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.

Liquiritigenin alleviates doxorubicin-induced chronic heart failure via promoting ARHGAP18 and suppressing RhoA/ROCK1 pathway.

Chronic heart failure (CHF) is one of the most common chronic diseases with increasing incidence and mortality. Liquiritigenin (LQG) is shown to protect mice from cardiotoxicity. However, its underlying mechanism remains unclear. Our study aimed to reveal the role of ARHGAP18 in LQG-mediated cardioprotective effects in CHF. In the current study, CHF cell model and rat model were established by the application of doxorubicin (DOX). The reactive oxygen species (ROS) level and cell apoptosis were determined by flow cytometry. The cardiac function of rats was evaluated by measuring left ventricular systolic pressure, left ventricular end diastolic pressure, and serum level of lactate dehydrogenase and brain natriuretic peptide. The expression of active RhoA was elevated and that of ARHGAP18 was decreased in DOX-induced CHF cell model. ARHGAP18 could reduce DOX-induced RhoA activation, ROS elevation, and cell apoptosis. Meanwhile, the knockdown of ARHGAP18 could promote the activation of RhoA, the level of ROS, and the rate of cell apoptosis, which could be reversed by the application of RhoA inhibitor. LQG promoted the expression of ARHGAP18 and exerted similar effects of ARHGAP18 in CHF cell model. The application of LQG could also reverse the effects mediated by ARHGAP18 knockdown. Moreover, LQG significantly improved cardiac function and ameliorated DOX-induced cardiotoxicity of CHF rats. In conclusion, LQG could alleviate DOX-induced CHF via promoting ARHGAP18 and suppressing RhoA/ROCK1 pathway. LQG was a potential agent for CHF treatment.

Design, synthesis, and biological evaluation of urea-based ROCK2 inhibitors.

A series of urea-based ROCK2 inhibitors were design and synthesized. The inhibitory activity on ROCK2 was screened by enzyme-linked immunosorbent assay (ELISA). The study results showed that the urea derivatives exhibited certain ROCK2 inhibitory activity. The most potent compound 10p showed ROCK2 inhibitory activity with the IC50  value of 0.03 µM. A preliminary structure-activity relationship was then summarized. The molecular docking studies showed that further optimization needs to conduct to obtain more potent ROCK inhibitors.

Immortalization and Characterization of Rat Lingual Keratinocytes in a High-Calcium and Feeder-Free Culture System Using ROCK Inhibitor Y-27632.

Cultured keratinocytes are desirable models for biological and medical studies. However, primary keratinocytes are difficult to maintain, and there has been little research on lingual keratinocyte culture. Here, we investigated the effect of Y-27632, a Rho kinase (ROCK) inhibitor, on the immortalization and characterization of cultured rat lingual keratinocyte (RLKs). Three Y-27632-supplemented media were screened for the cultivation of RLKs isolated from Sprague-Dawley rats. Phalloidin staining and TUNEL assay were applied to visualize cytoskeleton dynamics and cell apoptosis following Y-27632 removal. Label-free proteomics, RT-PCR, calcium imaging, and cytogenetic studies were conducted to characterize the cultured cells. Results showed that RLKs could be conditionally immortalized in a high-calcium medium in the absence of feeder cells, although they did not exhibit normal karyotypes. The removal of Y-27632 from the culture medium led to reversible cytoskeletal reorganization and nuclear enlargement without triggering apoptosis, and a total of 239 differentially expressed proteins were identified by proteomic analysis. Notably, RLKs derived from the non-taste epithelium expressed some molecular markers characteristic of taste bud cells, yet calcium imaging revealed that they rarely responded to tastants. Collectively, we established a high-calcium and feeder-free culture method for the long-term maintenance of RLKs. Our results shed some new light on the immortalization and differentiation of lingual keratinocytes.

Simultaneous Use of ROCK Inhibitors and EP2 Agonists Induces Unexpected Effects on Adipogenesis and the Physical Properties of 3T3-L1 Preadipocytes.

