Modified Liuwei Dihuangtang Combined with Losartan Potassium Regulates ACE1/AngⅡ/AT1R Axis and Intestinal Flora in Rat Model of Diabetic Kidney Disease / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of modified Liuwei Dihuangtang in preventing and treating renal injury in diabetic kidney disease （DKD） via the angiotensin-converting enzyme 1 （ACE1）/angiotensin Ⅱ （AngⅡ）/angiotensin Ⅱ type 1 receptor （AT1R） axis. MethodFifty male SD rats were randomized into a normal group （n=8） and a modeling group （n=42）. The rats in the modeling group were fed with a high-sugar and high-fat diet for 6 weeks and intraperitoneally injected with 35 mg·kg-1 streptozotocin （STZ） to establish the model of DKD. After successful modeling， the rats were randomized into model， traditional Chinese medicine （modified Liuwei Dihuangtang granules 21 g·kg-1）， western medicine （losartan potassium， 33 mg·kg-1）， and integrated Chinese and western medicine （losartan potassium 33 mg·kg-1 combined with modified Liuwei Dihuangtang granules 21 g·kg-1） groups. The levels of fasting blood glucose （FBG）， urinary protein （Up）， blood urea nitrogen （Bun）， and serum creatinine （SCr） were measured in each group after 8 consecutive weeks of drug intervention. Enzyme-linked immunosorbent assay was employed to determine the serum levels of ACE1， AngⅡ， and AT1R. Western blot was employed to measure the protein levels of ACE1， AngⅡ， and AT1R in the renal tissue. The pathological and morphological changes of the renal tissue were observed after hematoxylin-eosin （HE） staining， Masson staining， and periodic acid Schiff 's （PAS） staining. The fecal samples of rats in each group were collected for 16S rDNA high-throughput sequencing. ResultCompared with the normal group， the model group showed elevated levels of Up， FBG， Bun， SCr， ACE1， AngⅡ， and AT1R （P<0.01）， serious lesions in the renal tissue， up-regulated protein levels of ACE1， AngⅡ， and AT1R （P<0.01）， increased Firmicutes/Bacteroidetes （F/B） ratio， decreased relative abundance of Lactobacillus， and increased relative abundance of Moralella and Bifidobacteria. Compared with the model group， drug intervention lowered the levels of Bun， SCr， ACE1， AngⅡ， and AT1R （P<0.01） and alleviated the pathological changes in the renal tissue. Chinese medicine and integrated Chinese and western medicine lowered the levels of Up and FBG （P<0.01）， and western medicine and integrated Chinese and western medicine down-regulated the protein levels of ACE1， AngⅡ， and AT1R. In addition， Chinese medicine down-regulated the protein levels of AngⅡ （P<0.01） as well as ACE1 and AT1R （P<0.05）. Chinese medicine and integrated Chinese and western medicine decreased the F/B ratio， and western medicine and Chinese medicine increased the relative abundance of Blautia. Chinese medicine and integrated Chinese and western medicine increased the relative abundance of Lactobacillus， Ruminococcus undetermined genera， and Bifidobacteria， decreased the relative abundance of Moralella， and increased the Chao 1 and Ace indexes （P<0.05）. Compared with the western medicine group， the integrated Chinese and western medicine group showed lowered levels of Up （P<0.01）， Bun （P<0.05）， and ACE1 and AT1R （P<0.01）， down-regulated protein levels of ACE1， AngⅡ， and AT1R （P<0.05）， alleviated pathological changes in the renal tissue， increased relative abundance of Bifidobacteria， and increased Chao 1 and Ace indexes （P<0.05）. ConclusionModified Liuwei Dihuangtang combined with losartan potassium can mitigate renal fibrosis by regulating the ACE1/AngⅡ/AT1R axis， increasing the relative abundance of Lactobacillus and Bifidobacterium， reducing the relative abundance of Moralella， improving the richness and evenness of intestinal flora， and alleviating pathological damage in the renal tissue.

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.

