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OBJECTIVE To investigate the inhibitory effects of Ginkgo biloba extract （GBE） on renal inflammation in diabetic nephropathy （DN） model mice， and its potential mechanism. METHODS KK/Ay mice were fed with high fat and high sugar to induce DN model. They were divided into model group， positive control group [metformin 200 mg/（kg·d）]， GBE low-dose and high-dose groups [100， 200 mg/（kg·d）]， with 6 mice in each group. Six C57BL/6J mice were fed with a regular diet as the control group. Administration groups were given relevant liquid intragastrically， control group and model group were given constant volume of normal saline intragastrically， once a day， for 8 consecutive weeks. The body weight， fasting blood glucose， 24-hour food intake， 24-hour urine output， monocyte chemoattractant protein-1 （MCP-1）， interleukin-12 （IL-12）， IL-10， advanced glycation end products （AGEs）， blood urea nitrogen （BUN） and serum creatinine （Scr） of mice were measured， and the ratio of bilateral kidneys to body weight was also calculated. The pathological injury and fibrotic changes of the renal cortex were observed， and the expressions of macrophage polarization marker proteins [type M1： inducible nitric oxide synthase （iNOS）； type M2： arginase-1 （Arg-1）] and AGEs-the receptor of advanced glycation end products （RAGE）/Ras homolog gene pharm_chenjing@163.com family member A （RhoA）/Rho-associated coiled-coil forming protein kinase （ROCK） signaling pathway-related proteins were determined in renal cortex. RESULTS Compared with the model group， the symptoms such as renal cortical hyperplasia， vacuoles， infiltration of inflammatory cells， and renal cortical fibrosis had been improved in GBE low-dose and high-dose groups； body weight， serum level of IL-10， the expression of Arg-1 in the renal cortex were significantly higher than model group （P＜ 0.01）； fasting blood glucose， 24-hour food intake， 24-hour urine output， serum levels of MCP-1， IL-12， BUN， Scr and AGEs， the ratio of bilateral kidneys to body weight， renal injury score， the proportion of renal interstitial fibrosis， the protein expressions of iNOS， RAGE， RhoA and ROCK1 （except for GBE low-dose group） in renal cortex were significantly lower than model group （P＜0.01）. CONCLUSIONS GBE could improve kidney damage and alleviate inflammatory response in DN model mice， the mechanism of which may be related to inhibiting the AGEs-RAGE/RhoA/ROCK signaling pathway and regulating macrophage polarization.
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ObjectiveTo explore the intervention mechanism of Xiangsha Liujunzi Tang in rats with functional dyspepsia （FD） based on the Ras homolog gene family member A （RhoA）/Rho-associated coiled-coil containing protein kinase 2 （ROCK2）/Myosin phosphatase target Subunit 1 （MYPT1） pathway. MethodSixty male SD suckling rats in SPF grades were randomly divided into blank group （n=10） and model group （n=50）. The comprehensive modeling method （gavage administration of iodoacetamide+exhaustion of swimming+disturbance of hunger and satiety） was used to replicate the rat model of FD. After successful replication of the model， the rats in the model group were randomly divided into model group， mosapride group， and high， middle， and low-dose Xiangsha Liujunzi Tang groups， with 10 rats in each group. Rats in the blank group and model group were given 10 mL kg-1·d-1 normal saline， those in the mosapride group were given 1.35 mg·kg-1·d-1 mosapride， and those in the high， middle， and low-dose Xiangsha Liujunzi Tang groups were given 12， 6， and 3 g·kg-1·d-1 Xiangsha Liujunzi Tang， respectively. The intervention lasted 14 days. The general living conditions of rats were observed before and after modeling and administration， and the 3-hour food intake and body mass of rats were measured. After intervention， the intestinal propulsion rate of rats was measured， and the pathological changes in the gastric tissue were observed by hematoxylin-eosin （HE） staining. The content of choline acetyl transferase （ChAT） and vasoactive intestinal peptide （VIP） in the medulla oblongata and gastric tissue homogenate was determined by enzyme-linked immunosorbent assay （ELISA）， the distribution of adenosine triphosphate （ATP） enzyme in gastric antrum smooth muscle was observed by frozen section staining， and the protein expression levels of RhoA， ROCK2， and phosphorylated-myosin phosphatase target subunit 1 （p-MYPT1） in the gastric tissue were detected by Western blot. ResultCompared with the blank group， the model group had withered hair， lazy movement， slow action， poor general living condition， lower 3-hour food intake， body mass， and lower intestinal propulsion rate （P<0.05）， whereas no obvious abnormality in gastric histopathology. In the model group， the content of ChAT in the medulla oblongata and gastric tissue decreased， the content of VIP in gastric tissue increased， the distribution of ATP enzyme in gastric antrum smooth muscle decreased significantly， and the protein expression levels of RhoA， ROCK2， and p-MYPT1 in the gastric tissue decreased significantly （P<0.05）. As compared with the model group， the general living condition of rats in each intervention group was significantly improved， and the 3-hour food intake， body mass， and intestinal propulsion rate were significantly increased （P<0.05）. There was no significant difference in gastric pathology in the intervention groups. The content of ChAT in the medulla oblongata and gastric tissue increased significantly， the content of VIP in the gastric tissue decreased， the distribution of ATP enzyme in gastric antrum smooth muscle increased significantly， and the protein expression levels of RhoA， ROCK2， and p-MYPT1 in the gastric tissue increased significantly （P<0.05）. The intervention effect of Xiangsha Liujunzi Tang group on the above indexes was dose-dependent. ConclusionXiangsha Liujunzi Tang can effectively improve the general living condition and gastric motility of rats with FD， and its specific mechanism may be related to the activation of the RhoA/ROCK2/MYPT1 pathway in the gastric tissue to regulate smooth muscle relaxation and contraction and promote gastric motility.