To elucidate the additive effects of an EP2 agonist, omidenepag (OMD) or butaprost (Buta) on the Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, ripasudil (Rip) on adipose tissue, two- or three-dimension (2D or 3D) cultures of 3T3-L1 cells were analyzed by lipid staining, the mRNA expression of adipogenesis-related genes, extracellular matrix (ECM) molecules including collagen (Col) -1, -4 and -6, and fibronectin (Fn), and the sizes and physical properties of 3D organoids, as measured by a micro-squeezer. The results indicate that adipogenesis induced (1) an enlargement of the 3D organoids; (2) a substantial enhancement in lipid staining as well as the expression of the Pparγ, Ap2 and Leptin genes; (3) a significant softening of the 3D organoids, the effects of which were all enhanced by Rip except for Pparγ expression; and (4) a significant downregulation in Col1 and Fn, and a significant upregulation in Col4, Col6, the effects of which were unchanged by Rip. When adding the EP2 agonist to Rip, (1) the sizes of the 3D organoids were reduced substantially; (2) lipid staining was increased (OMD), or decreased (Buta); (3) the stiffness of the 3D organoids was substantially increased in Buta; (4-1) the expression of Pparγ was suppressed (2D, OMD) or increased (2D, Buta), and the expressions of Ap2 were downregulated (2D, 3D) and Leptin was increased (2D) or decreased (3D), (4-2) all the expressions of four ECM molecules were upregulated in 2D (2D), and in 3D, the expression of Col1, Col4 was upregulated. The collective findings reported herein indicate that the addition of an EP2 agonist, OMD or Buta significantly but differently modulate the Rip-induced effects on adipogenesis and the physical properties of 2D and 3D cultured 3T3-L1 cells.

Xuesaitong exerts long-term neuroprotection for stroke recovery by inhibiting the ROCKII pathway, in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Xuesaitong (XST) is a traditional Chinese medicine injection with neuroprotective properties and has been extensively used to treat stroke for many years. The main component of XST is Panax notoginseng saponins (PNS), which is the main extract of the Chinese herbal medicine Panax notoginseng. AIM OF THE STUDY: In this study, we investigated whether XST provided long-term neuroprotection by inhibiting neurite outgrowth inhibitor-A (Nogo-A) and the ROCKII pathway in experimental rats after middle cerebral artery occlusion (MCAO) and in SH-SY5Y cells exposed to oxygen-glucose deprivation/reperfusion (OGD/R). MATERIALS AND METHODS: Rats with permanent MCAO were administered XST, Y27632, XST plus Y27632, and nimodipine for 14 and 28 days. Successful MCAO onset was confirmed by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. Neurological deficit score (NDS) was used to assess neurological impairment. Hematoxylin-eosin (HE) staining and immunohistochemical (IHC) analysis of synaptophysin (SYN) and postsynaptic density protein-95 (PSD-95) were performed to evaluate cerebral ischemic injury and the neuroprotective capability of XST. Nogo-A levels and the ROCKII pathway were detected by IHC analysis, western blotting, and quantitative real-time polymerase chain reaction (qRT-PCR) to explore the protective mechanism of XST. OGD/R model was established in SH-SY5Y cells. Cell counting kit 8 (CCK8) was applied to detect the optimum OGD time and XST concentration. The expression levels Nogo-A and ROCKII pathway were determined using western blotting. RESULTS: Our results showed that XST reduced neurological dysfunction and pathological damage, promoted weight gain and synaptic regeneration, reduced Nogo-A mRNA and protein levels, and inhibited the ROCKII pathway in MCAO rats. CCK8 assay displayed that the optimal OGD time and optimal XST concentration were 7 h and 20 µg/mL respectively in SH-SY5Y cells. XST could evidently inhibit OGD/R-induced Nogo-A protein expression and ROCKII pathway activation in SH-SY5Y cells. CONCLUSIONS: The present study suggested that XST exerted long-term neuroprotective effects that assisted in stroke recovery, possibly through inhibition of the ROCKII pathway.

