Urotensin Ⅱinduces pulmonary arterial smooth muscle cell proliferation and up-regulates Egr-1 expression through ERK1/2 pathway in rats

AIM: To investigate the effect of urotensinⅡ (UⅡ) on the proliferation of cultured rat pulmonary arterial smooth muscle cells (PASMCs), and to explore whether mitogen-activated protein kinase (MAPK) signaling pathways and early growth response factor-1 (Egr-1) involved in the regulation of the PASMCs proliferation stimulated by UⅡ. METHODS: The rat PASMCs were isolated and cultured in vitrowith explant culture technique. The proliferation of cultured PASMCs stimulated by different doses of UⅡwas detected by BrdU incorporation. The mRNA expression of extracellular signal-regulated kinase 1/2 (ERK1/2), stress-activated protein kinase (SAPK), p38 MAPK and Egr-1 in cultured PASMCs treated with UⅡ, UⅡ-specific antagonist urantide, and ERK1/2 inhibitor PD98059 was detected by real-time PCR. The protein levels of phosphorylated ERK1/2 (p-ERK1/2), p-SAPK, p-p38 and Egr-1 in cultured PASMCs were determined by Western blotting. RESULTS: UⅡ at concentrations of 1 μmol/L, 0.1 μmol/L and 0.01 μmol/L increased the proliferation of cultured PASMCs in a dose-dependent manner (P<0.01 or P<0.05), with the maximal effect at a concentration of 1 μmol/L. However, urantide inhibited the promotion effect of UⅡ on PASMC proliferation (P<0.05). UⅡ up-regulated the mRNA expression of ERK1/2, SAPK and Egr-1 (P<0.01 or P<0.05), but not the p38 MAPK. However, the up-regulatory effect of UⅡ on ERK1/2 and Egr-1 expression was inhibited by PD98059 and/or urantide (P<0.01 or P<0.05). UⅡ also increased the protein levels of p-ERK1/2, p-SAPK and Egr-1 (P<0.01 or P<0.05), but the promotion effect was also inhibited by PD98059 and/or urantide (P<0.01 or P<0.05).CONCLUSION: UⅡ increases the proliferation of PASMCs, and U Ⅱand Egr-1 participates in UⅡ-mediated proliferation of cultured PASMCs through activation of ERK1/2 signal pathway.     

Expression of urotensinⅡ receptor GPR14 in aorta of apoE knockout mice

AIM: To investigate the expression of the urotensin Ⅱ (UⅡ) receptor GPR14 in the aorta of apoE knockout mouse. METHODS: The expression of GPR14 in the aorta of apoE knockout C57BL/6J mice at various ages (18 weeks, 28 weeks, and 38 weeks old, respectively) was determined with competitive RT-PCR. A binding assay of ［125I］-UⅡ on the aortic tissue was also performed in 28 weeks group. RESULTS: We found significant upregulation of GPR14 mRNA at all three ages. Compared with wild type group at the same age, the GPR14 mRNA level in apoE knockout mice increased 54.2% in 18 week group (P<0.05), 50.0% in 28 weeks group (P<0.05) and 97.0% in 38 weeks group (P<0.01). In the knockout group or in the wild type group, expressions of GPR14 in the 28 weeks time point were significantly higher than that in other two age groups, and there was no difference between the 18 weeks and 38 weeks group. In the binding assay, the Bmax of ［125I］-UⅡ to the aorta of apoE knockout mouse at 28 weeks increased 64% compared with the wild type (P<0.01), and no difference about the Kd between the two groups was observed. CONCLUSION: UⅡ and its receptor probably play an important role in the development of atherosclerosis.     

Effects of sodium hydrosulfide on cardiac function and activity of renin-angiotensin-aldosterone system in rats with chronic heart failure