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Objective @#To investigate the effect of isoprene cysteine carboxymethyltransferase (ICMT) gene on the migration and invasion of salivary adenoid cystic cancer cells (SACC) and the related mechanism, to provide experimental evidence for molecular targeted therapy of SACC.@*Methods@# Adenoid cystic cancer cells SACC-LM and SACC-83 were cultured in vitro, and siRNA was transfected into human SACC-LM and SACC-83 cells (experimental group) by transient transfection of a liposome vector. A blank control group and negative control group were set up respectively (transfected NC-siRNA). qRT-PCR was peformed to measure the mRNA expression of ICMT and RhoA in each group after transfection and to determine the silencing efficiency. The expression of ICMT, membrane RhoA, total RhoA, matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and Rho associated with coiled helical binding protein kinase 1 (ROCK1) in each group was detected by Western blot. The proliferation abilityies of SACC cells was detected by CCK-8 assay. The migration and invasion ability of SACC cells were detected by comparing the relative healing area of cell scratch assay and the number of Transwell assay cells. @*Results@#After transfection of ICMT-siRNA into SACC-LM and SACC-83 cells, the expression of ICMT gene and protein in the experimental group was significantly decreased compared with the negative control group and blank control group (P<0.05), but there were no significant differences in the expression of RhoA gene and total protein among all groups (P>0.05). The expression of RhoA membrane proteins, ROCK1, MMP-2, MMP-9 in the experimental group was significantly decreased compared with that in the negative control group and blank control group (P<0.05). Cell proliferation ability was significantly decreased (P<0.05). The migration and invasion abilities were significantly decreased (P<0.05). @*Conclusion @#In vitro silencing of ICMT gene can effectively inhibit the migration and invasion of human SACC-LM and SACC-83 cells, and the mechanism may be related to RhoA-ROCK signaling pathway.
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ObjectiveTo investigate the effects of Yishen Daluo prescription （YSDL） on Ras homolog（Rho）/Rho-associated coiled-coil containing protein kinase（ROCK）signaling pathway in mice with experimental autoimmune encephalomyelitis （EAE） based on the silencing of β-arrestin1 gene. MethodSixty C57BL/6 female mice were randomly divided into a blank group， a model group， a virus group， a YSDL group， a virus + YSDL group， and a prednisone acetate group （hormone group）. The EAE model was induced in mice except for those in the normal group. Adeno-associated virus（AAV）solution （150 μL， 1×1011 vg·mL-1） was injected into the tail vein of each mouse in the virus group and the virus + YSDL group on the 4th day of immunization. Drugs were administered on the 8th day of modeling. Specifically， normal saline was given to the mice in the normal group，the model group，and the virus group at 10 mL∙kg-1， prednisone acetate suspension to those in the hormone group at 3.9 g∙kg-1，and YSDL to those in other groups at 20 g∙kg-1 for 14 consecutive days. The mice were weighed and scored every day. The neurological function scores of mice in each group were recorded every day after immunization. Hematoxylin-eosin （HE） staining was used to determine the inflammatory response and lesion location in the brain tissues and spinal cord tissues of mice. The protein expression of β-arrestin1，Ras homolog gene family member A（RhoA）， and Rho-associated coiled-coil forming protein kinase Ⅰ（ROCK Ⅰ） in spinal cord and brain tissues of EAE mice was determined by Western blot. ResultCompared with the model group， the virus group and the virus + YSDL group showed decreased neurological function scores （P<0.01），and the YSDL group also showed decreased neurological function scores（P<0.05）. HE results showed that there was obvious inflammatory reaction in the central nervous system （CNS） of the model group， which was alleviated to varying degrees in other groups compared with the model group. Western blot results showed that compared with the blank group， the model group showed increased protein expression levels of β-arrestin1， RhoA， and ROCK Ⅰ in the spinal cord tissues （P<0.01）. Compared with the model group， the virus group， the YSDL group， the virus + YSDL group， and the hormone group showed decreased protein expression levels of β-arrestin1， RhoA， and ROCKⅠ in the spinal cord tissues （P<0.01）. Compared with the blank group， the model group showed increased protein expression levels of β-arrestin1， RhoA， and ROCK Ⅰ in the brain tissues （P<0.01）. Compared with the model group， the virus group， the YSDL group， the virus + YSDL group， and the hormone group showed decreased protein expression level of β-arrestin1 in the brain tissues （P<0.01）， and the virus group and the YSDL group showed decreased protein expression levels of RhoA， and ROCKⅠ in the brain tissues （P<0.05）. Additionally， the virus + YSDL group and the hormone group showed decreased protein expression levels of RhoA and ROCKⅠ in the brain tissues （P<0.01）. ConclusionYSDL can improve the clinical symptoms of EAE mice and improve the inflammatory response of CNS. The mechanism is presumably attributed to the fact that YSDL inhibits the expression of β-arrestin1 in CNS，thereby reducing the expression of Rho/ROCK signaling pathway. Furthermore， YSDL may have a synergistic effect with the inhibition of β-arrestin1 gene expression.