YiQiFuMai Lyophilized Injection ameliorates tPA-induced hemorrhagic transformation by inhibiting cytoskeletal rearrangement associated with ROCK1 and NF-κB signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Thrombolytic therapy with tissue plasminogen activator (tPA) after ischemic stroke exacerbates blood-brain barrier (BBB) breakdown and leads to hemorrhagic transformation (HT). YiQiFuMai Lyophilized Injection (YQFM) is a modern preparation derived from Sheng-mai San (a traditional Chinese medicine). YQFM attenuates the BBB dysfunction induced by cerebral ischemia-reperfusion injury. However, whether YQFM can suppress tPA-induced HT remains unknown. AIM OF THE STUDY: We investigated the therapeutic effect of YQFM on tPA-induced HT and explored the underlying mechanisms in vivo and in vitro to improve the safety of tPA use against stroke. METHODS: Male C57BL/6J mice were subjected to 45 min of ischemia and 24 h of reperfusion. tPA (10 mg/kg) were infused 2 h after occlusion and YQFM (0.671 g/kg) was injected 2.5 h after occlusion. The in vitro effect of YQFM (100, 200, 400 µg/mL) on tPA (60 µg/mL)-induced dysfunction of the microvascular endothelial barrier in the brain following oxygen-glucose deprivation/reoxygenation (OGD/R) was observed in bEnd.3 cells. RESULTS: YQFM suppressed tPA-induced high hemoglobin level in the brain, mortality, neurologic severity score, BBB permeability, expression and activation of matrix metalloproteinase (MMP)-9 and MMP-2, and degradation of tight-junction proteins. Furthermore, YQFM significantly blocked tPA-induced brain microvascular endothelial permeability and phosphorylation of Rho-associated kinase (ROCK)1, myosin light chain (MLC), cofilin and p65 in vivo and in vitro. CONCLUSION: YQFM suppressed tPA-induced HT by inhibiting cytoskeletal rearrangement linked with ROCK-cofilin/MLC pathways and inhibiting the nuclear factor-kappa B pathway to ameliorate BBB damage caused by tPA.

ER stress induced by ER calcium depletion and UVB irradiation regulates tight junction barrier integrity in human keratinocytes.

BACKGROUND: Endoplasmic reticulum (ER) calcium depletion-induced ER stress is a crucial signal for keratinocyte differentiation and barrier homeostasis, but its effects on the epidermal tight junction (TJ) have not been characterized. Ultraviolet B (UVB) causes ER calcium release in keratinocytes and disrupts epidermal TJ, however, the involvement of ER stress in the UVB-induced TJ alterations remains unknown. OBJECTIVES: To investigate the effect of ER stress by pharmacological ER calcium depletion or UVB on the TJ integrity in normal human epidermal keratinocytes (NHEK). METHODS: NHEK were exposed to ER calcium pump inhibitor thapsigargin (Tg) or UVB. ER stress markers and TJ molecules expression, TJ and F-actin structures, and TJ barrier function were analyzed. RESULTS: Tg or UVB exposure dose-dependently triggered unfolded protein response (UPR) in NHEK. Low dose Tg induced the IRE1α-XBP1 pathway and strengthened TJ barrier. Contrary, high dose Tg activated PERK phosphorylation and disrupted TJ by F-actin disorganization. UVB disrupted TJ and F-actin structures dose dependently. IRE1α RNase inhibition induced or exacerbated TJ and F-actin disruption in the presence of low dose Tg or UVB. High dose Tg increased RhoA activity. 4-PBA or Rho kinase (ROCK) inhibitor partially prevented the disruption of TJ and F-actin following high dose Tg or UVB. CONCLUSIONS: ER stress has bimodal effects on the epidermal TJ depending on its intensity. The IRE1α pathway is critical for the maintenance of TJ integrity during mild ER stress. Severe ER stress-induced UPR or ROCK signalling mediates the disruption of TJ through cytoskeletal disorganization during severe ER stress.