AIM: To investigate the effect of sodium hydrosulfide (NaHS) on cardiac function and activity of renin-angiotensin (Ang)-aldosterone (ALD) system (RAAS) in the rats with chronic heart failure (CHF).METHODS: The CHF rat model was established by abdominal aortic coarctation. SD rats were randomly divided into sham operation group, model group, low dose of NaHS group and high dose of NaHS group (n=6). The left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD) and left ventricular ejection fraction (LVEF) were measured before and after treatment by echocardiography in each group. The levels of renin, AngⅡ and ALD in the plasma were measured by ELISA. The expression of angiotensin-converting enzyme (ACE) and angiotensin Ⅱ type 1 receptor (AT1R) at mRNA and protein levels in the myocardium tissues was determined by qPCR and Western blot, respectively.RESULTS: After treatment with NaHS, compared with model group and before treatment, LVEDD and LVESD in low dose of NaHS group and high dose of NaHS group were decreased significantly, while LVEF was increased significantly (P<0.05). Compared with low dose of NaHS group, LVEDD and LVESD were decreased, while LVEF was increased in high dose of NaHS group (P<0.05). Compared with sham operation group, the levels of renin, AngⅡ and ALD in the plasma of model group were significantly increased (P<0.05), and the expression of ACE and AT1R at mRNA and protein levels in the myocardium tissues of model group were significantly increased (P<0.05). Compared with model group, the plasma levels of renin, AngⅡ and ALD in low dose of NaHS group and high dose of NaHS group were significantly decreased (P<0.05), and the myocardial expression of ACE and AT1R at mRNA and protein levels was significantly down-regulated (P<0.05). The plasma levels of renin, AngⅡ and ALD, and the myocardial expression of ACE and AT1R at mRNA and protein levels in high dose of NaHS group were significantly lower than those in low dose of NaHS group (P<0.05).CONCLUSION: NaHS inhibits the activation of RAAS, thus improving the cardiac function of CHF rats, and the effect of high-dose NaHS is better than that of low-dose NaHS.     

Role of vascular remodeling for urotensin Ⅱ in rat thoracic aorta after balloon injury

AIM: To explore the possible effect of UII in the process of remodeling after vascular injury. METHODS: The rat model of balloon injury in thoracic aorta was established. Male rats were randomized to 4 groups (n=5), including sham injury group, injury group, UII group (UII pumped into the rats after thoracic aorta balloon injury at 1.0 nmol·kg-1·h-1) and urantide group (urantide pumped into the rats after thoracic aorta balloon injury at 10 nmol·kg-1·h-1). At 21 days, the thoracic aortas were taken out to measure the changes of pathology, the expression of UII, the proliferation of VSMC and the expression of collagen. RESULTS: (1) At the 21 days after operations, the systolic blood pressure was higher in UII group than that in injury group ［(140.0±10.0) mmHg vs (132.0±3.4) mmHg, P>0.05］. The systolic blood pressure was also obviously higher than that in urantide group ［(140±10.0) mmHg vs (128.0±2.4) mmHg, P<0.05］. (2) Urotensin Ⅱ was expressed strongly in the injured area after thoracic aorta injury. (3) In contrast to injury group, the intimal thicken in urotensin Ⅱ group enhanced, the decrease in lumen area was marked (0.13±0.05 vs 0.07±0.02, P<0.05), the cell proliferation index was markedly increased (0.74±0.16 vs 0.40±0.11, P<0.01), and the expression of collagen was also markedly increased (counted as IOD, 318±127 vs 78±26, P<0.01). (4) In contrast to injury group, the decrease in lumen area was not abolished (0.09±0.03 vs 0.07±0.02, P>0.05) after chronic infusion of urotensin Ⅱ receptor antagonist urantide, the cell proliferation index was markedly increased (0.73±0.15 vs 0.40±0.11, P<0.01) and the expression of collagen was not statistically increased (counted as IOD, 200±79 vs 78±26, P>0.05). CONCLUSION: Urotensin Ⅱ expresses strongly in the myointimal cells after thoracic aorta injury in rat. The extra UII enhances the proliferation of VSMC and expression of collagen in the myointimal, increases the stenosis of injured vasculature, indicating that UII might take part in the process of repairing after vessel injury.     

Effect of nitric oxide on urotensin-Ⅱ expression in pulmonary arterioles of rats chronically exposed to hypoxia-hypercapnia

AIM: To investigate the roles of nitric oxide/L-arginine (NO/L-Arg) pathway and urotensin-Ⅱ (UⅡ) in the development of pulmonary hypertension induced by chronic hypoxia-hypercapnia in rats.METHODS: Forty male Sprague-Dawley rats were randomly divided into four groups (n=10): normal control group (A), hypoxia-hypercapnia+saline group (B), hypoxia-hypercapnia+L-Arg liposome group (C) and hypoxia-hypercapnia+N-nitro-L-arginine methyl ester (L-NAME) group (D).Contents of UⅡ, UⅡ mRNA and receptor of UⅡ (UT) mRNA in pulmonary arterioles were measured with immunohistochemistry analysis and in situ hybridization, respectively.Change of small pulmonary vascular microstructure was also investigated.RESULTS: (1) The mean pulmonary artery pressure (mPAP) and the weight ratio of right ventricle to left ventricle plus septum ［RV/(LV+S)］ in B and D groups were all higher than those in A group (respectively, P<0.05), with C group significantly lower than those in B group (respectively, P<0.01).(2) Light microscopy showed that the ratio of vessel wall area to total area (WA/TA) and the media thickness of pulmonary arterioles (PAMT) in B group were higher than those in A group (P<0.05), with C group significantly lower than those in B group.(3) The contents of UⅡ, UⅡ mRNA and UT mRNA in pulmonary arterioles in B and D groups were all higher than those in A group (respectively, P<0.01), while the expression of UⅡ and UⅡ mRNA in C group were lower than those in B group (P<0.01).CONCLUSION: The pathological process of pulmonary hypertension induced by chronic hypoxia-hypercapnia might be related to upregulation of UⅡ located in pulmonary arterioles, which might be partially inhibited by exogenous NO in rats.     