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Abstract Introduction: Coronary artery disease (CAD) is an ischemic condition that occurs as a result of partial or complete interruption of blood flow by narrowing or complete blockage of the vessels supplying the heart, which are called coronary arteries. Our objective in this study is to investigate the RhoA/Rho-associated kinase (ROCK)-1 signaling pathway and oxidative stress in CAD patients. Methods: A total of 81 individuals aged between 40-70 years - including 45 patients (15 females and 30 males) who were admitted to the Artvin State Hospital Cardiovascular Surgery Clinic and were diagnosed with CAD and 36 healthy volunteers (15 females and 21 males) - participated in this study. Serum samples were tested for total cholesterol, triglyceride, low-density lipoprotein, high-density lipoprotein, malondialdehyde (MDA), superoxide dismutase (SOD), RhoA, and ROCK-1 values. Results: Serum RhoA, MDA levels, and ROCK-1 activity in the CAD group were found to be statistically significantly higher than in the control group (P<0.001). Concordantly, serum SOD activity was found to be statistically significantly lower in the CAD group than in the control group (P<0.001). Conclusion: Inhibition of the activity of RhoA/ROCK-1 pathway would be beneficial in treating cardiovascular diseases since this pathway plays an important role in the development of these diseases.
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Objective@# To explore whether RhoA plays a role in the migration and invasion of the salivary adenoid cystic carcinoma cell lines SACC-LM and SACC-83.@*Methods@#Total RNA and total protein were extracted from 20 salivary adenoid cystic carcinoma (SACC) and normal adjacent tissues frozen in liquid nitrogen to detect RhoA expression. RhoA-siRNA was constructed to transfect two cell lines (SACC-LM and SACC-83) for cytological experiments. The research included an experimental group (RhoA-siRNA transfection), negative control group (siRNA-NC transfection) and blank group by transient transfection with liposomes. Expression of RhoA mRNA and protein as well as the protein expression of biomarkers of epithelial-mesenchymal transition (EMT) were analyzed, including E-cadherin, N-cadherin, and Vimentin. Furthermore, the changes in invasion and migration of cells in each group were analyzed by comparing the number of transmembrane cells in the Transwell assay and the results of the scratch test.@*Results@#Compared with normal adjacent tissues, RhoA protein and mRNA levels increased in SACC tissues. Compared with the control group, the relative expression levels of RhoA mRNA and protein decreased (P < 0.01), the relative expression levels of E-cadherin protein increased, and the relative expression levels of N-cadherin and vimentin protein increased in the experimental group (P < 0.01). Additionally, the trial results revealed that RhoA knockdown restrained cell migration and invasion (P < 0.01).@*Conclusion @#RhoA expression increased in SACC tissue. Silencing RhoA in vitro could effectively restrain cell migration and invasion in SACC-LM and SACC-83 cells through the regulation of EMT signaling pathways.
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Objective:To determine the protective effect of fasudil on acute lung injury in septic mice.Methods:Forty-five 4-6-week-old male C57BL mice were randomly(random number) assigned to three groups ( n=15 each group): control group, lipopolysaccharide (LPS) group and Fasudil intervention group (FAS+LPS). Acute lung injury model of septic mice was established with an intraperitoneal injection and intratracheal infusion of LPS. The mice in the FAS+LPS group were injected with fasudil hydrochloride intraperitoneally 30 min before intraperitoneal LPS injection and 1 h after intratracheal LPS infusion, respectively. All mice were sacrificed at 4 h after modeling, and lung tissues were collected. Hematoxylin-eosin staining was preformed to observe the morphological changes in the lung tissue. The wet /dry weight (W/D) ratio, malondialdehyde (MDA) content and the activity of myeloperoxidase (MPO) in the lung tissues were detected. Caspase-3 expression was examined by immunohistochemical (IHC) staining. Western blot was employed to detect the expression of RhoA, ROCK1, endothelial nitric oxide synthase (eNOS), and p-eNOS. Results:Inflammatory cell infiltration and erythrocyte exudation were significantly reduced, and the degree of interstitial oedema and derangement of alveolar structure appeared in a decreasing degree after FAS intervention. Compared with the LPS group, the W/D ratio, MDA content, MPO activity and the expression of Caspase-3 in the FAS+LPS group were significantly reduced (all P<0.01). Meanwhile, the expression of RhoA and ROCK1 of the LPS group were obviously higher than those in the control group ( P<0.05), and p-eNOS was obviously lower than that in the control group ( P<0.05). Furthermore, the expression of RhoA and ROCK1 of the FAS+LPS group were obviously lower than those in the LPS group, and p-eNOS was obviously higher than that in the LPS group. There was no significant difference on the expression of eNOS among the three groups. Conclusions:Fasudil can alleviate the degree of inflammatory cell infiltration, reduce apoptosis in lung tissue, inhibit the RhoA/ROCK1 signaling activity, and promote the phosphorylation expression of eNOS in septic mice.
