Inducible nitric oxide synthase and arterial pressure regulation

AIM and METHODS:To clarify the role of inducible nitric oxide synthase (iNOS) in the regulation of blood pressure,in the present study, we examined the effect of aminoguanidine (AG), a selective inhibitor of iNOS on the hemodinamical response of Dahl salt-sensitive (DS) and Dahl salt-resistant (DR) rats to low (0.3%) or high (8%) sodium chloride (NaCl) infusion by chronical in vivo hemodynamic experiment, and the effect of NaCl or NaCl plus AG infusion on urinary nitrate (NO₃)/nitrite (NO₂), the end product of nitric oxide (NO), excretion by Greiss Reaction. Furthermore, NOS activity assay was also carried out to probe the effect of NaCl and AG on calcium-dependent or independent NOS activity in renal tissue. RESULTS:1. High or low NaCl-infused DR rats and low NaCl-infused DS rats have no hemodinamical response to AG, however, the hypertensive effect of high NaCl (8%) infusion on DS rats were greatly amplified by co-infusion of AG. 2. Administration of high NaCl significantly elevated the iNOS activity of renal tissue, and greatly increased urinary NO₃/NO₂ excretion. CONCLUSION:Inducible NOS is an important modulator of arterial pressure, especially in case of higher blood pressure.     

Change of synapsins in hippocampal formation during spacial learning and memory in rats

AIM and METHODS: The immunohistochemical method was used to study the on synapsin express in hippocampal formation during spatial learning and memory in rats.RESULTS: (1) In control group there are diffuse brown granules without any obvious granular product in hippocampal formation and in model group there are synapsin products in the hippocampal formation ,especially in the subregion CA3 , CA4 and the dentate gyrus after water maze trained for one week. After two weeks water maze trained the hippocampal formation appear the same distribution of the product granules with darker staining. (2) The light dendity of synapsin products between the model and the control groups showed significant differences, that in the model group was highter than that in the control group (P＜0.05). The light dendity of synapsin products increased with the training time from the first week to the second week in the model group. And there is no increase with the training time from the second week to the third week . The light dendity is higher in the CA3, CA4 subregion and the dentate gyrus than the CA1 and CA2 subregion (P＜0.05).CONCLUSIONS: (1) As mentioned above, it is believed that the new synapes are formed in the hippocampal formation of the rat by spacial learning and memory training. (2) The CA3, CA4 subregion and the dentate gyrus are correlated with the spacial learning and memory.     

Effect of 2, 3-butanedione monoxime (BDM) on myocardial protection in cold cardioplegia

AIM: The effects of BDM on isolated rat heart in cold cardioplegia were studied. METHODS: Rat heart were subjected to cold cardioplegia at 4℃ for 8, 18 and 24 h.Then each heart was perfused (90 cm H₂O) in Langendorff model at 37℃ for 40 min. In the high K⁺ group(n=24) the hearts were preserved in St.Thomas cardioplegic solution, in BDM group(n=24) hearts were preserved in K-H solution with BDM 30 mmoL/L. RESULTS: After 18 h, heart rate and the coronary flow in BDM group were significantly higher than in high K⁺ group(P＜0.05). Activity of Na⁺-K⁺-ATPase in cell membrane and in mitochondrial membrane in the BDM group was significantly higher than high K⁺ group(P＜0.01). After 24 h, all hearts in high K⁺ group were dead, but were alive in BDM group. CONCLUSION: Under given experiment conditions, BDM did enhance the tolerance to cold ischemia significantly. The results showed that BDM may become a useful agent for prolong the storage period of heart in cold cardioglegia.     

Mitochondrial DNA deletion of the brain tissue of aged rats with learning and memory deficit

AIM and METHODS: The ratio of mitochondrial DNA (mtDNA) deletion was measured to find the relationship between mtDNA deletion and aged learning and memory deficit. The aged rats were divided into two groups, aged learning and memory deficit group and aged learning and memory normal group. The ratio of mtDNA deletion was measured by dilution polymerase chain reaction. RESULTS: There are deleted mtDNA (about 4834 bp) in the cerebral cortex, hippocampus and cerebellum of both young and aged rats. The ratios of deleted mtDNA were similar in the cerebral cortex,hippocampus and cerebellum of young rats (about 0.00018%). The ratio mtDNA of aged learning and memory normal rats had increased by five-fold in the cerebral cortex and hippocampus, or one-fold in the cerebellum over young rats. The ratio of aged learning and memory dificit rats had increased by one-fold in the cerebral cortex or 0.8-fold in the hippocampus or two-fold in the cerebellum over aged learning and memory normal rats.CONCLUSIONS: There was really the increase of mtDNA in aging rat brain. And this increase was double in amount in aged learning and memory deficit rats compared to the normal learning and memory aged rats. It is suggested that the mtDNA deletions in the brain regions associated with learning and memory may be contributed to the cellular and molecular mechanism of learning and memory deicit with aged rats.     

