhibitor was employed to investigate the contribution of the RhoA/ROCK signaling pathway in SHLI-induced hypersensitivity reactions, both in HUVECs and in mice. Our results indicate that SHLI was able to cause immediate dose-dependent vascular leakage, edema, and exudates in the ears, lungs, and intestines, and histamine release in mice. These were pseudo-allergic reactions, as SHLI-specific IgE was not elicited during sensitization. In addition, SHLI induced reorganization of actin cytoskeleton and disrupted the endothelial barrier. The administration of SHLI directly activated the RhoA/ROCK signaling pathway both in HUVECs and in the ears, lungs, and intestines of mice. Fasudil hydrochloride, a ROCK inhibitor, ameliorated the SHLI-induced hypersensitivity reactions in both endothelial cells and mice indicating its protective effect. SHLI-induced pseudo-allergic reactions were mediated by the activation of the RhoA/ROCK signaling pathway. : This study presents a novel mechanism of SHLI-induced immediate hypersensitivity reactions and suggests a potential therapeutic strategy to prevent the associated adverse reactions.

Preterm birth (PTB) remains a challenging issue in the reproductive field, and cervical maturation is an essential physiological prerequisite for parturition. The cervix is rich in smooth muscle cells, and their abnormal contractility is a key trigger for premature cervical remodeling, which may further lead to spontaneous PTB. Herein, we found that high serum arachidonic acid (AA) expression in PTB mice may predict potential harms through untargeted metabolomics analysis. After AA intervention, immunofluorescence/qPCR/WB revealed that the expression of cervical smooth muscle contraction indexes calponin/oxytocin receptor (OR)/connexin‐34 and premature birth‐related factors cyclooxygenase‐2 (COX‐2) increased significantly, indicating that AA acting on cervical smooth muscle cells may lead to premature birth. WB results showed that the expression of phosphorylated myosin light chain (p‐MLC) in cervical smooth muscle cells treated with AA increased significantly, and myosin light chain (MLC) protein was closely related to smooth muscle contraction. After adding the Ras homolog gene family member A (RhoA)/Rho‐associated coiled‐coil forming protein kinase (ROCK) pathway inhibitor, the expression of p‐MLC decreased significantly, indicating that AA could induce MLC phosphorylation through the RhoA/ROCK signaling pathway to cause cervical smooth muscle shrinkage and lead to premature birth. In summary, our findings provided evidence that AA enhanced cervical smooth muscle contraction and led to PTB by inducing MLC phosphorylation through the RhoA/ROCK signaling pathway. Hence, our study provided new insights into mechanisms linking cervical smooth muscle contraction to PTB muscle shrinkage, suggesting that AA could be a potential novel drug intervention target for PTB therapy.