“Baihui”(DU20)-penetrating “Qubin”(GB7) acupuncture on blood–brain barrier integrity in rat intracerebral hemorrhage models via the RhoA/ROCK Ⅱ/MLC 2 signaling pathway

Background: Blocking the Rho A/ROCK Ⅱ/MLC 2(Ras homolog gene family member A/Rho kinase Ⅱ/myosin light chain 2) signaling pathway can initiate neuroprotective mechanisms against neurological diseases such as stroke, cerebral ischemia, and subarachnoid hemorrhage. Nevertheless, it is not clear whether and how disrupting the Rho A/ROCK Ⅱ/MLC 2 signaling pathway changes the pathogenic processes of the blood–brain barrier(BBB) after intracerebral hemorrhage(ICH). The present investigation included the injection of rat caudal vein blood into the basal ganglia area to replicate the pathophysiological conditions caused by ICH. Methods: Scalp acupuncture(SA) therapy was performed on rats with ICH at the acupuncture point “Baihui”-penetrating “Qubin,” and the ROCK selective inhibitor fasudil was used as a positive control to evaluate the inhibitory effect of acupuncture on the Rho A/ROCK Ⅱ/MLC 2 signaling pathway. Post-assessments included neurological deficits, brain edema, Evans blue extravasation, Western blot, quantitative polymerase chain reaction, and transmission electron microscope imaging. Results: We found that ROCK Ⅱ acts as a promoter of the Rho A/ROCK Ⅱ/MLC 2 signaling pathway, and its expression increased at 6 h after ICH, peaked at 3 days, and then decreased at 7 days after ICH, but was still higher than the preintervention level. According to some experimental results, although 3 days is the peak, 7 days is the best time point for acupuncture treatment. Starting from 6 h after ICH, the neurovascular structure and endothelial cell morphology around the hematoma began to change. Based on the changes in the promoter ROCK Ⅱ, a 7-day time point was selected as the breakthrough point for treating ICH model rats in the main experiment. The results of this experiment showed that both SA at “Baihui”-penetrating “Qubin” and treatment with fasudil could improve the expression of endothelial-related proteins by inhibiting the Rho A/ROCK Ⅱ/MLC 2 signaling pathway and reduce neurological dysfunction, brain edema, and BBB permeability in rats. Conclusion: This study found that these experimental data indicated that SA at “Baihui”-penetrating “Qubin” could preserve BBB integrity and neurological function recovery after ICH by inhibiting Rho A/ROCK Ⅱ/MLC 2 signaling pathway activation and by regulating endothelial cell–related proteins.

Research progress of RhoA/ROCK pathway in diabetes-related diseases

RhoA is a small GTPase protein.Its downstream effector protein Rho kinase(ROCK)regulates a variety of cell functions,including cell growth,gene expression and cytoskeleton recombination.Studies have demonstrated that this pathway plays an important pathophysiological role in diabetic nephropathy and other complications,hypertension,stroke,tumor,osteoarthritis,acute lung injury and other diseases.The occurrence and development of diabetes-related disease is closely related to the activation or up-regulation of RhoA/ROCK pathway.As a key target of drug development,ROCK inhibitors have been widely concerned by scholars.This article reviews the relationship between RhoA/ROCK pathway and diabetesrelated disease,and offers a new and effective strategy for the prevention and treatment of this series of diseases.

Research progreess on relevant diseases of RhoA/ROCK signaling pathway

RhoA (Ras homolog gene family member A) belongs to the Rho subfamily of GTPases. ROCK (Rho—associated coiled—coil forming protein kinase) is downstream of the active RhoA and affects the generation and secretion of cellular element, which will result in relevant biologic effects. The RhoA/ROCK signaling pathway consists of these serious reactions. Therefore, the activation and inhibition of this pathway are closely related to the occurrence and development of many diseases. The research on the molecular mechanism of these diseases may be instructive and helpful to the clinical treatmen and prognosis of diseases. Recent studies of these typical diseases related to RhoA/ROCK signaling pathway are viewed in this article.