Rho-Kinase inhibitors ameliorate diclofenac-induced cardiotoxicity in chloroquine-treated adjuvant arthritic rats.

AIMS: Although chloroquine and diclofenac are not cardiovascular drugs, their chronic administration may trigger cardiotoxicity. We, therefore, evaluated the cardiotoxic impact of diclofenac in chloroquine-treated adjuvant arthritic rats and the protective role of Rho-kinase inhibitors. METHODS: 90 male rats were equally distributed into 9 groups including control. Arthritis was induced by S.C injection of Complete Freund's adjuvant in hind paw plantar surface. Arthritic rats were subdivided into 8 groups, orally treated with: no drug, chloroquine (50 mg/kg), diclofenac sodium (1 mg/kg) and chloroquine + diclofenac. To study the role of Rho-kinase in chloroquine/diclofenac-triggered cardiotoxicity, four arthritic groups were also co-treated with Rho-kinase inhibitors (fasudil or atorvastatin) along with diclofenac and chloroquine + diclofenac. KEY FINDINGS: All treatments significantly elevated serum cardiac injury and dysfunction markers as well as left ventricular malondialdehyde but depleted antioxidants with the greatest effect in the combination group. Chloroquine and/or diclofenac; in particular, their combination shifted the balance between left ventricular pro- and anti-apoptotic proteins towards myocardial apoptosis. Surprisingly, treatment with diclofenac or chloroquine/diclofenac markedly up-regulated cardiac RhoA and Rho-kinase1. Such up-regulation was coupled with a greater increase in cardiac oxidative damage biomarkers in the combination group than in individually-treated ones. However, Rho-kinase inhibition protected against diclofenac-induced increase in myocardial oxidative damage markers. SIGNIFICANCE: Diclofenac greatly amplified cardiac oxidative damage in chloroquine-treated arthritic rats via up-regulation of Rho-kinase1. However, Rho-kinase inhibitors provided cardioprotection against diclofenac toxicity. Overall, they could be used as safer adjuvants to diclofenac during the treatment of rheumatoid arthritis with chloroquine.

Tetrandrine Suppresses Transient Receptor Potential Cation Channel Protein 6 Overexpression- Induced Podocyte Damage via Blockage of RhoA/ROCK1 Signaling.

OBJECTIVE: Podocyte damage is common in many renal diseases characterized by proteinuria. Transient receptor potential cation channel protein 6 (TRPC6) plays an important role in renal function through its regulation of intracellular Ca2+ influx and RhoA/ROCK pathways. Chinese herb Stephania tetrandra, with the main active component being tetrandrine, has been used for the treatment of various kidney diseases for several years and has shown a positive effect. This study aimed at investigating the effect and mechanism of tetrandrine in podocyte damage induced by high expression of TRPC6. METHODS: Immortalized, differentiated murine podocytes, MPC5 were treated with valsartan (0-800 µM) and tetrandrine (0-40 µM) for 48 h. The maximum safe concentrations of valsartan and tetrandrine were selected using a cell viability assay. MPC5 podocytes stably expressing TRPC6 were constructed using a lentivirus packaging system, followed by treatment with valsartan, tetrandrine, and Y-27632 for 48 h and U73122 (10 µM) for 10 min. The RhoA/ROCK pathway and podocyte-specific proteins (nephrin and synaptopodin) levels were quantified. Podocyte apoptosis and intracellular Ca2+ concentration were measured. RESULTS: Maximum safe concentrations of 100 µM valsartan and 10 µM tetrandrine showed no observable toxicity in podocytes. MPC5 podocytes stably expressing TRPC6 had higher intracellular Ca2+ influx, apoptotic percentages, and expression of RhoA/ROCK proteins, but lower expression of nephrin and synaptopodin proteins. U73122 treatment for 10 min did not inhibit TRPC6, but suppressed RhoA/ROCK protein. Y-27632 decreased ROCK1 expression, but did not influence the expression of TRPC6 protein. Both 100 µM valsartan and 10 µM tetrandrine for 48 h significantly inhibited intracellular Ca2+ influx, apoptosis, and RhoA/ROCK pathway, and increased nephrin and synaptopodin proteins in podocytes stably expressing TRPC6. CONCLUSION: Elevated TRPC6 expression can lead to podocyte injury by inducing intracellular Ca2+ influx and apoptosis of podocytes, and this effect may be mediated by activation of the RhoA/ROCK1 pathway. Tetrandrine can alleviate podocyte injury induced by TRPC6 expression through inhibition of the RhoA/ROCK pathway, suggesting a protective role in podocyte damage.

