Effect of Fushengong Prescreption on Regulation-antagonism Effect of ACE-AngⅡ-AT1R Axis and ACE2-Ang(1-7)-MASR Axis of Rats with Chronic Renal Failure / 中国实验方剂学杂志

Objective:To study the effect of Fushengong prescreption on the regulation-antagonism effect of angiotensin converting enzyme-angiotensin Ⅱ-angiotensin Ⅱ 1 receptor （ACE-AngⅡ-AT1R） axis and angiotensin converting enzyme 2-angiotensin （1-7）-Mas receptor［ACE2-Ang（1-7）-MASR］ axis of rats with chronic renal failure（CRF）， and to explore its mechanism of delaying the development of CRF. Method:The 65 male SD rats were randomly divided into normal group （<italic>n</italic>=10） and modeling group （<italic>n</italic>=55）. The normal group was routinely reared， while the modeling group were administered by gavage with 0.25 g·kg^-1d^-1adenine suspension for 28 days. After the model was successfully established， the survival model rats were randomly divided into model group， benazepril group（0.01 g·kg^-1·d^-1）and low，medium and high dose of Fushengong prescreption groups （4，8，16 g·kg^-1·d^-1）. The normal group and model group were administered the same volume of normal saline by gavage， lasted for 28 days. After the experiment， systolic blood pressure （SBP） and diastolic blood pressure （DBP） of caudal artery were measured， and 24-hour urine was collected to determine 24-hour urine protein （24 h U-pro）. The content of serum creatinine（SCr） and blood urea nitrogen （BUN） in the serum were measured， the histological morphology was observed by hematoxylin eosin（HE）staining， and the degree of renal interstitial fibrosis was observed by Masson staining. Enzyme linked immunosorbent assay （ELISA） was used to determine the contents of AngⅡ， Ang （1-7） and Cystatin C （CysC） in serum and renal homogenate. The protein level of ACE， ACE2， AT1R and MASR were detected by Western blot. The expression of ACE and ACE2 protein in renal tissues were detected by immunohistochemistry. Result:Compared with normal group， the expression levels of SCr， BUN and CysC in model group were significantly increased（<italic>P</italic><0.05）， the content of AngⅡ in serum and kidney tissues were significantly increased， the content of Ang （1-7） were significantly decreased（<italic>P</italic><0.05）， the expression of ACE and AT1R protein in renal tissues were significantly increased（<italic>P</italic><0.05）， and the expression of ACE2 and MASR protein were significantly decreased（<italic>P</italic><0.05）. Compared with model group and benazepril group， after the intervention with Fushengong prescreption， the serum SCr，BUN and CysC decreased（<italic>P</italic><0.05），the content of AngⅡ in serum and kidney tissues decreased significantly，Ang（1-7） increased significantly（<italic>P</italic><0.05）， the expression of ACE and AT1R protein in renal tissues decreased significantly（<italic>P</italic><0.05）， ACE2 and MASR protein increased significantly（<italic>P</italic><0.05）. The high-dose Fushengong prescreption has the best effect. The high， medium and low-dose effects of Fushengong prescreption were dose-dependent. Conclusion:Fushengong prescreption improved renal function and pathological change of kidney in adenine-induced rats with chronic renal failure. The mechanism may be related to the inhibition of ACE-AngⅡ-AT1R axis and promotion of ACE2-Ang（1-7）-MASR axis ，which leads to the delaying of the progression of chronic renal failure.

Effect of Mas receptor agonist activation on pancreatic α cell function of diabetic db/db mice by RAS non-classical pathway / 医学研究生学报

Objective The imbalance of glucagon secretion plays an important role in the development of diabetes. The newly discovered ACE2/Ang(1-7)/Mas pathway is an vital branch of the RAS system and has regulatory effects on islet function, but its effect on glucagon secretion is still unknown. The article aimed to investigate the effect and possible mechanisms of AVE0991, a Mas receptor agonist on glucagon secretion of diabetic db/db mice. Methods A tolal of 30 db/db mice were randomized to AVE group and model group (n=15), and their age-matched nondiabetic db/m mice were selected as the normal group (n=15). The mice in AVE group were treated with AVE0991 20mg/kg/d and those in model group were treated with placebo via gavage for 6 weeks. The metabolic indicators were measured every week. After 6 weeks of treatment, intraperitoneal glucose tolerance test (IPGTT) and islet perifusion were performed to evaluate glucagon release kinetics in vivo and vitro. Double-label immunofluoresence assay was adoppted to assess the content of α and β cells. Moreover, qRT-PCR and western blot were employed to detect the GCK expression in islets. Results The fasting blood sugar[(19.1±0.8)mmol/L] and glucose tolerance [(14.1±0.5) mU/L]of AVE group were significantly lower than those of the model group[(25.3±3.0)mmol/L,(17.3±1.8)mU/L](P＜0.05) and still higher than those of normal group[(6.3±0.5)mmol/L,(5.7±0.3)mU/L](P＜0.05). At the 30, 60, and 120 min after IPGTT, the blood glucose level and glucagon level in AVE group were lower than model group, but still higher than the normal group (P<0.05). During low glucose perfusion, the glucagon secretion level of the islets of normal group [(20.6±0.5 pmol/L)] was lower than that of model group [(29.1±0.7) pmol/L)] and AVE group [(27.6±0.8) Pmol/L], and the difference was statistically significant (P<0.05). During high glucose perfusion, there was a statistically significant difference in glucagon level between AVE group and model group at 30 and 60 min(P<0.05). Semi-quantitative analysis showed that the islet α-cell content of model group [(3.3±0.7) mg] was significantly higher than that of normal group [(1.2±0.3) mg] (P<0.05), and the β cell content [(2.4±0.6) mg] was significantly lower than that of normal group [(4.8±0.3) mg] (P<0.05); While compared with the model group, the islet α-cell content [(1.8±0.4) mg] decreased significantly (P<0.05) and the β-cell content [(4.2±0.5) mg] increased significantly in AVE group (P<0.05). The expression levels of GCK mRNA and protein in model group were lower than those in normal group (P<0.05). Conclusion Activation of Mas receptors can improve glucose metabolism by reducing the secretion of glucagon after glucose load in db/db diabetic mice. The mechanism may be related to the decrease of islet α cell content and the increase of islet GCK expression.

