ABSTRACT
This study aimed to investigate the effect and underlying mechanism of Poria cocos polysaccharides(PCP) on myocardial cell apoptosis in the rat model of myocardial ischemia-reperfusion injury(MI/RI). Male SPF-grade SD rats were randomly divided into a sham group(saline), a model group(saline), low-and high-dose PCP groups(100 and 200 mg·kg~(-1)), and a fasudil group(10 mg·kg~(-1)), with 16 rats in each group. Except for the sham group, the other four groups underwent left anterior descending coronary artery ligation for 30 min followed by reperfusion for 2 h to establish the MI/RI model. The myocardial infarct area was assessed by TTC staining. Histological changes were observed through HE staining. Myocardial cell apoptosis was evaluated using TUNEL staining. Serum lactate dehydrogenase(LDH), creatine kinase MB(CK-MB), interleukin-1β(IL-1β) and IL-18 levels, myocardial superoxide dismutase(SOD) activity and malondialdehyde(MDA) levels were detected by ELISA. Protein expression of B-cell lymphoma 2(Bcl-2), Bcl-2 associated X protein(Bax), cleaved caspase-3, Ras homolog gene A(RhoA), myosin phosphatase target subunit 1(MYPT-1), phosphorylated MYPT-1(p-MYPT-1), and Rho-associated coiled-coil forming kinase 1(ROCK 1) were measured by Western blot. Pathological staining of myocardial tissue revealed that in the model group, there was focal necrosis of myocardial tissue, myocardial cell swelling, unclear boundaries, and neutrophil infiltration. These pathological changes were alleviated in the low-and high-dose PCP groups and the fasudil group. Compared with the model group, the low-and high-dose PCP groups and the fasudil group showed significantly reduced myocardial infarct area and myocardial cell apoptosis rate. Compared with the sham group, the model group exhibited elevated serum LDH, CK-MB, IL-1β and IL-18 levels, increased MDA levels, relative protein expression of Bax, cleaved caspase-3, RhoA, ROCK1 and p-MYPT-1, and decreased myocardial SOD levels and Bcl-2 protein expression. Compared with the model group, the PCP groups and the fasudil group showed lowered serum LDH, CK-MB, IL-1β and IL-18 levels, decreased MDA levels, relative protein expression of Bax, cleaved caspase-3, RhoA, ROCK1 and p-MYPT-1, and increased myocardial SOD levels and Bcl-2 protein expression. PCP exhibited a certain preventive effect on myocardial tissue pathological damage and myocardial cell apoptosis in MI/RI rats, possibly related to the inhibition of the Rho-ROCK signaling pathway activation, thereby reducing oxidative stress and inflammatory responses.
Subject(s)
Rats , Male , Animals , Myocardial Reperfusion Injury/drug therapy , bcl-2-Associated X Protein/metabolism , Rats, Sprague-Dawley , Caspase 3/metabolism , Interleukin-18 , Wolfiporia , Signal Transduction , Myocardial Infarction/drug therapy , Proto-Oncogene Proteins c-bcl-2/metabolism , Creatine Kinase, MB Form , Apoptosis , Polysaccharides/pharmacology , Superoxide Dismutase/metabolism , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivativesABSTRACT
@#The occurrence and development of ophthalmic diseases are closely related to the abnormality of cellular regulation and ocular tissue function, in which the relevant signaling pathways play an important role in the regulation of physiological functions. The Rho/ROCK signaling pathway is involved in a variety of cellular events, including cytoskeletal reorganization, cell adhesion, cell proliferation and angiogenesis, and also plays a significant role in cell cycle progression, cell differentiation and apoptosis. Studies have shown that the Rho/ROCK signaling pathway is widely distributed in ocular tissues, and its aberrant activation can affect the normal physiological function of ocular tissues, demonstrating it is closely related to the occurrence and development of ophthalmic diseases. This paper briefly reviews the role of Rho/ROCK signaling pathway in the occurrence and development of ophthalmic diseases, providing new insights into the clinical treatment of ophthalmic diseases.
