Antihypertensive effects of the combined extract of Sorghum bicolor, Vigna angularis, and Eleusine coracana in spontaneously hypertensive rats.

This study investigated the antihypertensive effects of the combined extract of sorghum, adzuki bean, and finger millet (SAFE) on spontaneously hypertensive rats. The rats were divided into four groups (n = 8): WKY, SHR, SAFE (500 mg/kg SAFE), and CAP (50 mg/kg captopril). SAFE significantly decreased the lean-to-fat mass ratio with no notable changes in body weight, food intake, or food efficiency ratio, and it effectively lowered both systolic and diastolic blood pressures, comparable to CAP. Moreover, it significantly reduced the cardiac mass index and alleviated cardiac fibrosis. SAFE did not induce hepatotoxicity, as indicated by the maintenance of aspartate aminotransferase and alanine aminotransferase levels in the normal range, confirming its safety. Taken together, these findings suggested that SAFE can be used as a dietary supplement for blood pressure regulation and cardiovascular disease prevention.

Chronic Intermittent Hypobaric Hypoxia Reduces Hypothalamic N-Methyl-d-Aspartate Receptor Activity and Sympathetic Outflow in Spontaneously Hypertensive Rats.

Guo, Xinqi, Hongyu Ma, Ziye Cui, Qiyue Zhao, Ying Zhang, Lu Jia, Liping Zhang, Hui Guo, Xiangjian Zhang, Yi Zhang, Yue Guan, and Huijie Ma. Chronic intermittent hypobaric hypoxia reduces hypothalamic N-Methyl-d-Aspartate Receptor activity and sympathetic outflow in spontaneously hypertensive rats. High Alt Med Biol. 25:77-88, 2024. Objective: This study aims to determine the role of hypothalamic renin-angiotensin system (RAS) in the antihypertensive effect of chronic intermittent hypobaric hypoxia (CIHH). Methods: Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs) received 35 days of hypobaric hypoxia simulating an altitude of 4,000 m, 5 h/day. The levels of RAS, blood pressure, and N-methyl-d-aspartate receptor (NMDAR) activities of hypothalamic paraventricular nucleus (PVN) presympathetic neurons from each group of rats were determined. Results: The systolic blood pressure, diastolic blood pressure, and mean arterial blood pressure (MAP) of SHRs significantly decreased from the third week of CIHH treatment. This blood pressure reduction effect could be maintained for at least 2 weeks after stopping the CIHH treatment. CIHH treatment also attenuated the decrease in MAP and renal sympathetic nerve activity induced by hexamethonium administration in SHRs, but not in WKY rats. Furthermore, CIHH reversed the increase in serum angiotensin (Ang)II concentration and the expression of PVN angiotensin-converting enzyme (ACE) and AngII type 1 (AT1) receptors, as well as the decrease in serum Ang1-7 concentration and the expression of PVN ACE2 and Mas receptors in SHRs. In addition, the administration of CIHH resulted in a reduction in the frequency of miniature excitatory postsynaptic currents and amplitude of NMDAR current in PVN presympathetic neurons of SHRs, which means that CIHH decreased the pre- and postsynaptic NMDAR activity of PVN presympathetic neurons in SHRs. However, pretreatment with A779 (a Mas receptor blocker) or AngII abrogated the above effects. Meanwhile, Ang1-7 pretreatment mimicked the CIHH effect on pre- and postsynaptic NMDAR activity of presympathetic neurons in SHRs. Conclusions: Our data indicate that CIHH reduces pre- and postsynaptic NMDAR activity of PVN presympathetic neurons, sympathetic outflow, and blood pressure by decreasing the activity of the ACE/AngII/AT1 axis and increasing the activity of ACE2/Ang1-7/Mas axis in the hypothalamus in hypertension.

The therapeutic effects of fermented milk with lactic acid bacteria from traditional Daqu on hypertensive mice.