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Objective:To observe the effect of astragaloside Ⅳ on lysophosphatidic acid(LPA)- induced neurite retraction of N1E-115 cells and its potential mechanism.Methods:N1E-115 cells were divided into blank group, model group, the low, medium and high dose groups of astragaloside Ⅳ. The blank group and model group was not intervened by astragaloside; while the low, medium and high dose groups were treated with 20,40 and 80 μg/ml astragaloside Ⅳ for 24 h. Each group was cultured with serum-free medium for 12 h. The model group and astragaloside Ⅳ groups were intervened by 40 μmol/L LPA for 10 min. Each group was observed and photographed with the inverted microscope, and the number of neurites in N1E-115 cells was counted by Image J software. The fluorescence expression of recombinant ras homolog gene family member A (RhoA), rho associated coiledcoil protein kinase 2 (ROCK2), phospho-rho associated coiledcoil protein kinase 2 (p-ROCK2) and phospho-myosin light chain 2 (p-MLC2) proteins was detected by immunohistochemistry. Real-time fluorescent quantitative polymerase chain reaction was used to detect the mRNA expression levels of RhoA and ROCK2 ; the protein expression levels of RhoA, ROCK2, p-MLC2 and myosin light chain 2 (MLC2) were detected by Western blotting.Results:Compared with 20 μg/ml astragaloside Ⅳ group, the inhibition rate of neurite retraction in 40 and 80 μg/ml astragalosideⅣ groups increased ( P<0.05). Compared with model group, the average fluorescence intensity of RhoA, p-ROCK2, p-MLC2 in 20, 40, 80 μg/ml astragaloside Ⅳ groups and the ROCK2 average fluorescence intensity in 40 μg/ml astragaloside Ⅳ group were decreased ( P<0.05, P<0.01); the expression of RhoA mRNA (0.89±0.09, 0.41±0.01, 0.09±0.03 vs. 1.50±0.01) and ROCK2 mRNA (0.89±0.09, 0.14±0.01, 0.20±0.01 vs. 1.62±0.17) decreased in 20, 40, 80 μg/ml astragaloside Ⅳ groups ( P<0.05, P<0.01); the ROCK2 protein (0.75±0.06, 0.57±0.02, 0.66±0.01 vs. 1.08±0.02), p-MLC2 protein (1.72±0.03, 1.40±0.04, 1.29±0.03 vs. 2.19±0.11), MLC2 protein (1.13±0.02, 0.68±0.03, 0.75±0.03 vs. 1.60±0.03) in 20, 40, 80 μg/ml astragaloside Ⅳ groups and the RhoA protein (0.35±0.01, 0.40±0.03 vs. 0.57±0.08) in 20, 40 μg/ml astragaloside Ⅳ groups were decreased ( P<0.05, P<0.01). Conclusion:Astragaloside Ⅳ can prevent LPA-induced neurite retraction and promote damaged nerve regeneration. The mechanism may down-regulae the protein expression levels of RhoA, ROCK2, p-ROCK2, p-MLC2 and MLC2 in RhoA-ROCK2 signaling pathway, and inhibite nerve growth cone collapse.
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ObjectiveTo observe the effect of icariin on the recombinant Ras homolog family member A （RhoA）/Rho-associated coiled-coil forming protein kinase （ROCK） signaling pathway in rats with Alzheimer's disease （AD）， and to explore the mechanism of icariin in ameliorating the neuronal and dendritic damage. MethodThe β-amyloid 1-42 （Aβ1-42， 2.5 g·L-1） was used to induce AD in rats via lateral ventricle injection， and the rats were divided into a model group， a low-dose icariin group （0.03 g·kg-1）， a middle-dose icariin group （0.06 g·kg-1）， a high-dose icariin group （0.09 g·kg-1）， and a control group. The control group and the model group were given an equal volume of normal saline at a dose of 10 mL·kg-1. The cognitive function of rats was assessed by the Morris water maze. The pathological morphology of the rat hippocampal CA1 area was observed by Nissl staining. Dendritic spine density and dendritic length in the CA1 region of the hippocampus were observed by Golgi-Cox staining. Real-time quantitative polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression levels of tumor necrosis factor-α （TNF-α）， interleukin （IL）-1β， IL-6， RhoA， ROCK1， and ROCK2 in the hippocampus. Western blot assay was used to detect the protein expression levels of TNF-α， IL-1β， IL-6， RhoA， ROCK1， and ROCK2 in the hippocampus. ResultAs compared with the control group， the escape latency of the rats in the model group was increased （P<0.01）， while the number of crossing the platform and the dwelling time in the target quadrant were decreased （P<0.01）. As compared with the model group， the escape latency of the rats in the middle and high-dose icariin groups was decreased （P<0.05， P<0.01）， while the number of crossing the platform and the dwelling time in the target quadrant were increased （P<0.05， P<0.01）. As compared with the control group， the number of neurons， dendritic spine density， and dendritic length in the hippocampal CA1 area of the rats in the model group were decreased （P<0.01）. As compared with the model group， the number of neurons， dendritic spine density， and dendritic length in the hippocampus of the rats in the middle and high-dose icariin groups were increased （P<0.05， P<0.01）. As compared with the control group， the mRNA and protein expression levels of TNF-α， IL-1β， IL-6， RhoA， ROCK1， and ROCK2 in the hippocampus of the rats in the model group were increased （P<0.01）. As compared with the model group， the mRNA and protein expression levels of TNF-α， IL-1β， IL-6， RhoA， ROCK1， and ROCK2 in the hippocampus of the rats in the middle and high-dose icariin groups were decreased （P<0.05， P<0.01）. ConclusionIcariin improves cognitive function and neuronal and dendritic damage in AD by inhibiting the RhoA/ROCK signaling pathway.