Salvianolic acid B protects against oxLDL-induced endothelial dysfunction under high-glucose conditions by downregulating ROCK1-mediated mitophagy and apoptosis.

Diabetes is related to alterations in glucose and lipid metabolism, which are linked to endothelial cell (EC) dysfunction. Salvianolic acid B (Sal B), one of the major ingredient of Danshen (Salvia miltiorrhiza), possesses many of the biological activities. However, protective effect of Sal B against oxLDL induced ECs dysfunction under high glucose condition (high Glu) is not well known. Thus, in this study, we investigated the protective effects of Sal B against EC dysfunction induced by oxLDL and high Glu and examined the associated mechanisms. Our results showed that Sal B significantly and dose-dependently decreased oxLDL- and high Glu-mediated induction of lectin-like oxLDL receptor-1 and significantly decreased oxLDL- and high Glu-induced mitochondrial ROS (mtROS) production and mitochondrial DNA (mtDNA) expression. In addition, oxLDL stimulation under high-Glu conditions activated the intrinsic apoptosis pathway in ECs. These effects were abolished by Sal B through reductions in mtROS and mtDNA. Furthermore, Sal B inhibited oxLDL- and high Glu-induced increases in fission protein (p-DRP 1 and FIS 1) levels. OxLDL and high Glu activated the ROCK1 pathway, which is involved in apoptosis and mitophagy, while Sal B significantly reduced ROCK1 protein levels. The protective effects of Sal B against oxLDL- and high Glu-induced endothelial dysfunction may be mediated by reductions in apoptosis-related proteins and fission proteins through suppression of the ROCK1-mediated pathway.

ROCK Y-27632 Inhibitor, Ascorbic Acid, and Trehalose Increase Survival of Human Wharton Jelly Mesenchymal Stem Cells After Cryopreservation.

OBJECTIVES: Wharton jelly mesenchymal stem cells are good candidates for application in different aspects of regenerative medicine, and their long-time banking is important. In this study, the effects of trehalose, ascorbic acid, and Y-27632 on proliferation and survival rate of these cells after cryopreservation were investigated. MATERIALS AND METHODS: Mesenchymal stem cells were isolated from human umbilical cord Wharton jelly and frozen using a slow-rate cooling process. Different concentrations of trehalose (35, 75, and 125 mM), ascorbic acid (0.06, 0.125, 0.25, and 0.5 mM), and Y-27632 (10 µM) were used to treat culture medium and/or to supplement freezing medium. Assessment of cell viability after thawing was performed using Trypan blue staining, and MTT assay was performed to measure the cell proliferation rate. RESULTS: We observed significantly increased postthaw viability, increased cell proliferation, and decreased doubling time of cells when 75 mM trehalose, 0.25 and 0.5 mM ascorbic acid, and 10 mM Y-27632 were used. In addition, increased viability, proliferation, and attachment were observed after 24 hours of pretreatment with these cryoprotective agents and when they were added to conventional freezing medium. CONCLUSIONS: The use of different cryoprotective agents in culture and freezing media could be useful for long-term storage of Wharton jelly mesenchymal stem cells.

RKI-1447 suppresses colorectal carcinoma cell growth via disrupting cellular bioenergetics and mitochondrial dynamics.