Lactic acid bacteria (LAB), a type of microorganism widely used in functional foods, has gained notable research attention in recent years. Certain strains possess the proteolytic ability to release potentially antihypertensive peptides from dairy proteins, which prompted us to explore the LAB strains from an understudied and unique ingredient, Daqu. We screened for 67 strains of LAB strains from traditional Daqu using the calcium dissolution ring method. Sixteen strains exhibiting angiotensin-converting enzyme inhibition (ACE-I) activity exceeding 50% were chosen for 16S rDNA sequencing and safety assessment. It is noteworthy that Enterococcus faecium CP640 and Lacticaseibacillus rhamnosus CP658 exhibited significant ACE-I activity, which was the result of strain fermentation in reconstituted skim milk. These 2 strains did not exhibit hemolytic activity or antibiotic resistance. They also did not produce biogenic amines and showed high survival rates in the gastrointestinal tract. Additionally, Enterococcus faecium CP640 and Lacticaseibacillus rhamnosus CP658 fermented milk exhibited a notable reduction in blood pressure levels in spontaneously hypertensive rats (SHR) compared with negative controls in SHR. Importantly, no adverse effect was observed in normal Wistar-Kyoto rats. Through the analysis of physiological, serum, and urine-related indicators, it was observed that Enterococcus faecium CP640 and Lacticaseibacillus rhamnosus CP658 have the potential to promote weight gain in SHR, alleviate excessive heart rate, improve renal function indicators, and effectively regulate blood sugar and uric acid levels in SHR. These 2 strains showed optimal properties in lowering blood pressure and have the potential to be used in functional dairy products in the future.

DNA demethylation in the hypothalamus promotes transcription of Agtr1a and Slc12a2 and hypertension development.

Increased expression of angiotensin II AT1A receptor (encoded by Agtr1a) and Na+-K+-Cl- cotransporter-1 (NKCC1, encoded by Slc12a2) in the hypothalamic paraventricular nucleus (PVN) contributes to hypertension development. However, little is known about their transcriptional control in the PVN in hypertension. DNA methylation is a critical epigenetic mechanism that regulates gene expression. Here, we determined whether transcriptional activation of Agtr1a and Slc12a2 results from altered DNA methylation in spontaneously hypertensive rats (SHR). Methylated DNA immunoprecipitation and bisulfite sequencing-PCR showed that CpG methylation at Agtr1a and Slc12a2 promoters in the PVN was progressively diminished in SHR compared with normotensive Wistar-Kyoto rats (WKY). Chromatin immunoprecipitation-quantitative PCR revealed that enrichment of DNA methyltransferases (DNMT1 and DNMT3A) and methyl-CpG binding protein 2, a DNA methylation reader protein, at Agtr1a and Slc12a2 promoters in the PVN was profoundly reduced in SHR compared with WKY. By contrast, the abundance of ten-eleven translocation enzymes (TET1-3) at Agtr1a and Slc12a2 promoters in the PVN was much greater in SHR than in WKY. Furthermore, microinjecting of RG108, a selective DNMT inhibitor, into the PVN of WKY increased arterial blood pressure and correspondingly potentiated Agtr1a and Slc12a2 mRNA levels in the PVN. Conversely, microinjection of C35, a specific TET inhibitor, into the PVN of SHR markedly reduced arterial blood pressure, accompanied by a decrease in Agtr1a and Slc12a2 mRNA levels in the PVN. Collectively, our findings suggest that DNA hypomethylation resulting from the DNMT/TET switch at gene promoters in the PVN promotes transcription of Agtr1a and Slc12a2 and hypertension development.

[Effect of electroacupuncture on myocardial fibrosis in spontaneously hypertensive rats based on cholinergic anti-inflammatory pathway].