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Objective@#To investigate the influence of the Rho/ROCK signaling pathway on the anti-cryodamage ability of human sperm and provide some theoretical evidence for the development of high-efficiency semen cryoprotectants.@*METHODS@#We collected semen samples from 25 healthy males, each divided into a fresh, a normal cryopreservation control and an Rho-inhibition group. Before and after freezing, we detected sperm motility, viability, membrane integrity, morphology, DNA fragmentation index (DFI), acrosomal enzyme activity (AEA) and mitochondrial membrane potential (MMP) and determined the expressions of RhoA and ROCK proteins in the sperm by immunofluorescence staining.@*RESULTS@#Compared with the normal cryopreservation control, the frozen-thawed sperm of the Rho-inhibition group showed significantly increased sperm motility （ ［51.20 ± 7.70］% vs ［57.50 ± 6.83］%, P = 0.002), survival rate ( ［52.87 ± 5.07］% vs ［60.24 ± 5.53］%, P = 0.001), membrane integrity (［59.78±5.56］% vs ［67.10 ± 4.43］%, P = 0.001), percentage of morphologically normal sperm (［4.83 ± 1.11］% vs ［7.46 ± 1.28］, P = 0.001) and MMP (56.30 ± 4.28 vs 63.11 ± 2.97, P = 0.001), but decreased DFI (［27.64 ± 6.64］% vs ［18.87 ± 4.07］%, P = 0.001). There was no statistically significant difference in the AEA of the frozen-thawed sperm between the control and Rho-inhibition groups (97.65 ± 9.31 vs 98.30 ± 11.33, P > 0.05). Immunofluorescence staining revealed extensive expressions of RhoA and ROCK proteins in the head and neck of the sperm.@*CONCLUSIONS@#The Rho/ROCK signaling pathway plays a role in the cryodamage to human sperm, and inhibiting the activity of Rho/ROCK can significantly improve the ability of sperm to resist cryodamage.
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Objective To investigate the role of Rho/Rock signaling pathways in ventilator-in-duced lung injury in rats.Methods Ninety-six male SD rats,weighing 300-350 g,were equally and randomly divided into four groups using a random number table (n =24 each):control group (group C),fasudil group (group F),high tidal volume group (group H)and high tidal volume + fasudil group (group HF).Rats in group C and group F received no mechanical ventilation,rats in group H and group HF were intubated and mechanically ventilated (VT = 40 ml/kg,RR = 40 beats per minutes,FiO 2 =40%)for 4 h.The animals in group F and group HF were given intraperitoneal in-jection of fasudil 10 mg/kg at the time 1 h before mechanically ventilated.Six rats were chosen in each group at the time before ventilation (T0 )and at 4,8,24 h after ventilation (T1-T3 ),and blood sam-ples were taken for determination of the levels of serum tumor necrosis factor-α (TNF-α),IL-6 and IL-10,lungs were removed,bronchoalveolar lavage fluid (BALF)was collected to examine protein content,wet/drying (W/D)ratio was determined,which were then stained with haematoxylin and e-osin and examined under microscope,the pathological changes of lungs were scored.Myeloperoxidase (MPO)activity in lung tissue was determined by spectrophptometry.Protein and gene expression of RhoA and Rock2 in the lung tissue were detected by Western blot and reverse transcription PCR (RT-PCR).Results Compared with group C,the serum TNF-α,IL-6 and IL-10,BALF protein content, W/D ratio,the pathological scores,MPO activity,the expression of RhoA,Rock2 protein and mR-NA were significantly increased in group H and HF at T1-T3 (P <0.05 ).Compared with group H, the serum TNF-αand IL-6,BALF protein content,W/D ratio,the pathological scores,MPO activi-ty,the expression of RhoA,Rock2 protein and mRNA were significantly decreased,and the serum IL-10 was significantly increased in group HF at T1-T3 (P <0.05).Conclusion Fasudil can attenuate ventilator-induced lung injury in rats,and the mechanism may be associated with inhibition of the Rho/Rock signaling pathways reducing the inflammatory response.