Accumulated evidence suggested the importance of the Rho/Rho-kinase (ROCK) signaling pathway in cancer proliferation and invasion. However, its role in colorectal carcinoma (CRC) is not well understood. This study evaluated the effect of ROCK signaling pathway on CRC behavior on the basis of a novel Rho/ROCK inhibitor RKI-1447. Here, we found RKI-1447 could drastically suppress HCT-8 and HCT-116 cell growth and promoted apoptosis. Our in vitro data indicated suppressed cytoskeletal dynamics induced by RKI-1447 inhibition on mitochondrial respiration, which was evidenced by basal and maximal respiration rates, and ATP production. Simultaneously, cellular basal and maximal glycolytic rates, and glycolytic capacity were also reduced in response to RKI-1447. Moreover, RKI-1447 caused excessive reactive oxygen species generation and membrane depolarization as well as activated ER-stress. We also demonstrated CHOP is essential for RKI-1447 induced cell apoptosis. Finally, we proved inhibition of ROCK by RKI-1447 could effectively inhibit CRC growth in vivo. Taken together, this study demonstrated that inhibition of ROCK signaling pathway by RKI-1447 could suppress CRC via cytoskeleton associated mitochondrial dysfunction and cellular bioenergetics disruption. Our data suggest RKI-1447 may be an attractive antitumor drug candidate for the treatment of CRC.

Catharmus tinctorius volatile oil promote the migration of mesenchymal stem cells via ROCK2/Myosin light chain signaling.

MSC transplantation has been explored as a new clinical approach to stem cell-based therapies for bone diseases in regenerative medicine due to their osteogenic capability. However, only a small population of implanted MSC could successfully reach the injured areas. Therefore, enhancing MSC migration could be a beneficial strategy to improve the therapeutic potential of cell transplantation. Catharmus tinctorius volatile oil (CTVO) was found to facilitate MSC migration. Further exploration of the underlying molecular mechanism participating in the pro-migratory ability may provide a novel strategy to improve MSC transplantation efficacy. This study indicated that CTVO promotes MSC migration through enhancing ROCK2 mRNA and protein expressions. MSC migration induced by CTVO was blunted by ROCK2 inhibitor, which also decreased myosin light chain (MLC) phosphorylation. Meanwhile, the siRNA for ROCK2 inhibited the effect of CTVO on MSC migration ability and attenuated MLC phosphorylation, suggesting that CTVO may promote BMSC migration via the ROCK2/MLC signaling. Taken together, this study indicates that C. tinctorius volatile oil could enhance MSC migration via ROCK2/MLC signaling in vitro. C. tinctorius volatile oil-targeted therapy could be a beneficial strategy to improve the therapeutic potential of cell transplantation for bone diseases in regenerative medicine.

Microengineered human amniotic ectoderm tissue array for high-content developmental phenotyping.

During early post-implantation human embryogenesis, the epiblast (EPI) within the blastocyst polarizes to generate a cyst with a central lumen. Cells at the uterine pole of the EPI cyst then undergo differentiation to form the amniotic ectoderm (AM), a tissue essential for further embryonic development. While the causes of early pregnancy failure are complex, improper lumenogenesis or amniogenesis of the EPI represent possible contributing factors. Here we report a novel AM microtissue array platform that allows quantitative phenotyping of lumenogenesis and amniogenesis of the EPI and demonstrate its potential application for embryonic toxicity profiling. Specifically, a human pluripotent stem cell (hPSC)-based amniogenic differentiation protocol was developed using a two-step micropatterning technique to generate a regular AM microtissue array with defined tissue sizes. A computer-assisted analysis pipeline was developed to automatically process imaging data and quantify morphological and biological features of AM microtissues. Analysis of the effects of cell density, cyst size and culture conditions revealed a clear connection between cyst size and amniogenesis of hPSC. Using this platform, we demonstrated that pharmacological inhibition of ROCK signaling, an essential mechanotransductive pathway, suppressed lumenogenesis but did not perturb amniogenic differentiation of hPSC, suggesting uncoupled regulatory mechanisms for AM morphogenesis vs. cytodifferentiation. The AM microtissue array was further applied to screen a panel of clinically relevant drugs, which successfully detected their differential teratogenecity. This work provides a technological platform for toxicological screening of clinically relevant drugs for their effects on lumenogenesis and amniogenesis during early human peri-implantation development, processes that have been previously inaccessible to study.