Role and regulation of calcineurin in angiotensin Ⅱ-stimulated cardiac myocyte hypertrophy of rats

AIM: To study the role and regulation of calcineurin(CaN) in angiotensin II(AngⅡ)-stimulated cardiacmyocyte hypertrophy of rats. METHODS: Using AngⅡ to induce the cultured cardiac myocyte hypertrophy of rats, and investigating the effect of CaN inhibitor on [³H]-leucine incorporation of AngⅡ-stimulated cardiomyocytes and the regulation of various factors on CaN activity in cardiomyocytes.RESULTS: AngⅡ can stimulate the CaN activity in cultured neonatal rat cardiomyocytes in a dose- and time-dependent manner. In cardiac myocytes incubated with 10, 100, 1000 nmol·L^-1 of AngⅡ for 12h, the CaN activities increased respectively by 13%,57%(P＜0.05) and 228%(P＜0.01) compared with that in non-stimulated cardiomyocytes. The CaN activities in AngⅡ-stimulated cardiomyocytes were significantly inhibited by losartan(50 μmol·L^-1), H₇(50 μmol·L^-1)and Fura-2/AM(4 μmol·L^-1),while no effect was observed with PD98059(50 μmol·L^-1).The [³H]-leucine incorporation in AngⅡ-stimulated cardiomyocytes increased by 46%(P＜0.01) compared with that in control group, which was dramatically inhibited by cyclosporin A(0.5~5μg/mL). CONCLUSIONS: Calcineurin, a Ca²⁺/calmodulin-dependent protein phosphatase, may play an important role in AngⅡ-induced cardiac myocyte hypertrophy. The activation of CaN may dependent on the sustained increases of [Ca²⁺]i and be regulated by some protein kinases (such as PKC,etc.).     

Experimental research on cardiac fibrosis induced by recombinant mouse interleukin-11 in mice

AIM To observe the effect of recombinant mouse interleukin-11 （rmIL-11）injected subcutaneously into mice on heart structure and function and to determine its pro-fibrotic effect. METHODS C57BL/6 mice were randomly divided into experimental group and control group.The mice in experimental group were injected subcutaneously with recombinant mouse IL-11 at the dose of 100 μg·kg^-1·d^-1 for 3 consecutive weeks, while the control group were given equal volume of normal saline in the same way. After the experiment was finished, the parameters of heart function were measured by echocardiography.The heart weight was weighed and the cardiac weight index (CWI) was calculated. HE staining and Masson's trichrome staining were performed to observe the pathological changes and the extent of myocardial fibrosis in mouse myocardia respectively, and the cardiac collagen volume fraction (CVF) was calculated. The expression levels of extracellular matrix proteins in the myocardial tissues of mice, including type Ⅰ collagen, type Ⅲ collagen and fibronectin, were determined by Western blot. RESULTS Left ventricular ejection fraction and left ventricular fraction shortening in experimental group were obviously lower than those in control group (P<0.01), however left ventricular end-diastolic diamension and left ventricular end systolic dimension were significantly higher than those in control group (P<0.05).Compared with control group, the CWI was increased (P<0.01), the myocardial arrangement was disorder, the necrosis of cardiac myocytes was increased, and excessive deposition of collagen was observed in the myocardial tissues in experimental group. Correspondingly, the CVF and protein levels of type Ⅰ collagen, type Ⅲ collagen and fibronectin in the left ventricle in experimental group were increased significantly (P<0.05). CONCLUSION Injection of rmIL-11 into the mice subcutaneously induces fibrogenesis in the heart, which implies that IL-11 is likely a novel pro-fibrotic factor.     