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ObjectiveTo investigate the protective effect and mechanism of Achyranthis Bidentatae Radix-Paeoniae Radix Alba on dopaminergic neurons in Parkinson's disease mouse model with the syndrome of ascendant hyperactivity of liver Yang. MethodThe C57BL/6 mice were randomly assigned into normal group， a model group， low-， medium， and high-dose （3.25， 6.5， 13 g·kg-1） Achyranthis Bidentatae Radix-Paeoniae Radix Alba groups， and a selegiline group （0.01 g·kg-1）. The mouse model of Parkinson's disease with the syndrome of ascendant hyperactivity of liver yang was established by intragastric administration of Fuzitang combined with intraperitoneal injection of 1-methyl-4-phenyl-1，2，3，6-tetrahydropyridine （MPTP）. The behavioral changes were evaluated by rotarod test and pole test. The protein levels of Ras homolog gene family member A （RhoA）， Rho-associated coiled-coil containing protein kinase 2 （ROCK2）， myosin light chain 1 （MLC1）， and α-synuclein in the substantia nigra were determined by Western blot. Real-time fluorescence quantitative PCR （Real-time PCR） was employed to determine the mRNA levels of RhoA， ROCK2， and MLC1 in the substantia nigra. Enzyme-linked immunosorbent assay （ELISA） was used to measure the levels of tumor necrosis factor-α （TNF-α） and interleukin-1β （IL-1β）. The ultrastructural changes of mouse neurons were observed under a transmission electron microscope. ResultCompared with the normal group， the modeling shortened the latency to fall， increased the average total time in the pole test （P<0.01）， and up-regulated the levels of RhoA， ROCK2， MLC1， TNF-α， α-synuclein， and IL-1β in the substantia nigra （P<0.05）. Compared with the model group， different doses of Achyranthis Bidentatae Radix-Paeoniae Radix Alba and selegiline prolonged the latency to fall， shortened the average total time in the pole test （P<0.05， P<0.01）， and down-regulated the levels of ROCK2， MLC1， α-synuclein， TNF-α， and IL-1β in a dose-dependent manner （P<0.05）. Further， the modeling decreased the number of cytoplasmic organelles and caused mitochondrial swelling and abnormal shape of endoplasmic reticulum compared with the normal group. The neurons in high-dose Achyranthis Bidentatae Radix-Paeoniae Radix Alba and selegiline groups showed intact nuclei， clear cell boundary， and normal endoplasmic reticulum shape. ConclusionThe combination of Achyranthis Bidentatae Radix and Paeoniae Radix Alba may improve the motor coordination ability of Parkinson's disease mouse model with the syndrome of ascendant hyperactivity of liver yang by inhibiting the neuroinflammation mediated by the RhoA/ROCK2 signaling pathway in the brain.
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OBJECTIVES@#The effect of isoprenylcysteine carboxymethyltransferase (ICMT) silencing on the migration and invasion of tongue squamous cell carcinoma was investigated by constructing the small interfering RNA (siRNA) of ICMT.@*METHODS@#Through liposomal transfection, siRNA was transfected into human tongue squamous cell carcinoma CAL-27 and SCC-4 cells (ICMT-siRNA group) with a negative control group (transfected with NC-siRNA) and a blank control group (transfected with a transfection reagent but not with siRNA). Quantitative real-time polymerase chain reaction was performed to analyze the mRNA expression of ICMT and RhoA in each group of cells after transfection and to measure the silencing efficiency. Western blot was applied to examine the expression levels of ICMT, total RhoA, membrane RhoA, ROCK1, matrix metalloproteinase (MMP)-2, and MMP-9 proteins in each group. The migration and invasion abilities were evaluated via wound healing and Transwell motility assays.@*RESULTS@#After CAL-27 and SCC-4 cells were transfected with ICMT-siRNA, the expression levels of ICMT genes and proteins decreased significantly in the experimental group compared with those in the negative and blank control groups (@*CONCLUSIONS@#The migration and invasion abilities of CAL-27 and SCC-4 cells were reduced significantly after the transfection of ICMT-siRNA, and the involved mechanism might be related to the RhoA-ROCK signaling pathway.