Preventive effect of Sini decoction on expression of TGF-β1 in rat myocardiac fibrosis induced by isoproterenol

AIM: To explore the protective effects of Sini decoction (SD) on myocardial fibrosis induced by isoproterenol (Iso) in rats.METHODS: Nineteen Wistar rats were divided into Iso group, SD treatment group and control group. The rats in Iso group were injected with Iso and were then fed with saline. The rats in SD treatment group were injected with Iso and were then fed with SD. The rats in control group were injected with saline and were then fed with saline. The level of hydroxyproline (Hyp), the contents of angiotensin Ⅱ (AngⅡ) and transforming growth factor beta-1 (TGF-β1) in plasma were measured 4 weeks after administration. TGF-β1 at mRNA and protein levels were measured by the techniques of ELISA and RT-PCR. RESULTS: The plasma levels of TGF-β1 and AngⅡ were lower in control group than those in Iso group and SD treatment group (P<0.05). The plasma levels of TGF-β1 and AngⅡ in SD treatment group were lower than those in Iso group (P<0.05). Compared to Iso group, the cardiac diastolic function was significantly improved in SD treatment group (P<0.05). The results of immunohistochemistry and RT-PCR showed that the mRNA and protein expressions of TGF-β1 were lower in SD group than those in Iso group (P<0.05). CONCLUSION: SD alleviates myocardial fibrosis induced by Iso in rats by decreasing TGF-β1 expression at mRNA and protein levels.     

Effect of ginsenoside Rb1 on liver cell pyroptosis in hyperlipidemia rats and its possible mechanism

AIM:To discuss the mechanism of ginsenoside Rb1 against liver lipid deposition by observing the effect of ginsenoside Rb1 on liver cell pyroptosis in hyperlipidemia rats. METHODS:Totally 32 healthy SPF rats were randomly divided into control group, model group, ginsenoside Rb1 group and simvastatin group. The rats in control group was given the basic feed, while the others were given high-fat diet. The rats in ginsenoside Rb1 group and simvastatin group were given corresponding drugs. The rats in control group and model group were intraperitoneal injected with equal volume of saline. Eight weeks later, the serum levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were tested by the automatic biochemistry analyzer. The pathological changes of the liver tissues were observed with HE staining. The protein and mRNA expression levels of pyroptosis-related factors NLRP3, caspase-1, IL-1β, IL-18 and GSDMD were detected by Western blot and RT-qPCR. RESULTS:Compared with control group, the serum levels of TC, TG and LDL-C in model group were increased significantly (P<0.01), and the HDL-C content was decreased significantly (P<0.05). The steatotic liver cells covered the visual field. The mRNA and protein expression levels of NLRP3, caspase-1, IL-1β, IL-18 and GSDMD were increased significantly (P<0.01). Ginsenoside Rb1 significantly decreased the serum levels of TC, TG and LDL-C (P<0.05), and significantly increased the content of HDL-C (P<0.01). Ginsenoside Rb1 also significantly decreased the degree of steatosis, and the number and size of lipid droplets. The mRNA and protein expression levels of NLRP3, caspase-1, IL-1β, IL-18 and GSDMD were decreased significantly (P<0.05 or P<0.01). CONCLUSION:Ginsenoside Rb1 atte-nuates liver injury and inhibits liver lipid deposition in hyperlipidemia rats by reducing the expression of hepatic pyroptosis-related factors.     

Effects of gypenosides on PCSK9 gene expression and blood lipids lowered by simvastatin