Study on the relationship between melatonin and hepatic encephalopathy

AIM:To explore the relationship between change of serum melatonin (MT) and pathogenesis of hepatic encephalopathy (HE). METHODS: Changes of MT level in sera of cirrhosic patients with HE and without HE were determined by ELISA, normal serum served as control. The change of serum MT level in exacerbation and remission in HE was also determined.RESULTS:MT level in patients with HE was higher than that withour HE (P＜0.01). MT levels of both groups were higher than that of normal group (P＜0.01). They were (308.53±59.07) ng/L, (139.85±34.59)ng/L,(77.73±28.41)ng/L, respectively. Serum MT level in exacerbation was higher than that in remission (P＜0.01), they were (301.52±66.42)ng/L and (147.81±23.31) ng/L, respectively. CONCLUSION: The elevation of MT content in sera may be closely related to the onset of hepatic coma.     

Role of miR-219 and TGFBR2 in pathogenesis of renal fibrosis

AIM:To study the role of microRNA-219 (miR-219) in regulation of transforming growth factor-β receptor type 2 (TGFBR2) in renal fibrosis. METHODS:The renal fibrosis patients (n=70) were selected in this stu-dy, and 20 cases of healthy people were selected as control group. RT-qPCR was used to detect the expression of miR-219 in the serum of the patients with renal fibrosis and control group, and the expression of miR-219 in NRK49F cells after stimulation with angiotensin Ⅱ(AngⅡ) was detected. The protein expression of α-smooth muscle actin (α-SMA) in the NRK49F cells transfected with miR-219 mimics after stimulation with AngⅡ was determined by Western blot. The potential target gene TGFBR2 of miR-219 was screened and verified by the method of luciferase reporter gene. RT-qPCR and Western blot were used to detected the effect of miR-219 mimics on the expression of TGFBR2 at mRNA and protein levels, and the mRNA expression of α-SMA, connective tissue growth factor (CTGF), type I collagen α1 (COL1A1) and COL3A1 in the NRK49F cells was also detected, respectively. The unilateral ureteral occlusion (UUO) mouse model was established and the expression of miR-219 in the renal tissue was monitored. The morphological change of renal fibrosis was observed in the UUO mice after injection of miR-219, and the mRNA expression levels of COL1A1 and COL3A1 were detected. RESULTS:The expression level of miR-219 in the patients with renal fibrosis was significantly lower than that in control group, and the expression of miR-219 in the UUO mice was decreased significantly (P<0.01). The expression level of miR-219 was significantly decreased in the NRK49F cells after AngⅡ stimulation, and miR-219 mimics inhibited the protein expression of α-SMA(P<0.01). miR-219 mimics had a targeted regulatory effect on TGFBR2 gene, which inhibited the mRNA and protein expression of TGFBR2. miR-219 mimics inhibited the mRNA expression of α-SMA, CTGF, COL1A1 and COL3A1. miR-219 also down-regulated the mRNA expression of COL1A1 and COL3A1 in the UUO mice and inhibited the process of renal fibrosis. CONCLUSION:miR-219 inhibits the development of renal fibrosis by inhibiting the expression of TGFBR2, which may become a new target for the diagnosis and treatment of renal fibrosis.     

Effect of miR-29b-mediated TGF-β/Smad signaling pathway on progression of hepatic fibrosis in rats

AIM: To investigate the role of microRNA-29b (miR-29b)-mediated TGF-β/Smad signaling pathway in the activation of hepatic stellate cells (HSC) and its effect on the progression of hepatic fibrosis in rats.METHODS: Hepatic liver fibrosis rat model was established, and its HSC were isolated. Normal rat HSC were also obtained and identified in vitro. RT-qPCR and Western blot were used to detect the alterations of miR-29b, TGF-β/Smad signaling pathway-related proteins and liver fibrosis marker proteins in the acquired cells. Finally, the direct targeting binding of miR-29b to TGF-β1 was identified by dual-luciferase reporter assay system.RESULTS: With the activation of HSC, the expression of miR-29b gradually decreased (P<0.01), while the expression of collagen type I and α-smooth muscle actin gradually increased (P<0.01). At the same time, the expression of Smad2/3/4 was significantly increased, and the expression of Smad7 was significantly decreased (P<0.01). Dual-luciferase reporter assay showed that miR-29b bound directly to "UCUCUCCGU" in the 3'UTR of TGF-β1, indicating that TGF-β1 was a downstream target gene of miR-29b.CONCLUSION: miR-29b may be involved in the inhibition of HSC activation and migration, thereby inhibiting the process of liver fibrosis. The biological function of miR-29b may be through the direct targeting of TGF-β1, thus regulating and inhibiting the TGF-β/Smad signaling pathway.