TNF-a induce the F-actin arrangement and permeability increase in endothelial cells by RhoA-ERK1/2 pathway

Background This study aimed to determine the effects of tumor necrosis factor(TNF-a) on endothelial cytoskeleton morphology and permeability,and to detect the underlying signaling mechanisms involved in these responses. Methods Cultured endothelial cells(ECs) were exposed to TNF-a,and EC cytoskeletal changes were evaluated by observing fluorescence of F-actin following ligation with labeled antibodies.Endothelial permeability was detected by measuring the flux of HRP-albumin across the EC monolayers.To explore the signaling pathways behind TNF-a-induced EC alteration, ECs were treated with either the RhoGTPase inhibitor Y27632 or the MAPK inhibitors PD98059 and SB203580 before TNF-a administration.To further elucidate possible involvement of the RhoA and ERK pathways in TNF-induced EC changes,retrovirus-carried recombinant dominant-negative forms and constitutive-activative forms of RhoA,namely T19NRhoA and Q63LRhoA,were pre-infect-ed into ECs prior to TNF-a exposure.Results TNF-a induced F-actin cytoskeleton rearrangement,as well as EC hyperpermeability in a dose and time-dependent manner.The effects were attenuated in cells pretreated with Y27632 or PD98059,respectively.EC pre-infection with T19NRhoA also alleviated the effects of TNF-a.Furthermore,retrovirus-mediated administration of activated forms of Q63LRhoA alone induced rearrangement of F-actin and hyperpermeability as well as induced the activation of pERK.Conclusions These results indicate that RhoA-ERK/MAPK signal pathway play important roles in the mediation of TNF-a induced EC barrier dysfunction associated with morphological changes of the Factin.

按部选穴针刺对糖尿病胃轻瘫大鼠胃窦平滑肌RhoA/ROCK信号表达的影响

目的:通过固定循经取穴配伍以不同部位选穴进行对比研究,以糖尿病胃轻瘫(diabetic gastroparesis,DGP)大鼠为观察对象,研究不同按部选穴针刺治疗对DGP大鼠Rho A/ROCK信号的表达差异,探讨按部选穴是影响腧穴配伍效应的主要影响因素.方法:将60只♂SPF级SD大鼠,适应性喂养1 w k后,随机分为空白对照组、模型组、足三里+中脘组、足三里+内关组、足三里+非经非穴组,每组12只.除空白对照组12只外,其余48只大鼠运用链脲佐菌素腹腔注射造糖尿病模型,普通喂养8 wk后建立DGP大鼠模型,针刺治疗4 wk,于13 w k末墨汁灌胃后处死,取胃窦组织.运用Western blot检测胃窦平滑肌组织Ras同源物基因组成员A(Ras homolog gene family,member A,Rho A)、Rho蛋白相关卷曲螺旋激酶(Rho-associatedc,oiled-coil containing protein kinase,ROCK)、肌球蛋白磷酸酶靶亚单位1(myosin phosphatase target subunit 1,MYPT1)、p-MYPT1蛋白的表达量;应用免疫组织化学检测胃窦平滑肌组织Rho A蛋白平均灰度值改变.结果:与空白对照组相比,模型组的小肠推进率、胃窦平滑肌组织Rho A、ROCK、MYPT1、p-MYPT1蛋白的表达量明显降低(P<0.01),胃窦平滑肌组织R h o A灰度值表达升高(P<0.05).与模型组相比,足三里+中脘组、足三里+内关组、足三里+非经非穴组的小肠推进率和胃窦平滑肌组织Rho A、ROCK、MYPT1、p-MYPT1蛋白的表达量明显升高(P<0.05),胃窦平滑肌组织Rho A灰度值表达均具有降低的趋势(P<0.05).与足三里+中脘组相比,足三里+内关、足三里+非经非穴组胃窦平滑肌组织R h o A、ROCK、MYPT1、p-MYPT1蛋白的表达量降低(P<0.05),胃窦平滑肌组织Rho A灰度值表达升高(P<0.05).在治疗期间,与模型组相比,足三里+中脘组的饮食量明显降低(P<0.05).结论:针刺能通过上调Rho A/ROCK信号的表达来促进胃平滑肌收缩,改善DGP的症状;证实按部选穴是影响腧穴配伍效应的重要因素,且配伍局部穴明显优于配伍远端穴及非经非穴.