Subject(s)
Humans , Carcinoma, Squamous Cell , Cell Line, Tumor , Cell Movement , Cell Proliferation , Neoplasm Invasiveness , Protein Methyltransferases , RNA, Small Interfering , Tongue , Tongue Neoplasms , Transfection , rho-Associated KinasesABSTRACT
OBJECTIVES@#This study aimed to explore the effects of silencing isoprenylcysteine carboxyl methyltransfe-rase (Icmt) through small interfering RNA (siRNA) interference on the proliferation and apoptosis of tongue squamous cell carcinoma (TSCC).@*METHODS@#Three siRNA were designed and constructed for the Icmt gene sequence and were then transfected into TSCC cells CAL-27 and SCC-4 to silence Icmt expression. The tested cells were divided as follows: RNA interference groups Icmt-siRNA-1, Icmt-siRNA-2, and Icmt-siRNA-3, negative control group, and blank control group. The transfection efficiency of siRNA was detected by the fluorescent group Cy3-labeled siRNA, and the expression of Icmt mRNA was screened by quantitive real-time polymerase chain reaction (qRT-PCR) selected the experimental group for subsequent experiments. The expression of Icmt, RhoA, Cyclin D1, p21, extracellular regulated protein kinases (ERK), and phospho-extracellular regulated protein kinases (p-ERK) were analyzed by Western blot. The proliferation abilities of TSCC cells were determined by cell counting kit-8 assay. The change in apoptosis was detected by AnnexinV-APC/propidium staining (PI) assay. Cell-cycle analysis was conducted by flow cytometry.@*RESULTS@#The expression of Icmt mRNA and protein in TSCC cells significantly decreased after Icmt-siRNA transfection (@*CONCLUSIONS@#Silencing Icmt can effectively downregulate its expression in TSCC cells, reduce the RhoA membrane targeting localization and cell proliferation, and induce apoptosis. Thus, Icmt may be a potential gene therapy target for TSCC.
Subject(s)
Humans , Apoptosis , Carcinoma, Squamous Cell , Cell Line, Tumor , Cell Proliferation , Protein Methyltransferases , RNA, Small Interfering , Tongue , Tongue NeoplasmsABSTRACT
Objective@#The underlying mechanism of Ezrin in ovarian cancer (OVCA) is far from being understood. Therefore, this study aimed to assess the role of Ezrin in OVCA cells (SKOV3 and CaOV3) and investigate the associated molecular mechanisms.@*Methods@#We performed Western blotting, reverse transcription-quantitative polymerase chain reaction, MTT, cell colony, cell wound healing, transwell migration and invasion, RhoA and Rac active pull down assays, and confocal immunofluorescence experiments to evaluate the functions and molecular mechanisms of Ezrin overexpression or knockdown in the proliferation and metastasis of OVCA cells.@*Results@#The ectopic expression of Ezrin significantly increased cell proliferation, invasiveness, and epithelial-mesenchymal transition (EMT) in OVCA cells. By contrast, the knockdown of endogenous Ezrin prevented OVCA cell proliferation, invasiveness, and EMT. Lastly, we observed that Ezrin can positively regulate the active forms of RhoA rather than Rac-1 in OVCA cells, thereby promoting robust stress fiber formation.@*Conclusion@#Our results indicated that Ezrin regulates OVCA cell proliferation and invasiveness by modulating EMT and induces actin stress fiber formation by regulating Rho-GTPase activity, which provides novel insights into the treatment of the OVCA.
Subject(s)
Female , Humans , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cytoskeletal Proteins/metabolism , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Neoplasm Invasiveness , Ovarian Neoplasms/pathology , Stress Fibers/metabolism , rhoA GTP-Binding Protein/metabolismABSTRACT
Objective:To investigate the potential mechanism and effects of LncRNA BBOX1-AS1 in radiosensitivity of ovarian cancer.Methods:qRT-PCR was used to explore BBOX1-AS1, miR-185-5p expression in OC tissue and cells. Dual luciferase reporter assay was used to confirm the interaction of BBOX1-AS1, miR-185-5p and RHOA. MTT assay and flow cytometry were used to detect the proliferation and apoptosis of OC cells.Results:BBOX1-AS1 was up-regulated in OC tissue and cells, compared with the cell proliferative activity at 2, 3, 4 day (0.89±0.07) (1.48±0.13) (1.69±0.15) in si-NC group, proliferative activity in si-BBOX1-AS1 group (0.59±0.06) (0.97±0.09) (1.21±0.10) was obviously down-regulated (all P<0.05) . Compared with si-NC group (6.24±0.28) , silencing of BBOX1-AS1 induced apoptosis (12.07±1.33) (all P<0.05) . miR-185-5p was down-regulated in OC tissue and cells, the targeting relationship between BBOX1-AS1 and miR-185-5p, RHOA and miR-185-5p was confirmed. Inhibition of miR-185-5p reversed the effects of BBOX1-AS1 on OC cells (all P<0.05) . Conclusion:LncRNA BBOX1-AS1 inhibits radiotherapy sensitivity of OC cells via regulating miR-185-5p/RHOA axis.