AIM: To explore the effect of gypenosides (GPs) on PCSK9 gene expression in hyperlipidemic rat liver and the blood lipids lowered by simvastatin.METHODS: Healthy male SD rats (n=60) were randomized into 5 groups:normal control group, hyperlipidemic model group, simvastatin group, GPs group and GPs combined with simvastatin group (combined group). The rats in all groups were fed high-fat diet except normal control group which were fed with ordinary diet. The rats in control group and hyperlipidemic model group were gavaged with 0.3% CMC-Na every day. The rats in GPs group were gavaged with GPs at 160 mg·kg^-1·d^-1. The rats in simvastatin group were gavaged with simvastatin at 5 mg·kg^-1·d^-1. The rats in combined group were gavaged with GPs and simvastatin. The experiment lasted for 8 weeks. The rats were anesthetized with chloral hydrate, and abdominal arterial blood samples were collected to detect the total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C). The body weight and the wet weight of the livers were measured, and the liver index was calculated. The pathological changes of the livers were observed under microscope with HE staining. The expression of PCSK9 and low-density lipoprotein receptor (LDLR) at mRNA and protein levels was determined by real-time PCR and Western blot. RESULTS: The model of hyperlipidemia rats was established successfully. Compared with model group, the levels of TC, TG and LDL-C in simvastatin group, GPs group and combined group were obviously decreased (P<0.05), and the HDL-C levels were obviously upregulated (P<0.05). Compared with model group, the liver indexes in simvastatin group, GPs group and combined group were obviously decreased (P<0.05). The pathological changes of the liver tissues showed that hepatic adipose appeared in model group, and that in simvastatin group and GPs group had different degrees of relief, especially in combined group. Compared with model group, the mRNA expression levels of PCSK9 and LDLR in simvastatin group were obviously increased, while the mRNA expression levels of PCSK9 in GPs group and combined group were obviously decreased (P<0.05), and the mRNA expression of LDLR in combined group was obviously increased (P<0.05). Compared with model group, the protein expression of PCSK9 and LDLR in simvastatin group was obviously increased, while the protein expression levels of PCSK9 in GPs group and combined group were obviously reduced, and the LDLR protein levels were obviously increased (P<0.05). CONCLUSION: Gypenosides inhibit the expression of PCSK9 and increase the expression of LDLR in the liver. The combination of gypenosides and simvastatin promotes the lipid-lowering effect of simvastatin and attenuates hepatic steatosis, which may be related to inhibiting the expression of PCSK9 in the liver.     

Effect of Qiliqiangxin granule on apoptosis of renal tissues in rats with cardiorenal syndrome

AIM: To investigate the effect of Qiliqiangxin granule on the apoptosis of renal tissues in rats with cardiorenal syndrome (CRS) and its possible mechanism. METHODS: A rat model of CRS was established by ligation of the left anterior descending coronary artery and acute renal ischemia/reperfusion injury. After operation, the rats were divided into 6 groups:2-week sham operation (2w sham) group, 2-week model (2w CRS) group, 2-week drug (2w CRS-Q) group, 4-week sham operation (4w sham) group, 4-week model (4w CRS) group and 4-week drug (4w CRS-Q) group. The rats in 2w CRS-Q group and 4w CRS-Q group were given Qiliqiangxin granule (4 g·kg^-1·d^-1) by gavage for 2 weeks and 4 weeks, respectively. The levels of serum cystatin C (Cys-C), plasma angiotensin Ⅱ (Ang Ⅱ), urine neutrophil gelatinase-associated lipocalin (NGAL) and urine microalbumin (UMA) were measured by ELISA. The serum level of creatinine (Cre) was detected by sarcosine oxidase method. The renal histopathological changes were observed by HE staining. The mRNA and protein expression levels of Ang Ⅱ, Bcl-2 and Bax were evaluated by RT-qPCR and Western blot, respectively. The apoptosis rate of renal cells was assessed by TUNEL staining. RESULTS: The levels of serum Cys-C, serum Cre, plasma Ang Ⅱ, urine NGAL and UMA were significantly increased in 2w CRS group and 4w CRS group compared with 2w sham group and 4w sham group after modeling (P<0.05). The mRNA and protein expression levels of Bax and Ang Ⅱ in the renal tissues of CRS rats were significantly up-regulated (P<0.05), while Bcl-2 was significantly down-regulated (P<0.05) compared with 2w sham group and 4w sham group. Compared with 2w sham group and 4w sham group, the damage of renal tissues in 2w CRS and 4w CRS group was severe, and the apoptotic rates of renal cells were significantly increased. Compared with 2w CRS group and 4w CRS group, Qiliqiangxin granule greatly decreased the levels of Cys-C, Cre, Ang Ⅱ, NGAL and UMA, down-regulated the mRNA and protein expression levels of Bax and Ang Ⅱ in the renal tissues, and up-regulated the expression of Bcl-2 at mRNA and protein levels at 2 and 4 weeks. In addition, Qiliqiangxin granule also greatly attenuated the damage and apoptosis of the renal tissues. CONCLUSION: Qiliqiangxin granule significantly inhibits the apoptosis of renal tissues and improves the renal function of CRS rats, and its mechanism may be related to the inhibition of Ang Ⅱ expression.     