EHMT2 promotes tumorigenesis in GNAQ/11-mutant uveal melanoma via ARHGAP29-mediated RhoA pathway

Constitutive activation of GNAQ/11 is the initiative oncogenic event in uveal melanoma(UM).Direct targeting GNAQ/11 has yet to be proven feasible as they are vital for a plethora of cellular functions.In search of genetic vulnerability for UM,we found that inhibition of euchromatic histone lysine methyltransferase 2(EHMT2)expression or activity significantly reduced the proliferation and migration capacity of cancer cells.Notably,elevated expression of EHMT2 had been validated in UM samples.Furthermore,Kaplan-Meier survival analysis indicated high EHMT2 protein level was related to poor recurrence-free survival and a more advanced T stage.Chromatin immunoprecipitation sequencing analysis and the following mechanistic investigation showed that ARHGAP29 was a downstream target of EHMT2.Its transcription was suppressed by EHMT2 in a methyltransferasedependent pattern in GNAQ/11-mutant UM cells,leading to elevated RhoA activity.Rescuing constitutively active RhoA in UM cells lacking EHMT2 restored oncogenic phenotypes.Simultaneously blocking EHMT2 and GNAQ/11 signaling in vitro and in vivo showed a synergistic effect on UM growth,suggesting the driver role of these two key molecules.In summary,our study shows evidence for an epigenetic program of EHMT2 regulation that influences UM progression and indicates inhibiting EHMT2 and MEK/ERK simultaneously as a therapeutic strategy in GNAQ/11-mutant UM.

Mertk Reduces Blood-Spinal Cord Barrier Permeability Through the Rhoa/Rock1/P-MLC Pathway After Spinal Cord Injury

Disruption of the blood-spinal cord barrier(BSCB)is a critical event in the secondary injury following spinal cord injury(SCI).Mertk has been reported to play an important role in regulating inflammation and cytoskeletal dynamics.However,the specific involvement of Mertk in BSCB remains elusive.Here,we demonstrated a distinct role of Mertk in the repair of BSCB.Mertk expression is decreased in endothelial cells following SCI.Overexpression of Mertk upregulated tight junction proteins(TJs),reducing BSCB permeability and subsequently inhibiting inflammation and apoptosis.Ultimately,this led to enhanced neural regeneration and functional recovery.Further experiments revealed that the RhoA/Rock1/P-MLC pathway plays a key role in the effects of Mertk.These findings highlight the role of Mertk in promoting SCI recovery through its ability to mitigate BSCB permeability and may provide potential targets for SCI repair.

The mevalonate pathway promotes the metastasis of osteosarcoma by regulating YAP 1 activity via RhoA

Osteosarcoma is the most common malignant bone tumour,and the metastasis of osteosarcoma is an important cause of death.Evidence has shown that the mevalonate pathway is highly activated and is expected to be a new target for tumour therapy.In this study,we investigated the effect of mevalonate signalling on osteosarcoma metastasis and its molecular mechanism.First,we found that the key rate-limiting enzyme of mevalonate signalling,3-hydroxy-3-methylglutaryl-CoA reductase(HMGCR),was highly expressed in osteosarcoma cells,and inhibition of HMGCR with simvastatin significantly inhibited the motility of 143B cells.Next,we found that YAP1 activity was significantly upregulated in osteosarcoma cells and that YAP1 knockdown inhibited the motility of 143B cells.We also found that the mevalonate pathway regulated the motility of 143B cells by modulating YAP1 phosphorylation and cellular localization.Moreover,we found that the activity of YAP1 was regulated by the mevalonate pathway by modulating the cell membrane localization of RhoA.Finally,we demonstrated that inhibition of the mevalonate pathway notably reduced the lung metastasis of 143B cells,as reflected by the decreased incidence and number of metastatic nodules and the increased survival time of the nude mice.Taken together,our findings suggest that the mevalonate pathway can promote the metastasis of osteosarcoma by activating YAP1 via RhoA.Inhibition of the mevalonate pathway may be a promising therapeutic strategy for osteosarcoma metastasis.

Inhibition of neurite outgrowth using commercial myelin associated glycoprotein-Fc in neuro-2a cells

Myelin-associated glycoprotein（MAG） inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially available MAG-Fc can replace endogenous MAG for research purposes. Immunofluorescence using specific antibodies against MAG, Nogo receptor（NgR） and paired immunoglobulin-like receptor B（PirB） was used to determine whether MAG-Fc can be endocytosed by neuro-2a cells. In addition, neurite outgrowth of neuro-2a cells treated with different doses of MAG-Fc was evaluated. Enzyme linked immunosorbent assays were used to measure RhoA activity. Western blot assays were conducted to assess Rho-associated protein kinase（ROCK） phosphorylation. Neuro-2a cells expressed NgR and PirB, and MAG-Fc could be endocytosed by binding to NgR and PirB. This activated intracellular signaling pathways to increase RhoA activity and ROCK phosphorylation, ultimately inhibiting neurite outgrowth. These findings not only verify that MAG-Fc can inhibit the growth of neural neurites by activating RhoA signaling pathways, similarly to endogenous MAG, but also clearly demonstrate that commercial MAG-Fc is suitable for experimental studies of neurite outgrowth.