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BACKGROUND: Hypoxic pulmonary hypertension is a key link in the progression from chronic obstructive pulmonary disease to cor pulmonale. Its severity is closely related to disease development and prognosis. Current treatments cannot prevent or reverse disease progression. Maxing Xiongting Mixture has significant effect on hypoxic pulmonary hypertension with the syndrome of intermingled phlegm and blood stasis. OBJECTIVE: To study how the Maxing Xiongting Mixture regulates relevant factors of lung reshaping and vascular remodeling of hypoxic pulmonary hypertension rats with the syndrome of intermingled phlegm and blood stasis. METHODS: Seventy Sprague-Dawley rats, 5 weeks old, were randomly divided into normal group (n=10) and model group (n=60), where acute cor pulmonale model was prepared by injecting 50 mg/kg monocrotaline solution (1%) intraperitoneally, followed by forced smoking and swimming 6 days a week lasting for 4 weeks. Except for 10 rats in the normal group, there were 46 model rats in the model group. According to the normal distribution of body mass, 40 rats were selected and randomly divided into 4 groups: model group, high-dose Maxing Xiongting Mixture group (MH), low-dose Maxing Xiongting Mixture group (ML) and fasudil group, with 10 rats in each group. Rats in MH and ML groups were respectively given Maxing Xiongting Mixture at 20 g/(kg·d) and 5 g/(kg·d), respectively and those in the fasudil group were given fasudil at a dose of 10 mg/(kg·d). Other groups were given equal amount of saline. Administration was given intraperitoneally and intragastrically, once a day for 14 days in total. RT-PCR was used to test the expression of factors related to lung reshaping and vascular remodeling, including RhoA, stromelysin 1 and tumor necrosis factor-α mRNAs. An approval for the study was obtained from the Ethics Committee of Chengdu University of Traditional Chinese Medicine (approval No. 2017-03). RESULTS AND CONCLUSION: Compared with the model group, the expressions of RhoA, stromelysin 1, and tumor necrosis factor-α mRNAs were significantly lowered in the MH group (all P 0.05). To conclude, Maxing Xiongting Mixture, which is similar to fasudil, intervenes lung reshaping and vascular remodeling of hypoxic pulmonary hypertension rats with the syndrome of intermingled phlegm and blood stasis by inhibiting the expressions of RhoA, stromelysin 1, and tumor necrosis factor-α mRNAs.
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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 of α-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 marker α-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.
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Objective:To study the effect of icariin on renin homologous protein A (RhoA)/Rho-related kinase (ROCK) pathway in rats with nephrotic syndrome (NS) and its protective mechanism. Method:Totally 54 clean-grade male SD rats were tested and randomly divided into normal group, model group, RhoA inhibitor group (Rhosin, 40 mg·kg-1·d-1) and three doses of icariin groups (low, medium and high corresponding dose， 30, 60, 120 mg·kg-1·d-1). Adriamycin hydrochloride 6.5 mg·kg-1 was given in tail vein of rats to induce NS model in rats. After the model was established, peritoneal administration was carried out. The normal group and the model group were given saline 2.5 mL·d-1, and the inhibitor group and all of dose groups were given corresponding doses of Rhosin and icariin for intervention. Total urinary protein (Alb), creatinine (Cre), total urinary protein/creatinine ratio (A/C) kit were detected in rats, ultrastructure of kidney was identified by transmission electron microscopy (TEM), and Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR) and Western blot were used to detect the mRNA and proteins expressions of RhoA, ROCK1, ROCK2. Result:TEM showed that the basement membrane was intact and the foot process was regular in the normal group, in model group, basement membrane was damaged seriously, foot process disappeared, and fusion was serious, in the low-dose group, the basement membrane injury was alleviated, the number and density of foot process were improved, and the fusion was obvious， in the middle-dose group and the inhibitor group, the basement membrane thickening was alleviated, and the foot process was slightly fused, in the high-dose group, the basement membrane structure was more complete, and podocytes were longer and arranged tightly. Compared with the normal group, the levels of Alb, Cre and A/C in urine, and RhoA, ROCK1 and ROCK2 mRNA and protein expressions in kidney tissue of rats of the model group were significantly higher (P<0.05). Compared with model group, the levels of Alb, A/C in urine and RhoA, ROCK1, ROCK2 mRNA and protein expressions in kidney tissue in the inhibitor group and low, medium and high-dose groups, and Cre in urine in inhibitor group and high-dose group decreased significantly (P<0.05). Compared with the inhibitor group, the levels of Alb, Cre in urine and RhoA protein in kidney tissue in the high-dose group were significantly decreased (P<0.05), the levels of Alb, Cre, A/C in urine and RhoA, ROCK1, ROCK2 mRNA and protein expressions in kidney tissue of the low-dose group, and the levels of RhoA, ROCK1 and ROCK2 mRNA expressions in kidney tissue of the middle-dose group were significantly increased (P<0.05). Compared with the low-dose group, the levels of Alb, A/C in urine, and RhoA, ROCK1, ROCK2 mRNA and protein expressions in kidney tissue in the middle and high-dose groups, Cre in urine of the high-dose group were significantly decreased (P<0.05). Compared with the middle-dose group, the levels of Alb, Cre in urine, and RhoA, ROCK1, ROCK2 mRNA and protein expressions, ROCK2 mRNA expression in kidney tissue in the high-dose group were significantly decreased (P<0.05). Conclusion:Icariin may protect glomerular endothelium and podocyte by affecting RhoA/ROCK pathway in the treatment of NS rats.