Effects of electroacupuncture on postoperative cognitive dysfunction and AngⅡ/AT1R in the hippocampus in aging rats induced by D-galactose / 中国针灸

<p><b>OBJECTIVE</b>To observe the effects of electroacupuncture (EA) on postoperative cognitive dysfunction (POCD) and AngⅡ/AT1R in the hippocampus in D-galactose-induced aging rats which received hepalobectomy, and to explore the possible mechanism of EA on POCD.</p><p><b>METHODS</b>Eighty male Sprague-Dawley rats were randomly divided into a young control group (10 rats), a D-Galactose-induced aged (Da) group (10 rats), a Da+hepatolobectomy group (30 rats) and an EA group (30 rats). The rats in the Da+hepatolobectomy group and EA group were further randomly divided into a 1 d subgroup, 3 d subgroup and a 7 d subgroup, 10 rats in each subgroup. The rats in the EA group were treated with EA at "Baihui" (GV 20) and "Dazhui" (GV 14) with continuous wave (15 Hz in frequency and 1 mA in intensity), and rats in each subgroup were treated for 1 d, 3 d and 7 d, respectively. The rats in the remaining groups were treated with immobilization, once a day. The Y-maze was used to observe the behavior change of rats, and ELISA was applied to measure the level of hippocampal AngⅡ, and RT-PCR and immunohistochemistry method were performed to detect AT1R mRNA expressions and AT1R positive expression in the hippocampus.</p><p><b>RESULTS</b>The number of rat initiative avoidance in the Da group was significantly less than that in the young control group (<0.05), and the mRNA expression and positive percentage of AT1R in the hippocampus in the Da group were significantly higher than those in the young control group (both<0.01). Compared with the Da group, the number of rat initiative avoidance in each subgroup of Da+hepatolobectomy group and EA group were significantly reduced (all<0.01), and the expression of AngⅡ, AT1R mRNA and AT1R positive cells percentage in the hippocampus were significantly increased (<0.05,<0.01). The number of rat initiative avoidance in each subgroup of EA group was higher than that in the subgroup of Da+hepatolobectomy group (<0.05,<0.01); and the expression of AngⅡ, AT1R mRNA, and AT1R positive percentage in the EA group were significantly less than that in the Da+hepatolobectomy group (<0.05,<0.01).</p><p><b>CONCLUSIONS</b>EA at "Baihui" (GV 20) and "Dazhui" (GV 14) could improve POCD in D-galactose-induced aging rats which received hepalobectomy, and it is likely to be related with the inhibition of AngⅡ, AT1R positive expression and AT1R mRNA in the hippocampus.</p>

Cambios en la función ventricular sistólica y diastólica en un modelo de sobreexpresión cardíaca del receptor AT-1 de angiotensina / Changes in Systolic and Diastolic Function in a Mouse Model Overexpressing Cardiac Angiotensin II AT-1 Receptor

La angiotensina II (Ang II) está involucrada en diferentes procesos fisiopatológicos, particularmente actuando sobre los receptores AT-1 de Ang II (AT1R). El objetivo de este trabajo fue evaluar la función ventricular sistólica y diastólica in vivo e in vitro en ratones con sobreexpresión cardíaca específica del receptor AT-1 de Ang II (AT1R). Un segundo objetivo fue determinar si el bloqueo agudo y crónico del AT1R revierte los cambios en la función ventricular. Se estudiaron ratones divididos en cuatro grupos experimentales. El primer grupo incluyó animales no transgénicos (NTG, n = 10), el segundo grupo ratones transgénicos (TG, n = 7) que sobreexpresan el AT1R solo a nivel cardíaco y el tercero y el cuarto grupos, animales TG tratados con losartán (L) durante 7 días (TG L7, n = 9) y 30 días (TG L30, n = 7), respectivamente. Los ratones TG presentaron hipertrofia ventricular izquierda (HVI). El tratamiento con losartán por 7 días no revirtió la HVI, lo que sí sucede cuando se extiende por 30 días. Los animales TG presentan una disminución significativa de la fracción de acortamiento, desde un valor de 47,1% ± 2,3% hasta 32,3% ± 1,3% (p < 0,05), y de la +dP/dt máx, que se reduce desde un valor de 7.073 ± 674 mm Hg hasta 3.897,5 ± 209,7 mm Hg/seg (p < 0,05). El tratamiento con losartán por 7 y 30 días revierte esta disfunción sistólica. El tiempo de relajación isovolúmica y el t1/2 fueron de 24,1 ± 1,3 mseg y de 5,1 ± 0,5 mseg, respectivamente, en los NTG. Estos índices se incrementaron a 33,1 ± 2,2 mseg y a 8,4 ± 0,4 mseg, respectivamente, en los ratones TG (p < 0,05). Esta alteración de la función diastólica fue revertida por completo con el tratamiento con losartán por 7 y 30 días. El análisis de la función ventricular in vitro con control de variables corroboró los hallazgos realizados in vivo. La sobreexpresión cardíaca de los AT1R induce una disfunción ventricular sistólica y diastólica que es revertida completamente por el bloqueo del AT1R. Este efecto beneficioso es independiente de modificaciones en la masa ventricular izquierda.