Inhibitory effect of recombinant human endostatin on angiogenesis in atherosclerotic plaque of rats by regulating Dll4/Notch pathway

AIM: To observe the inhibitory effect of recombinant human endostatin (rhES) on plaque angiogenesis, and to explore the regulatory mechanism of Dll4/Notch pathway in the anti-angiogenic effect of rhES. METHODS: Male Wistar rats were randomized into 3 groups:normal control group (N group), atherosclerotic model group (AS group), and rhES treated group (AS+rhES group). The rats in N group were fed a normal diet, while the remaining 2 groups were established to atherosclerotic rat model via high-cholesterol diet, intraperitoneal injection of vitamin D₃ and aortic balloon injury. The rats in AS+rhES group received intraperitoneal injection of rhES. The blood total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), C-reactive protein (CRP), interleukin-1 (IL-1) and troponin I (TnI) were measured. The atherosclerotic abdominal aortas were taken for pathological observation. Immunohistochemical staining was used to measure the density of neovessels in the plaques, which were marked by CD31. The protein levels of Dll4 and Notch1 in the aortas were analyzed by Western blot. RESULTS: The levels of blood TC, TG, LDL-C, CRP and IL-1 in AS group and AS+rhES group were much higher than those in N group (P<0.05), and no statistical difference between AS group and AS+rhES group was observed. The expression of CD31 in AS group was the highest among all groups. Compared with AS group, the density of neovessels in the plaques of AS+rhES group decreased significantly (P<0.05). The protein expression of Dll4 and Notch1 in AS group was lower than that in N group (P<0.05). Compared with AS group, the protein expression of Dll4 and Notch1 increased significantly (P<0.05). CONCLUSION: rhES has the ability to inhibit plaque angiogenesis in rats. The activation of Dll4/Notch pathway may be the mechanism of rhES in inhibiting plaque angiogenesis.     

PP2A activation in mesenteric arteries of angiotensin Ⅱ-induced hypertensive rats

AIM: To investigate the mechanism of protein phosphatase 2A (PP2A) activation in mesenteric arteries of angiotensinⅡ(AngⅡ)-induced hypertensive rats. METHODS: Adult male Sprague-Dawley (SD) rats were subjected to AngⅡinfusion (500 ng·kg^-1·min^-1) using osmotic minipump up to 14 d to established the hypertension model. The rats (n=40) were randomly divided into 4 groups:control group (n=10), AngⅡgroup (n=10), candesartan (CAN; AngⅡtype 1 receptor blocker)+AngⅡgroup (n=10) and CAN group (n=10). The rats in CAN+AngⅡgroup and CAN group were administered with candesartan ester at the dose of 10 mg·kg^-1·d^-1 by gavage on the first day after implantation of osmotic minipump. The rats were sacrificed on the 15th day after minipump implantation. Serum and mesenteric arteries were collected. Systolic blood pressure was measured by tail-cuff method. The serum levels of AngⅡ were measured by ELISA. The protein levels of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (Ser1177), PP2A catalytic subunit (PP2Ac), phosphorylated PP2Ac (Tyr307) and PP2A inhibitor 2 (I₂^PP2A) in the mesenteric arteries were determined by Western blot. The activity of PP2A in the arteries was detected using PP2A activity assay kit. RESULTS: Compared with control group, the systolic blood pressure in AngⅡgroup was significantly increased(P<0.05), while those in CAN+AngⅡgroup and CAN group were significantly decreased (P<0.05). The serum levels of AngⅡ in AngⅡ group and CAN+AngⅡ group were significantly higher than that in control group (P<0.05). Compared with control group, the phosphorylation levels of eNOS Ser1177 were decreased in AngⅡgroup (P<0.05), but the activity of PP2A was significantly increased (P<0.05), and Pearson correlation analyses showed a negative correlation between PP2A activity and eNOS S1177 phosphorylation (r=-0.842, P<0.05). Compared with AngⅡgroup, the phosphorylation levels of eNOS Ser1177 in CAN+AngⅡgroup were significantly increased (P<0.05), but the activity of PP2A was reduced (P<0.05). Compared with control group, the protein levels of phosphorylated PP2Ac (Tyr307) and I₂^PP2A in the mesenteric arteries were decreased in AngⅡgroup (P<0.05), but increased in CAN+AngⅡgroup (P<0.05). No significant difference in all above-mentioned measures between control group and CAN group, nor in the levels of total eNOS and PP2Ac protein expression among all the groups was observed. CONCLUSION: AngⅡmay reduce the protein levels of phosphorylated PP2Ac (Tyr307) and I₂^PP2A in the mesenteric arteries of AngⅡ-induced hypertensive rats through AngⅡ/AngⅡ type 1 receptor-mediated signaling pathway, resulting in the activation of PP2A, then leading to down-regulation of eNOS S1177 phosphorylation, which ultimately mediates the occurrence of vascular endothelial dysfunction.     