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OBJECTIVE@#To observe the effects of electroacupuncture (EA) at "Jiaji" (EX-B 2) points combined with nerve mobilization on protein and mRNA expression of RhoA in rabbits with sciatic nerve injury, and to provide theoretical basis for the treatment of peripheral nerve injury by EA at "Jiaji" (EX-B 2) points combined with nerve mobilization.@*METHODS@#A total of 180 New Zealand rabbits were randomly divided into a normal control group, a model control group, a nerve mobilization group, an EA group, an EA plus nerve mobilization group, 36 rabbits in each group. Each group was further divided into a 1-week subgroup, 2-week subgroup and 4-week subgroup, 12 rabbits in each subgroup. The sciatic nerve injury model was made by clamping method. The rabbits in the normal control group did not receive any intervention. The rabbits in the model control group was normally fed after operation. The rabbits in the nerve mobilization group were treated with nerve mobilization; the manipulation lasted for 1 s and relaxed for 5 s, 10 times per day, 6 days per week. The rabbits in the EA group were treated with EA at "Jiaji" (EX-B 2) points (L-L), once a day, 30 min each time, 6 times per week. The rabbits in the EA plus nerve mobilization group were treated with EA at "Jiaji" (EX-B 2) points, followed by nerve mobilization. The function of sciatic nerve on the injured side was evaluated by toe tension reflex and modified Tarlov score; the tissues of corresponding segments of spinal cord L-L and sciatic nerve were taken; the expression of RhoA gene was detected by real-time PCR and the expression of RhoA protein was detected by Western Blot.@*RESULTS@#① Toe tension reflex and modified Tarlov score: at 1, 2 and 4 weeks, the scores in the model control group were lower than those in the normal control group (all 0.05); at 2 weeks, the expression in the nerve mobilization group was higher than that in the EA group (all <0.01); at 4 weeks, the expression in the nerve mobilization group was lower than that in the EA group (all <0.01).@*CONCLUSION@#The nerve mobilization and EA at "Jiaji" (EX-B 2) points could both promote the repair of injured sciatic nerve, which may be related to the down-regulation of RhoA expression, and the combination of the two methods has better effects.
Subject(s)
Animals , Rabbits , Acupuncture Points , Chlorophenols , Electroacupuncture , Peripheral Nerve Injuries , RNA, Messenger , Metabolism , Sciatic Nerve , Wounds and Injuries , rhoA GTP-Binding ProteinABSTRACT
@# Objective: To investigate the expression of microRNA-380-5p (miR-380-5p) in cervical cancer tissues and cell lines, and to explore the mechanism of miR-380-5p inhibiting the proliferation and migration of cervical cancer cells. Methods: 16 pairs of cervical cancerous tissues and corresponding para-cancerous tissues were collected from the Department of Obstetrics and Gynecology, the Affiliated Wuhan Central Hospital of Tongji Medical College from December 2016 to July 2017; in addition, cervical cancer cell lines (HCC94, C33A, Hela, SiHa) and human cervical epithelial immortalized H8 cells were also collected for this study. The expression of miR-380-5p in above mentioned tissues and cell lines was detected by Real-time quantitative polymerase chain reaction (qPCR). miR380-5p mimic (experimental group) and miR-NC (negative control group) were transiently transfected into C33A cells by lipofection, and qPCR was used to detect the expression of miR-380-5p in the transfected cells. Cell proliferation and migration were evaluated by cell counting kit (CCK-8) and Transwell assay. Bioinformatics software TargetScan predicted the downstream genes of miR-380-5p, and dual luciferase reporter assay was used to verify the binding of miR-380-5p to the downstream gene RHOA (Ras homolog gene family member A). qPCR and Western blotting were used to detect the expression of miR-380-5p downstream gene-RHOA. Results: The expression level of miR-380-5p in cervical cancer tissues and cell lines was significantly lower than that in para-cancerous tissues and normal cervical epithelial H8 cells (P<0.01); and the expression in C33A cells was the lowest (P<0.01). Compared with the negative control group, the miR-380-5p mimic transfection singnificantly inhibited the proliferation (P<0.05) and migration ability of C33A cells (P<0.01), and down-regulated protein expressions of RHOA, ROCK1, ROCK2, CDK2 and N-cadherin (all P<0.01). Bioinformatics software predicted that RHOA may be a downstream gene of miR-380-5p, and dual luciferase reporter assay proved the specific binding of miR-380-5p to the 3'UTR of RHOA (P<0.01). miR-380-5p could significantly down-regulate RHOA gene expression (P< 0.01). Conclusion: miR-380-5p is low-expressed in cervical cancer cell lines. Over-expression of miR-380-5p may inhibit the proliferation and migration of cervical cancer C33Acells by down-regulating the expression of RHOAgene and its downstream proteins.
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Objective To observe the effect of different hypoxic time on hydrogen sulfide (H2S), nitric oxide (NO) and Ras homolog gene family,member A/Rho associated coiled coil-forming kinase(RhoA-ROCK) pathway in rat cerebrovascular endothelial cells(EC),and investigate the effect of dermatogenous H2S on the RhoA-ROCK pathway. Methods Rat brain vascular EC was cultured by collagenase digestion. The EC was measured for H2S and NO after hypoxia for 1,2,4,8 and 24 h respectively. G-LISA was used to detect RhoA activity. Proteins expression changes were detected by Western blot. Results After 1 hour of hypoxia,the content of H2S decreased significantly, the NO content decreased significantly after hypoxia of 4 hours,the activity of RhoA increased significantly after hypoxia of 8 h. The expression of CSE protein decreased significantly after 4 h of hypoxia,the expression level of eNOS protein decreased significantly after 8 h of hypoxia,and the expression of ROCK1 and ROCK2 increased significantly at 8 h of hypoxia. Both endogenous and exogenous H2S inhibited RhoA activity. Conclusion During the hypoxic injury of rat cerebrovascular endothelial cells. The decrease of endogenous H2S occurred first, followed by NO,and the activation of RhoA-ROCK pathway occurred later,which may be secondary to the decrease of H2S.