Angiotensin II (Ang II) is involved in various patho-physiological processes through the activation of Ang II AT-1 receptors. The purpose of this study was to assess in vivo and in vitro systolic and diastolic ventricular function in mice overexpressing the cardiac-specific AT-1 receptor (AT1R). A second objective was to deter­mine whether acute and chronic ATIR blockade revert the changes in ventricular function. Mice were divided into four experimental groups. The first group included non-transgenic animals (NTG, n=10), the second group consisted of transgenic mice (TG, n=7) with cardiac-specific AT1R overexpression and the third and fourth groups were TG animals treated with losartan (L) for 7 (TG L7, n=9) and 30 days (TG L30, n=7), respectively. Transgenic animals exhibited left ventricular hypertrophy (LVH) which was only regressed with losartan treatment for 30 days. They also presented a significant decrease in shortening fraction from 47.1 ± 2.3% to 32.3 ± 1.3% (p max from 7073 ± 674 to 3897.5 ± 209.7 mm Hg/sec (p Isovolumic relaxation time and t1/2 were 24.1 ± 1.3 and 5.1 ± 0.5 ms, respectively, in the NTG group. These indexes increased to 33.1 ± 2.2 and 8.4 ± 0.4 ms, respectively, in TG mice (p In conclusion, cardiac-specific AT1R overexpression induces systolic and diastolic ventricular dysfunction which is completely reversed by AT1R blockade. This beneficial effect is independent of left ventricular mass changes.

Exercício de força ativa a via AKT/mTor pelo receptor de angiotensina II tipo I no músculo cardíaco de ratos / Activation of AKT-mTor signaling pathways by angiotensin II receptor type 1 after a session of strength exercise in cardiac muscle of rats

O receptor de angiotensina II tipo I (AT1) tem uma importante participação no desenvolvimento da hipertrofia cardíaca. Em um trabalho publicado anteriormente, por nosso grupo, demonstramos que o bloqueio do receptor AT1 durante o treinamento de força inibiu a hipertrofia cardíaca em ratos. Por isso, o objetivo deste trabalho foi estudar a participação do receptor AT1 na ativação de vias de sinalização intracelular relacionadas com o aumento da síntese de proteína em ratos submetidos a uma sessão de exercício de força. Para isso, realizamos um experimento com seis grupos de animais (n = 6; cada): controle (Con), exercitado e sacrificado cinco minutos após o exercício (Exe 5), exercitado e sacrificado 30 minutos após o exercício (Exe 30), controle tratado com losartan (Con Los), tratado com losartan, exercitado e sacrificado cinco minutos após o exercício (Exe 5 Los), tratado com losartan, exercitado e sacrificado 30 minutos após o exercício (Exe 30 Los). Os resultados mostram que no grupo Exe 5 e Exe 30 ocorreu um aumento de 63 por cento (P < 0,05) e 62 por cento (P < 0,05), respectivamente, na fosforilação da proteína AKT comparado com o grupo controle. Enquanto a fosforilação da mTor foi aumentada 65 por cento (P < 0,05) somente no grupo Exe 30 comparado com o grupo controle, sendo estes efeitos bloqueados pelo uso do losartan nos grupos Exe 5 Los e Exe 30 Los. Portanto, esses resultados, juntamente com nossos resultados prévios, demonstram que o receptor AT1 tem participação na ativação da AKT e mTOR após uma sessão de exercício de força.