Angiotensin 1-7 attenuates angiotensin Ⅱ-induced human glomerular en-dothelial cell injury via Mas receptor / 中国病理生理杂志

AIM:To investigate the effect of angiotensin 1-7(Ang1-7)on the human glomerular endothelial cells(HGECs)injury induced by angiotensin Ⅱ(Ang Ⅱ)and its possible mechanism.METHODS: Cultured HGECs were divided into 6 groups randomly: control group, Ang Ⅱ group, Ang1-7 group, Ang Ⅱ +Ang1-7 group, Ang Ⅱ +Ang1-7+A779(an inhibitor of Mas receptor)group and A779 group.The apoptotic rate and reactive oxygen species (ROS)of HGECs were analyzed by flow cytometry and photographed by fluorescence microscopy.The levels of lactate de-hydrogenase(LDH),nitric oxide(NO),endothelin-1(ET-1),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α), transforming growth factor-β(TGF-β),monocyte chemoattractant protein-1(MCP-1)and intercellular adhesion molecule-1(ICAM-1)in the supernatant of cell cultures were measured.RESULTS:Compared with the control group,the apoptot-ic rate and the average fluorescence intensity of ROS were increased in the Ang Ⅱ group,IL-6,TNF-α,TGF-β,ICAM-1 and MCP-1 in cell supernatants were also increased in the Ang Ⅱ group(P<0.05).Compared with the Ang Ⅱ group,the apoptotic rate,ROS level, and the above inflammatory factors were decreased in Ang Ⅱ +Ang1-7 group(P<0.05). Compared with the Ang Ⅱ +Ang1-7 group,adding A779 increased the cell apoptotic rate,ROS production and the releases of the above inflammatory factors in cell supernatants(P<0.05).Compared with the Ang Ⅱ group,adding Ang1-7 inhibi-ted the LDH leakage, ET-1 secretion and promoted the release of NO in a dose-dependent manner(P<0.05).CON-CLUSION:Ang1-7 attenuates the HGECs injury induced by Ang Ⅱ by inhibiting the Mas receptor.

Changes of lung AT1R and Mas receptor protein expression in lung tissue of mice with lung injury after limb ischemia-reperfusion / 中国病理生理杂志

AIM: To explore the role of imbalance of local renin-angiotensin system (RAS) in lung injury by observing the changes of angiotensin Ⅱ type 1 receptor (AT1R) and Mas receptor protein expression in the lung and the degree of lung injury subject to limb ischemia-reperfusion (LIR) in the mice.METHODS: Male ICR mice (n=42, 8 weeks old) were randomly assigned into 7 groups (6 in each group), including control group and 6 model groups with LIR of 0.5 h, 1 h, 2 h, 4 h, 6 h and 12 h reperfusion.Tourniquets were used to block the blood flow of the hind limbs of the ICR mice and were released after 2 h ischemia to initiate reperfusion.The mice were sacrificed by eyeball blood withdrawal at different time points after reperfusion.The organ coefficient and wet/dry weight ratio (W/D) of the lung tissue were calculated.Bronchoalveolar lavage fluid (BALF) was taken for cell counting and protein concentration measurement.The histopathological changes of the lung tissues was observed, and the pathological score was calculated.The protein expression of AT1R and Mas receptor in the lung tissues was determined by Western blot.RESULTS: The organ coefficient, W/D of lung tissue, and cell number and protein concentration in BALF of model groups were significantly higher than those in control group after LIR.The pathological changes were found in the lung tissue of model mice, including alveolar capillary dilation and congestion, edema, inflammatory cell infiltration in peripheral vascular, alveolar and bronchial walls, alveolar septal thickening and inflammatory cell infiltration.The lung injury score was elevated gradually along with the extension of reperfusion time.The protein expression of AT1R began to increase at reperfusion time points of 0.5 h and 1 h.With the extension of reperfusion time, the protein expression of AT1R decreased gradually.Conversely, the protein expression of Mas receptor increased gradually with prolonged reperfusion.CONCLUSION: LIR induces acute lung injury gradually.The imbalance of AT1R and Mas receptor expression may be involved in the damage process.

