Mechanism of Xiangsha Liujunzi Tang in Regulation of RhoA/ROCK2/MYPT1 Pathway to Improve Gastric Motility in Rats with Functional Dyspepsia / 中国实验方剂学杂志

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.

Effect of Achyranthis Bidentatae Radix-Paeoniae Radix Alba on Neuroinflammation and RhoA/ROCK2 Signaling Pathway in Parkinson's Disease Mouse Model / 中国实验方剂学杂志

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.

Research of Taurine in improving the proliferation of neural stem cells in fetal rats with intrauterine growth restriction in fetal rats by inhibiting Rho/ROCK signaling pathway / 中华实用儿科临床杂志

Objective To investigate the effects of Taurine on the proliferation of neural stem cells(NSCs) with intrauterine growth restriction(IUGR) in fetal rats and the expressions of Ras homolog gene family member A(RhoA) and Rho-associated coiled-coil containing protein kinase (ROCK) Ⅱ in the process of proliferation.Methods IUGR fetal rats models were established with low protein diet.NSCs from subventricular zone were isolated and cultured in vitro.The NSCs were divided into 5 groups:normal control group(group Ⅰ),IUGR group(group Ⅱ),IUGR + Taurine group (group Ⅲ),IUGR + ROCK blockers group(group Ⅳ),IUGR + Taurine + ROCK blockers group(group Ⅴ).The cells were examined by immunofluorescence for counting fatty acid binding protein 7 (FABP7)-positive cells.mRNA and protein levels of RhoA and ROCK were detected by quantitative real time polymerase chain reaction (PCR) and Western blot,respectively.Results (1) Data of group Ⅰ were presented as standard,the number of FABP7 positive cells in group Ⅰ,group]Ⅱ,group Ⅲ,group Ⅳ,group Ⅴ was (100.0 ±1.6)％,(63.8 ±5.2)％,(82.8 ±4.3)％,(78.6 ± 2.7) ％,and (87.6 ± 3.2) ％,respectively.Compared with group Ⅰ,FABP7 positive cells in group Ⅱ decreased significantly by approximately 36％ (t =14.98,P ＜0.01).The number of FABP7 positive cells in group Ⅱ,group Ⅳ,group Ⅲ and group Ⅴ increased gradually and there were significant differences among the groups(F =66.53,P ＜0.01).(2) The levels of RhoA mRNA in group Ⅰ,group Ⅱ,group Ⅲ,group Ⅳ,group Ⅴ were 1,3.05 ± 0.36,2.15 ± 0.19,1.89 ± 0.11,and 1.66 ± 0.11 respectively;the levels of ROCK Ⅱ mRNA in group Ⅰ,group Ⅱ,group Ⅲ,group Ⅳ,group Ⅴ were 1,2.78 ± 0.27,2.12 ± 0.21,1.74 ± 0.12,and 1.65 ± 0.05,respectively.Cells of group Ⅱ showed a statistically higher level of RhoA and ROCK Ⅱ mRNA than those of control cells(t =-12.61,-14.67,all P ＜ 0.01).The levels of RhoA and ROCK Ⅱ mRNA in group Ⅱ,group Ⅲ,group Ⅳ,group Ⅴ decreased gradually,and there were significant differences among the groups(F =73.20,76.38,all P ＜ 0.01).(3)The levels of RhoA protein in group Ⅰ,group Ⅱ,group Ⅲ,group ⅢⅣ,group Ⅴ were 0.37 ± 0.03,0.87 ± 0.02,0.73 ± 0.04,0.72 ± 0.04,and 0.55 ± 0.04 respectively;the levels of ROCK Ⅱ protein in group Ⅰ,group Ⅱ,group Ⅲ,group Ⅳ,group Ⅴ were 0.46 ± 0.03,0.96 ± 0.02,0.74 ± 0.01,0.63 ± 0.02,and 0.55 ± 0.03,respectively.The levels of RhoA and ROCK Ⅱ protein were significantly higher in group Ⅱ than those in group Ⅰ (t =-27.60,-35.77,all P ＜ 0.01).The levels of ROCK Ⅱ protein in group Ⅱ,group Ⅲ,group Ⅳ,group Ⅴ reduced gradually,and there were significant differences among the groups(F =390.16,P ＜ 0.01).The levels of RhoA protein in group Ⅱ,group Ⅲ,group Ⅴ reduced gradually,and there were significant differences among the groups (F =130.51,P ＜ 0.01),but there was no significant difference between group Ⅲ and group Ⅳ (t =0.46,P ＞ 0.05).Conclusions Taurine supplementation can promote the proliferation of NSCs in IUGR fetal rats by inhibiting Rho/ROCK signaling pathway,thus to improve IUGR fetal brain development.