Rapamycin retards epigenetic ageing of keratinocytes independently of its effects on replicative senescence, proliferation and differentiation.

The advent of epigenetic clocks has prompted questions about the place of epigenetic ageing within the current understanding of ageing biology. It was hitherto unclear whether epigenetic ageing represents a distinct mode of ageing or a manifestation of a known characteristic of ageing. We report here that epigenetic ageing is not affected by replicative senescence, telomere length, somatic cell differentiation, cellular proliferation rate or frequency. It is instead retarded by rapamycin, the potent inhibitor of the mTOR complex which governs many pathways relating to cellular metabolism. Rapamycin, however, is also an effective inhibitor of cellular senescence. Hence cellular metabolism underlies two independent arms of ageing - cellular senescence and epigenetic ageing. The demonstration that a compound that targets metabolism can slow epigenetic ageing provides a long-awaited point-of-entry into elucidating the molecular pathways that underpin the latter. Lastly, we report here an in vitro assay, validated in humans, that recapitulates human epigenetic ageing that can be used to investigate and identify potential interventions that can inhibit or retard it.

Targeting ROCK/LIMK/cofilin signaling pathway in cancer.

Rho-associated coiled-coil-containing protein kinase (ROCK)/Lin11, Isl-1 and Mec-3 kinase (LIMK)/cofilin-signaling cascades are stimulated by receptor tyrosine kinases, G protein-coupled receptors, integrins and its ligands, growth factors, hormones, fibronectin, collagen, and laminin. Activated signaling cascades can cause transit from normal cells to cancer cells by modulating actin/filament dynamics. In various cancers including breast, prostate, and colorectal cancers, high expression or activity of each cascade protein is significantly associated with poor survival rate of patients as well as aggressive metastasis. Silencing ROCK, LIMK, or cofilin can abrogate their activities and inhibit cancer cell growth, invasion, and metastasis. Therefore ROCK/LIMK/cofilin signaling proteins might be good candidates to develop cancer prevention strategies or therapeutics. Currently, netarsudil, a ROCK inhibitor, is only used in clinical patients for glaucoma or ocular hypertension, but not for cancer. In this review, we will discuss comprehensive ROCK/LIMK/cofilin signaling pathway in cancers and its inhibitors for developing cancer therapy.

Fasudil alleviates brain damage in rats after carbon monoxide poisoning through regulating neurite outgrowth inhibitor/oligodendrocytemyelin glycoprotein signalling pathway.

Carbon monoxide (CO) poisoning can lead to many serious neurological symptoms. Currently, there are no effective therapies for CO poisoning. In this study, rats exposed to CO received hyperbaric oxygen therapy, and those in the Fasudil group were given additional Fasudil injection once daily. We found that the escape latency in CO poisoning group (CO group) was significantly prolonged, the T1 /Ttotal was obviously decreased, and the mean escape time and the active escape latency were notably extended compared with those in normal control group (NC group, P < 0.05). After administration of Fasudil, the escape latency was significantly shortened, T1 /Ttotal was gradually increased as compared with CO group (>1 week, P < 0.05). Ultrastructural damage of neurons and blood-brain barrier of rats was serious in CO group, while the structural and functional integrity of neuron and mitochondria maintained relatively well in Fasudil group. Moreover, we also noted that the expressions of neurite outgrowth inhibitor (Nogo), oligodendrocyte-myelin glycoprotein (OMgp) and Rock in brain tissue were significantly increased in CO group, and the elevated levels of the three proteins were still observed at 2 months after CO poisoning. Fasudil markedly reduced their expressions compared with those of CO group (P < 0.05). In summary, the activation of Nogo-OMgp/Rho signalling pathway is associated with brain injury in rats with CO poisoning. Fasudil can efficiently down-regulate the expressions of Nogo, OMgp and Rock proteins, paving a way for the treatment of acute brain damage after CO poisoning.