Effects of arginine vasopressin on subunit phosphorylation of GABAA receptors in rat preoptic area

AIM: To investigate the effects of arginine vasopressin (AVP) on the expression and phosphorylation of γ-aminobutyric acid (GABA) subtype A receptor (GABA_A receptor) subunits in the preoptic area (POA) of rats. METHODS: The rats were divided into AVP group (n=10), AVP+V1a receptor blocker group (n=10), V1a receptor blocker group (n=10) and control group (n=10). After intraperitoneal injection of AVP or V1a receptor antagonist for 0.5 h, the changes of the expression and phosphorylation of POA GABA_A receptor subunits (α, β and γ2) were analyzed by RT-qPCR and Western blot. RESULTS: Compared with control group, no significant change of GABA_A receptor subunit expression in the rats injected with AVP or V1a receptor antagonist was observed. AVP up-regulated the phosphorylation level of POA GABA_A receptor γ2 subunit (P<0.05), and significantly increased the expression and phosphorylation of protein kinase C (PKC) and calcium/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ). CONCLUSION: Exogenous AVP has no effect on expression of POA GABA_A receptor subunits (α, β and γ2) and is involved in modulation of GABAergic synaptic transmission in the POA by activation of PKC and CaMKⅡ and phosphorylation of γ2 subunit via V1a receptor.     

Influence of Salvia miltiorrhiza injection on lipid and ICAM-1 expression in rats with atherosclerosis

AIM: To study the effect of Salvia miltiorrhiza injection on rat atherosclerosis (AS), and elucidate the possible mechanism. METHODS: Wistar rats were fed with fat-rich diet and high dose of vitamin D₃ to induce AS, then treated with Salvia miltiorrhiza injection. Concentrations of triglyceride (TG) and total cholesterol (TC) in serum were measured by automatic serum biochemical assay. The level of ICAM-1 protein and mRNA were determined by Western blot and RT-PCR. RESULTS: Compared with the AS model group, the levels of TG and TC in serum were significantly lower in Salvia miltiorrhiza injection group (P<0.05). Western blot and RT-PCR showed that the level of ICAM-1 protein and mRNA were decreased in Salvia miltiorrhiza injection group compared with AS group. CONCLUSION: Salvia miltiorrhiza injection decreased blood lipid and reduced the ICAM-1 gene expression in rats with atherosclerosis.     

Effects of macrophage polarization during development of liver cirrhosis in rats

AIM: To explore the state of macrophage polarization and its relation with intestinal endotoxemia-endoplasmic reticulum stress in the development of liver cirrhosis induced by multiple pathogenic factors in rats. METHODS: The male SD rats (n=36) were randomly divided into normal control group and liver cirrhosis model group, and sacrificed at the end of the 4th, 6th and 8th weeks. The rat model of liver cirrhosis was induced by multiple pathogenic factors. The levels of alanine aminotransferase (ALT), endotoxin, homocysteine (Hcy) in the plasma, and inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), arginase-1 (Arg-1) and interleukin-10 (IL-10) in the liver tissues were detected by ELISA. Histopathological change of the liver was observed under microscope with the staining of hematoxylin and eosin (HE) and van Gieson (VG). The expression of glucose-regulated protein 78 (Grp78), nuclear factor-kappa B (NF-κB), interferon-regulatory factor 5 (IRF5), CD86, CD206 and transforming growth factor-β1 (TGF-β1) at mRNA levels in the liver tissues were detected by the method of real-time fluorescence quantitative PCR.RESULTS: Compared with the corresponding normal control group, the levels of ALT, endotoxin, Hcy in the plasma and Grp78 mRNA in the liver tissues in liver cirrhosis model group were significantly and gradually increased (P<0.05). The mRNA expression of NF-κB, IRF5 and CD86, and the protein levels of iNOS, TNF-α and IL-6 in the liver tissues were significantly increased (P<0.05), and they successively increased from the 4th week to the 6th week and decreased reversely at the 8th week. The mRNA expression of CD206, TGF-β1, Arg-1 and IL-10 in the liver tissues were significantly increased from the 6th week to the 8th week (P<0.05), and no significant difference at the 4th week was observed. The level of endotoxin in the plasma was correlated with the mRNA expression of Grp78 in the liver tissues (P<0.01). Both endotoxin in the plasma and Grp78 mRNA in the liver tissues were correlated with the mRNA expression of CD86 and CD206 in the liver tissues (P<0.01).CONCLUSION: The pathway of liver damage-intestinal endotoxemia-endoplasmic reticulum stress-macrophage polarization may be critical in the pathogenesis of liver cirrhosis induced by multiple pathogenic factors.     

