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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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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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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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Objective: To investigate the correlation between Ras homolog gene family member A (RhoA) gene in Wnt/PCP signaling pathway and acute exacerbation chronic obstructive pulmonary disease (AECOPD) traditional Chinese medicine(TCM)syndrome, attempting to provide an objective standard for the diagnosis of AECOPD TCM syndrome. Method: The 100 AECOPD patients were collected and divided into 5 groups:phlegm turbid obstructing lung syndrome,, phlegm-heat obstructing lung syndrome, syndrome of orifices confused by phlegm, deficiency of pulmonary and renal Qi, and edema due to yang deficiency, with 20 people in each group. 15 normal people were selected as a normal control group. All patients received fasting hemospasia, using a kit to extract blood total RibonucleicA(RNA) according to instructions. Real-time quantitative polymerase chain reaction (Real-time PCR) was adopted to detect the mRNA expression of RhoA gene in blood of patients with AECOPD TCM syndrome, and to explore the correlation. Result: There was no difference between phlegm-heat obstructing lung syndrome group and syndrome of orifices confused by phlegm group. The mRNA expression of RhoA gene in phlegm turbid obstructing lung syndrome group, phlegm-heat obstructing lung syndrome group, syndrome of orifices confused by phlegm group, deficiency of pulmonary and renal Qi group, and edema due to Yang deficiency group were significantly higher than that in normal group (PConclusion: The significant difference in mRNA relative expression of RhoA gene in Wnt/PCP signaling pathway among the five AECOPD TCM syndrome groups may provide some objective diagnostic criteria for AECOPD TCM syndromes and reveal their disease severity.