Rho Kinase Inhibition Blunts Lesion Development and Hemorrhage in Murine Models of Aggressive Pdcd10/Ccm3 Disease.

Background and Purpose- Previously, murine models Krit1 +/- Msh2 -/- and Ccm2 +/- Trp53 -/- showed a reduction or no effect on cerebral cavernous malformation (CCM) burden and favorable effects on lesional hemorrhage by the robust Rock (Rho-associated protein kinase) inhibitor fasudil and by simvastatin (a weak pleiotropic inhibitor of Rock). Herein, we concurrently investigated treatment of the more aggressive Pdcd10/Ccm3 model with fasudil, simvastatin, and higher dose atorvastatin to determined effectiveness of Rock inhibition. Methods- The murine models, Pdcd10 +/- Trp53 -/- and Pdcd10 +/- Msh2 -/-, were contemporaneously treated from weaning to 5 months of age with fasudil (100 mg/kg per day in drinking water, n=9), simvastatin (40 mg/kg per day in chow, n=11), atorvastatin (80 mg/kg per day in chow, n=10), or with placebo (n=16). We assessed CCM volume in mouse brains by microcomputed tomography. Lesion burden was calculated as lesion volume normalized to total brain volume. We analyzed chronic hemorrhage in CCM lesions by quantitative intensity of Perls staining in brain sections. Results- The Pdcd10 +/- Trp53 -/- /Msh2 -/- models showed a mean CCM lesion burden per mouse reduction from 0.0091 in placebos to 0.0042 ( P=0.027) by fasudil, and to 0.0047 ( P=0.025) by atorvastatin treatment, but was not changed significantly by simvastatin. Hemorrhage intensity per brain was commensurately decreased by Rock inhibition. Conclusions- These results support the exploration of proof of concept effect of high-dose atorvastatin on human CCM disease for potential therapeutic testing.

The novel protective effects of loganin against 1-methyl-4-phenylpyridinium-induced neurotoxicity: Enhancement of neurotrophic signaling, activation of IGF-1R/GLP-1R, and inhibition of RhoA/ROCK pathway.

Loganin, a major iridoid glycoside obtained from fruits of Cornus officinalis, possesses anti-inflammatory, antitumor, antidiabetic, and osteoporosis prevention effects. Loganin has been linked to neuroprotection in several models of neurodegeneration, including Parkinson's disease (PD). However, mechanisms underlying the neuroprotective effects of loganin are still mostly unknown. Here, we demonstrated the protective effects of loganin against PD mimetic toxin 1-methyl-4-phenylpyridinium (MPP+ ) and the important roles of insulin-like growth factor 1 receptor (IGF-1R) and glucagon-like peptide 1 receptor (GLP-1R) in the neuroprotective mechanisms of loganin. In primary mesencephalic neuronal cultures treated with or without MPP+ , loganin up-regulated expressions of neurotrophic signals including IGF-1R, GLP-1R, p-Akt, BDNF, and tyrosine hydroxylase. Loganin protected against MPP+ -induced apoptosis by up-regulating antiapoptotic protein and down-regulating proapoptotic protein. Moreover, loganin attenuated MPP+ -induced neurite damage via up-regulation of GAP43 and down-regulation of membrane-RhoA/ROCK2/p-LIMK/p-cofilin. Loganin also attenuated MPP+ -induced reactive oxygen species (ROS) production. However, both AG1024, an IGF-1R antagonist, and exendin 9-39, a GLP-1R antagonist, attenuated the protective effects of loganin on MPP+ -induced cytotoxicity, apoptosis, neurite length decrease, and ROS production. Our results suggest that loganin attenuates MPP+ -induced apoptotic death, neurite damage, and oxidative stress through enhancement of neurotrophic signaling, activation of IGF-1R/GLP-1R, and inhibition of RhoA/ROCK pathway, providing the evidence that loganin possesses novel neuroprotective effects.