Qishen-Yiqi dripping pills attenuate atherosclerosis by regulating Ca2+ homeostasis via TRPC1/STIM1 pathway

AIM:To explore the therapeutic effect of Qishen-Yiqi dripping pills (QS) on atherosclerosis (AS) and the mechanism. METHODS:AS rat model was established by high-fat diet, and SD rats were randomly divided into normal control group, AS model group, low-dose, middle-dose and high-dose QS groups, and positive group (n=6 each). After administration for 12 weeks, serum samples were collected to detect the serum lipid and Ca²⁺ levels. HE staining was used evaluated the histopathological changes of arterial tissue. The serum levels of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) were measured by ELISA. The nitric oxide (NO) level was detected by nitrate reductase method. The protein levels of transient receptor potential channel protein 1 (TRPC1), stromal interaction molecule 1 (STIM1) and endothelial NO synthase (eNOS) were determined by Western blot. RESULTS:QS significantly reduced the arterial damage via inhibiting the formation of atherosclerotic plaque and attenuated intimal thickening and vascular stenosis. Compared with AS group, the serum levels of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) were decreased significantly and the levels of high-density lipoprotein cholesterol (HDL-C) were increased significantly in high-dose QS group (P<0.05). The serum levels of IL-1β, IL-6 and TNF-α in high-dose QS group were lower than those in AS group (P<0.05). Compared with AS group, the serum Ca²⁺ level was lowered and the arterial tissue NO level was elevated in QS groups (P<0.05). Compared with AS rats, the protein levels of TRPC1 and STIM1 were decreased significantly and the protein level of eNOS was increased significantly in the rats treated with QS (P<0.05). CONCLUSION:QS regulate calcium homeostasis via TRPC1/STIM1 pathway, increase the production of NO and inhibit the inflammatory responses, thus exerting anti-AS effect.     

Expression of urotensin II and its receptor in nephridial tissues of rats with acute renal damage

AIM: To observe the mRNA expression of urotensin II (UII) and its receptor (G protein-coupled receptor 14,GPR14) in nephridial tissues of rats with acute renal damage. METHODS: Male Wistar rats were divided into 2 groups: the rats in control group (n=10) were administered with normal saline by gavage; the rats in model group (n=30) were administered with Caulis Aristolochiae manshuriensis (CAM) by gavage for 25 d to induce acute renal damage. Every 5 rats in model group were sacrificed on the 3rd, 7th, 15th and 25th days during CAM treatment and all rats in the 2 groups were killed 10 d after withdraw of CAM. The kidneys were collected for pathological observation and the UII and GPR14 mRNA examination.RESULTS: The degeneration, necrosis and disintegration in tubules were observed as major pathological changes in the rat kidneys after 3 d of CAM administration. The pathological changes were aggravated following the duration of in CAM administration, and were remained and even worsen when CAM was withdrawn for 10 d. Compared with control group, the mRNA expression of UII was significantly elevated (P<0.05) at the time point of CAM administration for 15 d,even obviously increased (P<0.01) at the time point of CAM administration for 25 d, and remained at the highest levels to the end of the observation. The mRNA expression of GPR14 was significantly increased (P<0.05) at the time point of CAM administration for 7 d, became higher (P<0.01) on the 15th day, and gradually increased as the experimental time went on. CONCLUSION: The mRNA levels of UII and its receptor are significantly elevated in CAM-induced renal lesion in rats, suggesting that UII plays a pathological role in the development of acute renal damage.     

Effect of high-fructose diet on adipose tissue renin-angiotensin system in rats

AIM: To observe the effects of high-fructose diet on adipose tissue inflammation and renin-angiotensin system (RAS), and to reveal the role of Toll-like receptor 2 (TLR2) in this process.METHODS: Male SD rats (n=16) were randomly divided into control group, high fructose group, high fructose+siRNA negative control group, and high fructose+TLR2-siRNA group. The rats in control group were fed with a standard chow diet. The rats in high fructose group were fed with a diet with 60% fructose, and the rats in high fructose+TLR2-siRNA group and high fructose+siRNA negative control group were transfected with TLR2 siRNA and scrambled siRNA, respectively. Serum uric acid was measured and visceral adipose tissue was weighed at the 14th week. The levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), angiotensinogen (AGT), and angiotensin Ⅱ (Ang Ⅱ) were measured by ELISA. Infiltrating macrophages in the adipose tissues were measured with immunohistochemistry. The mRNA expression of IL-6, TNF-α, monocyte chemoattractant protein-1 (MCP-1), AGT, angiotensin-converting enzyme 1 (ACE1), angiotensin Ⅱ type 1 receptor (AT1R), and angiotensin Ⅱ type 2 receptor (AT2R) was detected by RT-qPCR. The protein level of TLR2 was determined by Western blot.RESULTS: High fructose-fed rats showed elevated serum uric acid, raising fat content, higher serum concentrations of IL-6, TNF-α, AGT and AngⅡ, and more infiltrating macrophages in the adipose tissues (P<0.05). Moreover, the mRNA levels of IL-6, TNF-α,MCP-1, AGT, ACE1, AT1R and AT2R in the adipose tissues were increased (P<0.05). When high fructose-fed rats were transfected with TLR2-siRNA, the dramatic decreases in TLR2 protein level and number of infiltrating macrophages in the adipose tissues were found. Both in serum and adipose tissues, the mRNA levels of inflammatory cytokines and RAS components were all significantly decreased (P<0.05).CONCLUSION: High-fructose diet up-regulates RAS in adipose tissues via activation of TLR2 inflammation signaling pathway.     