The angiotensin II type I (AT1) receptor has an important participation in the development of cardiac hypertrophy. Previously, we have shown that AT1 receptor participates in the cardiac hypertrophy induced by resistance training in rats. Here, we studied the involvement of AT1 receptor in the activation of intracellular signaling pathways related to the concentric HC in rats submitted to a session of strength exercise. Male Wistar rats were divided into 6 groups (n= 6 each): control (Con); exercised and killed 5 minutes after exercise (Exe 5); exercised and killed 30 minutes after exercise (Exe 30); control treated with Losartan (Con Los); treated with Losartan, exercised and killed 5 minutes after the exercise (Exe Los 5); treated with Losartan, exercised and killed 30 minutes after training (Exe Los 30). The results show that phosphorylation activity of AKT in group Exe 5 and Exe 30 increased 63 percent (P < 0.05) and 62 percent (P < 0.05), respectively, compared with Con. Whereas the phosphorylation of mTOR was increased 65 percent (P < 0.05), compared to Con, only in the group Exe 30. Furthermore, these effects were blocked by losartan treatment in groups Exe Los 5 and Exe Los 30. These results, together with ours previous data shows that the AT1 receptor has an role in the activation of AKT and mTOR pathway after a session of strength exercise.

Analysis of A1166C Polymorphism in Type I Receptor for Angiotensin II Gene with Pregnancy Induced Hypertension(PIH) in Korean Women / 대한주산의학회잡지

BACKGROUND/AIM: Recent studies have suggested an association between genetic background of renin-angiotensin(RA) system and the pathogenesis of pregnancy induced hypertension(PIH). Even though various single nucleotide polymorphism(SNP) such as M235T, T174M polymorphism in angiotensinogen gene(AGT) and I/D polymorphism in angiotensin I-converting enzyme gene(ACE) have been studied extensively among essential hypertension and PIH in various population, its association was still inconclusive. Previous studies within Korean PIH patients also revealed that M235T or T174M single nucleotide polymorphism in AGT gene or I/D polymorphism in ACE gene were not linked tightly with PIH. However, recent studies on angiotensin II type I receptor(AT1) polymorphism A1166C with PIH in Polish or in Chinese suggested its possible correlations to a pathogenesis of PIH. Thus the aim of the present study was to determine the frequency of genotypes of A1166C mu-tation in women with PIH and to establish the role of this polymorphism on the susceptibility to the PIH development. PATIENTS AND METHODS: We have analysed 121 women with PIH and 98 healthy normotensive gravid women as a control. Genomic DNA was used for PCR. To genotype the A1166C polymorphism in angiotensin II receptor(AT1) gene, primers(sense 5'-CGA CTA CTG CTT AGC ATA-3', antisense 5'-GCA CCA TGT TTT GAG GTT-3') were employed to make 546bp PCR product. There was an initial denaturation at 94degrees C 5 minutes, followed by 30 cycles of one minute at 94degrees C, one minute at 58degrees C, and two minutes at 7degrees C. A 546bp PCR product was further digested with DdeI for 2 hour at 37degrees C and analysed through 2% agarose gel electrophoresis to determine genotype. Allele C1166 yielded 435bp, 111bp fragment and allele A1166 yielded intact 546 bp fragment RESULTS: We found that frequency of genotype A1166/C1166 and A1166/A1166 in PIH were 9.9% (12) and 90.1%(109), while in controls were 17.3%(17) and 82.7%(81). There was no statistical significance between development of PIH and A1166C polymorphism in type I receptor for Angiotensin II. Homozygous mutated genotype(C1166/C1166) was not detected in this study. CONCLUSION: Our results found no possible correlation of A1166C polymorphism in angiotensin II receptor(AT1) with PIH in Korean and found that allele C might behave as a protective factor rather than as a risk factor in the pathogenesis of PIH. We suggest a large-scale study to evaluate relevance to this polymorphism for PIH.