Moxibustion pretreatment inhibits RhoA/ROCK signaling to prevent lung inflammation in asthmatic rats

Objective:To determine whether pretreatment moxibustion prevents asthma by down-regulating the lung RhoA/ROCK pathway in rats with bronchial asthma and benignly mediating the lung inflammatory response.Methods:Twenty Sprague Dawley(SD)rats were randomly divided into normal control group(C),asthma model group(M),suspended moxibustion 40 min+asthma group(SM40),and suspended moxibustion 10 min+asthma group(SM10).Ovalbumin was used as a sensitizer.The two moxibustion groups completed moxibustion treatment lasted 40 min or 10 min respectively 30 min before modeling onset,and was repeated five times in each modeling cycle,for a total of 15 times.Samples were harvested on day 30.Results:Lung impairment was significant in the M group,whereas pretreatment with SM10 and SM40 dramatically attenuated the injury.After modeling,mRNA expression of RhoA and ROCK2 in the lung tissue was significantly higher than that in C group(both P<0.001),resulting in significant increase in protein levels of IL-17 A(P<0.001).Significant decrease in RhoA and ROCK2 mRNA expression was seen in the SM10(P<0.001,P<0.01)and SM40(both P<0.001)groups compared to that with M rats.The differential trend in the SM40 group was more evident than that in the SM10 group.Regarding IL-10 or IL-17 A protein concentration,an upregulation or down-regulation was observed in both SM10(P<0.05,P<0.01)and SM40 groups(both P<0.001)compared to that with the M group.Conclusions:Moxibustion pretreatment significantly prevented pulmonary inflammation in asthmatic rats,potentially via inhibition of the RhoA/ROCK pathway.The efficacty of moxibustion appeared to be significantly associated with the duration of intervention with moxibustion.

Effects of Bunao-Fuyuan decoction serum on proliferation and migration of vascular smooth muscle cells in atherosclerotic

Atherosclerosis(AS)is a chronic inflammatory disease,the main causes of which include abnormal lipid metabolism,endothelial injury,physical and chemical injury,hemodynamic injury,genetic factors and so on.These causes can lead to inflammatory injury of blood vessels and local dysfunction.Bunao-Fuyuan decoction(BNFY)is a traditional Chinese medicine compound that can treat cardiovascular and cerebrovascular diseases,but its effect on AS is still unknown.The aim of this study was to investigate the effect and mechanism of BNFY in proliferation and migration of vascular smooth muscle cells(VSMCs)on AS.At first,the expression ofα-SMA protein in ox-LDL-induced VSMCs,which was detected by immunofluorescence staining and western blot.CCK-8 technique and cloning technique were used to detect the cell proliferation of ox-LDL-induced VSMCs after adding BNFY.Meanwhile,the expression of proliferating protein Ki67 was detected by immunofluorescence staining.Western blot was also used to detect the expression of proliferation-related proteins CDK2,CyclinE1 and P27.Flow cytometry was used to detect the effect of BNFY on cell cycle.The effects of BNFY on proliferation and migration of cells were detected by cell scratch test and Transwell.Western blot was used to detect the expression of adhesion factors ICAM1,VCAM1,muc1,VE-cadherin and RHOA/ROCK-related proteins in cells.We found that the expression of AS markerα-SMA protein increased significantly and cells shriveled and a few floated on the medium after induction of ox-LDL on VSCMs.The proliferation rate of ox-LDL VSMCs decreased significantly after adding different doses of BNFY,and BNFY can inhibit cell cycle.Meanwhile,we also found that cell invasion and migration rate were significantly inhibited and related cell adhesion factors ICAM1,VCAM1,muc1 and VE-cadherin were inhibited too by BNFY.Finally,we found that BNFY inhibited the expression of RHOA,ROCK1,ROCK2,p-MLC proteins in the RHOA/ROCK signaling pathway.Therefore,we can summarize that BNFY may inhibit the proliferation and migration of atherosclerotic vascular smooth muscle cells by inhibiting the activity of RHOA/ROCK signaling pathway.