Effect of GLP-1 on insulin resistance and PKCε in rats with nonalcoholic fatty liver disease induced by high fat diet

AIM: To observe the therapeutic effect of glucagon-like peptide 1 (GLP-1) analog on nonalcoholic fatty liver disease of rats and to investigate the underlying mechanism.METHODS: SD rats (n=21) were used to establish a nonalcoholic fatty liver disease model by feeding a high fat diet for 12 weeks, and other 11 rats were fed with a normal diet for 16 weeks. The model rats were randomly divided into 2 equal groups:one group was treated with glucagon-like peptide 1 analog (0.6 mg·kg^-1·d^-1) by intraperitoneal injection for 4 weeks, the other group using saline as a control. After treatment, fasting blood glucose, serum insulin, blood lipids, liver function and the pathological changes of the hepatic tissues were evaluated and the expression of PKCε at mRNA and protein levels in the liver tissues was detected by real-time PCR and Western blot, respectively.RESULTS: Compared with model group, the intervention of GLP-1 significantly reduced insulin resistance index (HOMA-IR), improved the liver function (P<0.05), decreased the liver index and blood lipids (P<0.05). HE staining showed obvious pathological changes of the hepatic tissues in model group, and the intervention of GLP-1 significantly reduced lipid droplets in the hepatocytes and improved the structural damage of the liver. The expression of hepatic protein kinase Cε (PKCε) at mRNA and protein levels significantly decreased which were reversed by treating with GLP-1.CONCLUSION: GLP-1 shows good therapeutic effect on nonalcoholic fatty liver disease of rats, possibly by controlling lipid metabolism and reducing insulin resistance, which may be related to PKCε expression.     

Effects of antihistamine treatment on mast cell infiltration and c-Kit and SCF expression in liver tissues of rats with experimental hepatitis

AIM：To study the infiltration of mast cells and the expression of c-Kit and stem cell factor (SCF) in liver tissues of rats with experimental hepatitis and their changes after antihistamine (AH) treatment. METHODS：Thirty Wistar rats were divided into 3 groups at random: normal control (NC) group, chronic hepatitis (CH) group and AH group. The rat model of CH was established by composite factors (subcutaneous injection of carbon tetrachloride, accompanied by a diet containing high cholesterol, high alcohol, low protein and low choline). The rats in AH group were treated with ketotifen based on CH. At the end of the 4th week, blood samples were taken to determine plasma tryptase (TS) and histamine (HA) levels. Liver tissues were taken to detect HA content, observe the histological changes with HE staining and count the number of mast cells with toluidine blue (TB) staining. The mRNA and protein expression of c-Kit and SCF in liver tissues was detected by RT-PCR and immunohistochemistry. RESULTS：(1) The plasma TS and HA levels and liver HA content in CH group were significantly increased compared with NC group (P<0.05), while those in AH group were obviously decreased compared with CH group (P<0.05). (2) Fatty degeneration and fibrosis were observed in CH group under light microscope, but the hepatic injury was obviously attenuated in AH group. TB staining showed there were many degranulating and degranulated mast cells filled with purple granules around liver blood vessels and in fiber interval in CH group, and there were few purple granules in the cytoplasm of mast cells in AH group. The number of mast cells in CH group was increased compared with NC group (P<0.05), and that in AH group was reduced compared with CH group (P<0.05). (3) The results of RT-PCR showed that AH down-regulated the expression of c-Kit and SCF mRNA (P<0.05). The expression of c-Kit and SCF proteins in liver tissues increased in CH rats (P<0.05 vs NC group), decreased after AH treatment (P<0.05 vs CH group) and was positively correlated with liver HA content (P<0.05). CONCLUSION：These data suggest that an inflammatory pathway mediated by mast cell activation is involved in experimental hepatitis. Ketotifen can reduce mast cell degranulation by down-regulating the expression of mast cell membrane receptor c-Kit and its ligand SCF, thereby attenuating the liver inflammation.