OBJECTIVE: To observe the effect of electroacupuncture (EA) at "Neiguan" (PC 6) on myocardial fibrosis in spontaneously hypertensive rats (SHR), and explore preliminarily the mediating role of cholinergic anti-inflammatory pathway (CAP) and its downstream nuclear factor κB (NF-κB) signaling pathway. METHODS: Six 12-week-old WKY male rats were employed as the normal group. Eighteen 12-week-old SHR were randomly divided into 3 groups, i.e. a model group, an EA group and a blocking group (EA after blocking α7 nicotinic acetylcholine receptor [α7nAchR]), with 6 rats in each one. In the EA group, EA was delivered at "Neiguan"(PC 6) and the site 0.5 cm from its left side, with disperse-dense wave, 2 Hz/15 Hz in frequency and 1 mA in current intensity. One intervention took 30 min and was given once every 2 days, lasting 8 weeks. In the blocking group, prior to each EA, the α7nAchR specific blocker, α-bungartoxin was injected intravenously in the tails of the rats. After EA intervention, the systolic blood pressure (SBP), the diastolic blood pressure (DBP) and the mean arterial pressure (MAP) were measured with non-invasive blood pressure monitor. Using echocardiogram, the left ventricular (LV) anterior wall end-diastolic thickness (LVAWd) , LV posterior wall end-diastolic thickness (LVPWd) and the LV end-diastolic internal diameter (LVIDd) were measured. The level of hydroxyproline (Hyp) in the myocardial tissue was determined by using alkaline hydrolysis, and that of acetylcholine (Ach) was detected by ELISA. With the real-time PCR adopted, the mRNA expression of NF-κB p65, tumor necrosis factor α (TNF-α), interleukin (IL)-1ß and IL-6 were determined. RESULTS: Compared with the normal group, SBP, DBP, MAP, LVAWd and LVPWd were increased (P<0.01), and LVIDd was decreased (P<0.01) in the rats of the model group. SBP, DBP, MAP and LVAWd were dropped (P<0.01, P<0.05), and LVIDd rose (P<0.01) in the EA group when compared with those in the model group. The differences in the above indexes were not statistically significant between the blocking group and the model group (P>0.05). Compared with the normal group, Hyp level and the mRNA expression of NF-κB p65, TNF-α, IL-1ß and IL-6 in the myocardial tissue increased (P<0.01, P<0.05) and Ach level decreased (P<0.01) in the model group. Hyp level, the mRNA expression of NF-κB p65, TNF-α, IL-1ß and IL-6 in the myocardial tissue were reduced (P<0.05, P<0.01) and Ach level rose (P<0.01) in the EA group when compared with those in the model group. These indexes were not different statistically between the blocking group and the model group (P>0.05). CONCLUSION: CAP may be involved in ameliorating the pathological damage of myocardial fibrosis during EA at "Neiguan"(PC 6). The underlying effect mechanism is associated with up-regulating the neurotransmitter, Ach and down-regulating mRNA expression of NF-κB p65 and pro-inflammatory factors such as TNF-α, IL-1ß and IL-6 in myocardial tissue.

Blood pressure lowering effects of Vernonia amygdalina Del. Aqueous fraction on hypertensive Wistar Kyoto rats.

OBJECTIVES: Vernonia amygdalina (VA) is a plant that consumed as vegetable by Indonesians contained numerous secondary metabolites. VA's pharmacological action, including its antioxidant properties, anticancer, antidiabetic, and hepatoprotective. The purpose of this research is to reveal the activity of Vernonia amygdalina. leafs aqueous fraction (VALAF) as a blood pressure-lowering agent in hypertensive model. METHODS: Combination of prednisone and NaCl were used as hypertensive inducer. The animals were split into five different groups, normal control group treated with distilled water, treatment VALAF groups with dose of 10; 20 and 40 mg/kg BW respectively, while the last group was treated with captopril at dose of 2.25 mg/kg BW. All animals were given an oral treatment for 15 days. On days 5, 10, and 15, systolic and diastolic blood pressure, heart rate (HR), mean arterial pressure (MAP), and blood flow (BF) were all measured. On days 0 and 15, NO level were assessed. All data were analyzed using two-way ANOVA, and Duncan Multiple Range Test. RESULTS: The V. amygdalina leaf aqueous fraction has blood pressure lowering activity. The blood pressure parameter of the rats treated with VALAF were lower as compared to the normal control group (p<0.05). NO levels in the VALAF group were not significantly higher than in the normal control group (p>0.05). The VALAF 20 give the greatest percentage of decrease in blood pressure, heart rate and blood volume on the 15th day of examination. CONCLUSIONS: These study indicated that V. amygdalina leaf aqueous fraction has the potential to be an alternative therapy for managing blood pressure in hypertensive animal models.