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Objective To investigate whether ligustrazine can reduce endotoxemia and inhibit the increase of lipopolysaccharide (LPS)-induced vascular permeability by suppressing Rho/ROCK signaling pathway in guinea pigs. Methods Eighteen of white male healthy guinea pigs were divided into three groups randomly and pretreated with intravenous injection of ligustrazine(3 or 6 mg/kg) or saline. 30 min later, LPS (100, 300, 1 000 μg/0.1 mL/site) and saline (0.1 mL/site) were administered intracutaneously at the dorsum of guinea pigs. Vascular permeability was determined by leakage area and absorbance of 610 nm (OD610) of Evans Blue dye after intracutaneous injection of LPS (100-1 000 μg/site). Results Dye leakage in the skin began to increase at 5 min, and revealed significant increase 2 h after LPS injection. Significant differences in the dye leakage area(F = 5.77, P < 0.05) and the OD610 (F = 7.736, P < 0.05) induced by 100, 300 and 1,000 ug/site of LPS administration were demonstrated between the control and ligustrazine groups (3 or 6 mg/kg). Conclusion Ligustrazine has protective effect on the hypermeability of endothelial cells induced by lipopolysaccharide, and inhibition of Rho/ROCK signaling pathway is a possible mechanism.
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Objective To study the effect of Pingchuan Formula(PCF)on Rho/Rock signaling pathway in the mice of asthmatic model, explore the mechanism of PCF on asthma. Methods Forty BALB/C mice were randomly divided into four groups:a control group, a model group,a dexamethasone group and a PCF group. After treated with medicine for 4 weeks, albumen expression of RhoA and RockI in lung tissues were detected by using Western blot, mRNA expressions of RhoA and RockI were detected by using Real-time PCR. Results The albumen and the mRNA expression of RhoA and RockI in the model group were(1.05± 0.20),(1.06±0.08),(6.60±1.09), (6.53±1.84), the control group were(0.76±0.08), (0.84±0.14), (3.82± 1.77), (3.65±1.46),there was a significant difference between two groups(P0.05). Conclusion The expressions of Rho/Rock signaling pathway were controlled significantly by PCF, and the curative effect was similar to dexamethasone.
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Objective To investigate the role of Rho/ROCK signaling pathway in the process of human mesangial cells (HMCs) inflammation and fibrosis induced by high glucose. Methods Synchronized HMCs were divided into following groups: ( 1 ) Normal glucose control group ( NG, 5.5 mmol/L glucose); ( 2 ) High glucose group ( HG, 30 mmol/L glucose); (3) Mannitol group( Man,5.5 mmol/L glucose+ 24.5 mmol/L mannitol); (4) NG +Y-27632 group( 10 μ mmol/L Y-27632 ); ( 5 ) HG Y-27632 group ( 10 μmmol/L Y-27632 ). The supernatant and cells were collected at 0,12,24,36,48, and 72 h. Western blot was used to detect the active RhoA and total RhoA,while RhoA, ROCK-Ⅰ, CTGF, and TNF-α mRNA expressions were determined with realtime PGR method in the cells, then ELISA method was used to check protein levels of FN, CTGF, and TNF-α in the supernatant. Results ( 1 ) RhoA activation was stimulated after treatment for with 30 mmol/L glucose, peaked at 1 h, and then decreased ( P = 0. 02). (2) RhoA, ROCK-Ⅰ, CTGF, and TNF-α mRNA expressions in HMC cultured under high glucose were higher than those in the normal group ( P ＜ 0.05 ), and there was certain time-dependence. Besides, there was no statistical significance between Man and NG groups( P＞0. 05 ). ( 3 ) After Y-27632 pretreatment and being cultured with normal glucose and high glucose for24 h or48 h, RhoA, ROCK-Ⅰ, CTGF, and TNF-α mRNA expressions were significantly decreased ( P＜0.01 ) as compared with groups without treatment. (4) High glucose increased FN, CTGF,and TNF-α protein secretion of HMC in a time-dependent manner( P＜0. 05 ). ( 5 ) After Y-27632 pretreatment and being cultured with normal and high glucose for 12,24,36,48,72 h, FN, CTGF, and TNF-α protein secretions were significantly reduced( P＜0.05 ). Conclusion Rho/ROCK signaling pathway may mediate inflammation and fibrosis induced by high glucose in HMCs, supporting a potential role for inhibitors of Rho/ROCK in the treatment of diabetic nephropathy.