Angiotensin-(1-7)/Mas receptor axis protects cardiomyocytes against high glucose-induced injury by modulating nuclear factor-κB pathway / 中国病理生理杂志

AIM:Tostudywhe ther theangiotens in-(1-7)[Ang-(1-7)]/Mas receptor axis protects cardio-myocytes against high glucose (HG)-induced injury by inhibiting nuclear factor-κB (NF-κB) pathway.METHODS:The cell viability was measured by CCK-8 assay.The intracellular levels of reactive oxygen species ( ROS) were detected by DCFH-DA staining .The number of apoptotic cells was tested by Hoechst 33258 nuclear staining .Mitochondrial membrane potential ( MMP) was examined by JC-1 staining.The levels of NF-κB p65 subunit and cleaved caspase-3 protein were de-termined by Western blotting.RESULTS: Treatment of H9c2 cardiac cells with 35 mmol/L glucose (HG) for 30, 60, 90, 120 and 150 min significantly enhanced the levels of phosphorated ( p) NF-κB p65, peaking at 60 min.Co-treatment of the cells with 1 μmol/L Ang-(1-7) and HG for 60 min attenuated the up-regulation of p-NF-κB p65 induced by HG. Co-treatment of the cells with Ang-(1-7) at concentrations of 0.1~30μmol/L and HG for 24 h inhibited HG-induced cy-totoxicity, evidenced by an increase in cell viability .On the other hand, 1 μmol/L Ang-(1-7) ameliorated HG-induced apoptosis, oxidative stress and mitochondrial damage , indicated by decreases in the number of apoptotic cells , cleaved caspase-3 level, ROS generation and MMP loss .However, the above cardioprotective effects of Ang-(1-7) were markedly blocked by A-779, an antagonist of Ang-(1-7) receptor (Mas receptor).Similarly, co-treatment of H9c2 cardiac cells with 100 μmol/L PDTC ( an inhibitor of NF-κB) and HG for 24 h also obviously reduced the above injuries induced by HG.CONCLUSION:Ang-(1-7)/Mas receptor axis prevents the cardiomyocytes from the HG-induced injury by inhibiting NF-κB pathway .

Angiotensina-(1-7) durante o processo inicial na cascata da ovulação em bovinos / Angiotensin-(1-7) during the initial process of ovulatory cascade in cattle

O presente estudo teve como objetivo avaliar o efeito da Ang-(1-7) e de seu receptor (MAS) na regulação da ovulação. No experimento I, utilizando um modelo in vitro de cultivo de células foliculares, foi avaliado o efeito do tratamento com Ang-(1-7) ou do bloqueio do receptor MAS através do inibidor d-Ala7-Ang-(1-7) (A-779) na expressão de RNAm para epirregulina (Ereg; um marcador inicial do processo de ovulação) em células da granulosa. No experimento II, foi utilizado um modelo in vivo de injeção intrafolicular no qual vinte vacas tiveram o ciclo estral sincronizado e, quando os folículos atingiram um diâmetro mínimo de 12mm, foi realizada a injeção intrafolicular de A-779 ou solução salina 0,9%. No momento da injeção intrafolicular, foi realizada uma aplicação IM de análogo de GnRH. A suplementação com Ang-(1-7) ou o bloqueio de seu receptor MAS em sistema de cultivo de células da granulosa não alteraram o padrão de expressão de RNAm para Ereg. A aplicação intrafolicular de A-779 (10-5M) não bloqueou a ovulação quando realizada antes do início do pico esperado de LH (100% das vacas ovularam nos grupos A-779 e controle), sugerindo que a Ang-(1-7) não possui papel relevante no início da cascata ovulatória em bovinos.

This study aimed to evaluate the effect of Ang-(1-7) and its receptor (MAS) in the regulation of the ovulatory cascade. In the experiment I, the effect of Ang-(1-7) or d-Ala7-Ang-(1-7) (A-779; Ang-(1-7) antagonist) on the epirregulin (Ereg; initial marker of ovulation process) mRNA expression in granulosa cells was assessed using an in vitro model of follicular cell culture. In experiment II, it was used an in vivo intrafollicular injection model, in which twenty cows had their follicular waves synchronized and the ovarian follicular size was daily monitored by ultrasound. Follicles that reached a minimum diameter of 12mm were injected with A-779 or saline 0.9%. At the time of the intrafollicular injection, cows were challenged with an intramuscular application of GnRH analogue. Ang-(1-7) or the blockade of its receptor MAS had no effect in Ereg mRNA expression in granulosa cells cultured in vitro. Likewise, the intrafollicular injection of MAS receptor inhibitor (10-5M of A-779) did not block ovulation before the expected time of LH peak (100% of the cows ovulated after GnRH challenge in the treatment and control groups), suggesting that Ang-(1-7) has no role in the early ovulatory cascade in cattle.