ROCK1 and the relative signal molecules participate in proliferation of vascular smooth muscle cells induced by cyclic strain / 医用生物力学

Objective To investigate the role of Rho-associated coiled-coil containing protein kinase 1 (ROCK1) and the relative signal molecules in sensing the mechanical stimulation from tensile strain and regulating the proliferation of vascular smooth muscle cells (VSMCs).Methods Physiological cyclic strain with magnitude of 10％ and at frequency of 1.25 Hz was applied to VSMCs in vitro by using the strain loading system.The proliferation level of VSMCs was analyzed by BrdU ELISA;the expression level of ROCK1,phosphorylations of protein kinase C (PKC) α/β Ⅱ,protein kinase D (PKD) and extracellular regulated protein kinase (ERK) in VSMCs modulated by cyclic strain were detected with Western blotting;the expression of ROCK1 was specifically repressed by using RNA interference (RNAi).Results Compared with the static control,10％ cyclic strain significantly decreased the expression of ROCK1 and phosphorylations of PKD and ERK.The phosphorylation of PKCα/βⅡ decreased significantly under 10％ cyclic strain for 12 h,but returned to normal level after loading for 24 h.Repressed expression of ROCK1 with RNAi significantly down-regulated VSMC proliferation,suppressed phosphorylations of PKCα/βⅡ and PKD,but no obvious changes were found in phosphorylation of ERK.Conclusions Physiological cyclic strain with magnitude of 10％ may repress the phosphorylation of PKCα/βⅡ and PKD via inhibiting the expression of ROCK1,and subsequently affects VSMC proliferation and maintains vascular hemostasis.The investigation on intracellular mechanotransduction network of VSMCs under mechanical stimulation of cyclic strain may contribute to studying the physiological and pathological mechanisms of cardiovascular diseases.

ROCK1 and the relative signal molecules participate in proliferation of vascular smooth muscle cells induced by cyclic strain / 医用生物力学

Objective To investigate the role of Rho-associated coiled-coil containing protein kinase 1 (ROCK1) and the relative signal molecules in sensing the mechanical stimulation from tensile strain and regulating the proliferation of vascular smooth muscle cells (VSMCs).Methods Physiological cyclic strain with magnitude of 10％ and at frequency of 1.25 Hz was applied to VSMCs in vitro by using the strain loading system.The proliferation level of VSMCs was analyzed by BrdU ELISA;the expression level of ROCK1,phosphorylations of protein kinase C (PKC) α/β Ⅱ,protein kinase D (PKD) and extracellular regulated protein kinase (ERK) in VSMCs modulated by cyclic strain were detected with Western blotting;the expression of ROCK1 was specifically repressed by using RNA interference (RNAi).Results Compared with the static control,10％ cyclic strain significantly decreased the expression of ROCK1 and phosphorylations of PKD and ERK.The phosphorylation of PKCα/βⅡ decreased significantly under 10％ cyclic strain for 12 h,but returned to normal level after loading for 24 h.Repressed expression of ROCK1 with RNAi significantly down-regulated VSMC proliferation,suppressed phosphorylations of PKCα/βⅡ and PKD,but no obvious changes were found in phosphorylation of ERK.Conclusions Physiological cyclic strain with magnitude of 10％ may repress the phosphorylation of PKCα/βⅡ and PKD via inhibiting the expression of ROCK1,and subsequently affects VSMC proliferation and maintains vascular hemostasis.The investigation on intracellular mechanotransduction network of VSMCs under mechanical stimulation of cyclic strain may contribute to studying the physiological and pathological mechanisms of cardiovascular diseases.

ROCK1 and the relative signal molecules participate in proliferation of vascular smooth muscle cells induced by cyclic strain / 医用生物力学

Objective To investigate the role of Rho-associated coiled-coil containing protein kinase 1 (ROCK1) and the relative signal molecules in sensing the mechanical stimulation from tensile strain and regulating the proliferation of vascular smooth muscle cells (VSMCs).Methods Physiological cyclic strain with magnitude of 10％ and at frequency of 1.25 Hz was applied to VSMCs in vitro by using the strain loading system.The proliferation level of VSMCs was analyzed by BrdU ELISA;the expression level of ROCK1,phosphorylations of protein kinase C (PKC) α/β Ⅱ,protein kinase D (PKD) and extracellular regulated protein kinase (ERK) in VSMCs modulated by cyclic strain were detected with Western blotting;the expression of ROCK1 was specifically repressed by using RNA interference (RNAi).Results Compared with the static control,10％ cyclic strain significantly decreased the expression of ROCK1 and phosphorylations of PKD and ERK.The phosphorylation of PKCα/βⅡ decreased significantly under 10％ cyclic strain for 12 h,but returned to normal level after loading for 24 h.Repressed expression of ROCK1 with RNAi significantly down-regulated VSMC proliferation,suppressed phosphorylations of PKCα/βⅡ and PKD,but no obvious changes were found in phosphorylation of ERK.Conclusions Physiological cyclic strain with magnitude of 10％ may repress the phosphorylation of PKCα/βⅡ and PKD via inhibiting the expression of ROCK1,and subsequently affects VSMC proliferation and maintains vascular hemostasis.The investigation on intracellular mechanotransduction network of VSMCs under mechanical stimulation of cyclic strain may contribute to studying the physiological and pathological mechanisms of cardiovascular diseases.

ROCK1 and the relative signal molecules participate in proliferation of vascular smooth muscle cells induced by cyclic strain / 医用生物力学

Objective To investigate the role of rho-associated coiled-coil containing protein kinase 1 (ROCK1) and the relative signal molecules in sensing the mechanical stimulation from tensile strain and regulating the proliferation of vascular smooth muscle cells (VSMCs). Methods Physiological cyclic strain with magnitude of 10% and at frequency of 1.25 Hz was applied to VSMCs in vitro by using strain loading system. The proliferation level of VSMCs was analyzed by BrdU ELISA; the expression level of ROCK1, phosphorylations of protein kinase C (PKC) α/β II, protein kinase D (PKD) and extracellular regulated protein kinase (ERK) in VSMCs modulated by cyclic strain were detected with Western blotting; the expression of ROCK1 was specifically repressed by using RNA interference (RNAi). Results Compared with the static control, 10% cyclic strain significantly decreased the expression of ROCK1 and phosphorylations of PKD and ERK. The phosphorylation of PKCα/βII was decreased significantly under 10% cyclic strain for 12 h, but returned to normal level after 24 h-loading. Repressed expression of ROCK1 with RNAi significantly down-regulated VSMC proliferation, suppressed phosphorylations of PKCα/βII and PKD, but no obvious change was found in phosphorylation of ERK. Conclusions Physiological cyclic strain with magnitude of 10% may repress the phosphorylation of PKCα/βII and PKD via inhibiting the expression of ROCK1, which subsequently affect VSMC proliferation and maintain vascular hemostasis. The investigation on intracellular mechanotransduction network of VSMCs under mechanical stimulation of cyclic strain may contribute to the physiological and pathological mechanisms of cardiovascular diseases.

Inhibition of Rho-associated kinases disturbs the collective cell migration of stratified TE-10 cells

BACKGROUND: The collective cell migration of stratified epithelial cells is considered to be an important phenomenon in wound healing, development, and cancer invasion; however, little is known about the mechanisms involved. Furthermore, whereas Rho family proteins, including RhoA, play important roles in cell migration, the exact role of Rho-associated coiled coil-containing protein kinases (ROCKs) in cell migration is controversial and might be cell-type dependent. Here, we report the development of a novel modified scratch assay that was used to observe the collective cell migration of stratified TE-10 cells derived from a human esophageal cancer specimen. RESULTS: Desmosomes were found between the TE-10 cells and microvilli of the surface of the cell sheet. The leading edge of cells in the cell sheet formed a simple layer and moved forward regularly; these rows were followed by the stratified epithelium. ROCK inhibitors and ROCK small interfering RNAs (siRNAs) disturbed not only the collective migration of the leading edge of this cell sheet, but also the stratified layer in the rear. In contrast, RhoA siRNA treatment resulted in more rapid migration of the leading rows and disturbed movement of the stratified portion. CONCLUSIONS: The data presented in this study suggest that ROCKs play an important role in mediating the collective migration of TE-10 cell sheets. In addition, differences between the effects of siRNAs targeting either RhoA or ROCKs suggested that distinct mechanisms regulate the collective cell migration in the simple epithelium of the wound edge versus the stratified layer of the epithelium.

Suppression of hepatocellular carcinoma invasion and metastasis by Rho-kinase inhibitor Fasudil through inhibition of BTBD7-ROCK2 signaling pathway / 中南大学学报(医学版)

Objective: To explore the eff ect of Fasudil on the invasion and metastatic abilities of human high metastatic liver cancer cells (HCCLM3) and the underlying mechanisms. Methods: HCCLM3 cells were incubated with 100 μmol/L Fasudil. Fluorescence staining forF-actin and Transwell assay were performed to observe the invasion ability of HCCLM3 cells. HCCLM3 cells were divided into 3 groups: a negative control group, a Fasudil group and a BTB/POZ domain containing 7 (BTBD7)-siRNA group. Western blot assay was performed to detect the expression levels of BTBD7, ras homolog family member C (RhoC) and Rho-associated, coiled-coil containing protein kinase 2 (ROCK2), matrix metalloproteinases 2 (MMP2) and MMP9. Zymogram analysis method was performed to detect the expression activities of MMP2 and MMP9. hTe BTBD7-siRNA group was served as a positive control. Results: In HCCLM3 cells treated with Fasudil, the invasion ability was significant decreased compared with the control group, concomitant with the down-regulated expression levels of BTBD7, RhoC and ROCK2 protein as well as the decreased activities of MMP2 and MMP9. Conclusion: Fasudil plays an important role in interfering BTBD7-ROCK2 signaling pathway and suppressing the invasion and metastasis of hepatocellular carcinoma.