Qingda granule ameliorates vascular remodeling and phenotypic transformation of adventitial fibroblasts via suppressing the TGF-ß1/Smad2/3 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Qingda granule (QDG) exhibits significant therapeutic effects on high blood pressure, vascular dysfunction, and elevated proliferation of vascular smooth muscle cells by inhibiting multiple pathways. However, the effects and underlying mechanisms of QDG treatment on hypertensive vascular remodeling are unclear. AIM OF THE STUDY: The aim of this study was to determine the role of QDG treatment in hypertensive vascular remodeling in vivo and in vitro. MATERIALS AND METHODS: An ACQUITY UPLC I-Class system coupled with a Xevo XS quadrupole time of flight mass spectrometer was used to characterize the chemical components of QDG. Twenty-five spontaneously hypertensive rats (SHR) were randomly divided into five groups, including SHR (equal volume of double distilled water, ddH2O), SHR + QDG-L (0.45 g/kg/day), SHR + QDG-M (0.9 g/kg/day), SHR + QDG-H (1.8 g/kg/day), and SHR + Valsartan (7.2 mg/kg/day) groups. QDG, Valsartan, and ddH2O were administered intragastrically once a day for 10 weeks. For the control group, ddH2O was intragastrically administered to five Wistar Kyoto rats (WKY group). Vascular function, pathological changes, and collagen deposition in the abdominal aorta were evaluated using animal ultrasound, hematoxylin and eosin and Masson staining, and immunohistochemistry. Isobaric tags for relative and absolute quantification (iTRAQ) was performed to identify differentially expressed proteins (DEPs) in the abdominal aorta, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. Cell Counting Kit-8 assays, phalloidin staining, transwell assays, and western-blotting were performed to explore the underlying mechanisms in primary isolated adventitial fibroblasts (AFs) stimulated with transforming growth factor-ß 1 (TGF-ß1) with or without QDG treatment. RESULTS: Twelve compounds were identified from the total ion chromatogram fingerprint of QDG. In the SHR group, QDG treatment significantly attenuated the increased pulse wave velocity, aortic wall thickening, and abdominal aorta pathological changes and decreased Collagen I, Collagen III, and Fibronectin expression. The iTRAQ analysis identified 306 DEPs between SHR and WKY and 147 DEPs between QDG and SHR. GO and KEGG pathway analyses of the DEPs identified multiple pathways and functional processes involving vascular remodeling, including the TGF-ß receptor signaling pathway. QDG treatment significantly attenuated the increased cell migration, actin cytoskeleton remodeling, and Collagen I, Collagen III, and Fibronectin expression in AFs stimulated with TGF-ß1. QDG treatment significantly decreased TGF-ß1 protein expression in abdominal aortic tissues in the SHR group and p-Smad2 and p-Smad3 protein expression in TGF-ß1-stimulated AFs. CONCLUSIONS: QDG treatment attenuated hypertension-induced vascular remodeling of the abdominal aorta and phenotypic transformation of adventitial fibroblasts, at least partly by suppressing TGF-ß1/Smad2/3 signaling.

Effects and mechanism of Compound Qidan Formula on rats with HFpEF induced by hypertension and diabetes mellitus based on Ang â ¡/TGF-ß1/Smads signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Compound Qidan Formula is composed of traditional Chinese herbs and has a good curative effect in the clinical application of cardiovascular diseases such as heart failure. However, its potential molecular mechanisms of action remain highly unknown. AIM OF THE STUDY: To observe the effect of Compound Qidan Formula on cardiac function in rats with HFpEF induced by hypertension and diabetes mellitus, and to explore its mechanism from Ang Ⅱ/TGF-ß1/Smads signaling pathway. MATERIALS AND METHODS: A total of 50 SPF-grade spontaneously hypertensive rats (SHR) aged 14 weeks, fed with a high-fat and high-sucrose diet for 16 weeks, and after 2 weeks of a high-fat and high-sucrose diet, 1% streptozotocin (25 mg/kg body weight)was injected intraperitoneally to establish a rat model of HFpEF induced by hypertension and diabetes mellitus. After 8 weeks of intragastric administration, the changes in cardiac morphology and function were evaluated by echocardiography after anesthesia; the heart tissue was taken and embedded in paraffin for Masson staining, and the pathomorphological changes of left atrial tissue were observed under the optical microscope; the mRNA transcription levels of Ang Ⅱ, AT1R, TGF-ß1, Smad2, Smad3, MMP-9 and TIMP-1in left atrial tissue of rats were detected by RT-PCR; and the protein expressions were detected by Western blot. RESULTS: Compared with the SHR-DM group, the QD-Low and QD-High groups significantly decreased the left atrial (LA) anteroposterior diameter and interventricular septal thickness (IVST) and improved the peak velocity of mitral valve blood flow in early diastolic period (E), maximum mitral valve blood flow in systolic period (A), mitral ring myocardial movement velocity in early diastolic period (e') and E/e' ratio; the QD-High group significantly improved the E/A ratio, left atrial ejection fraction (LAEF) and left ventricular ejection fraction(LVEF). Masson staining showed that compared with the WKY group, the SHR-DM group had obvious myocardial histomorphological lesions. Compared with the SHR-DM group, the Compound Qidan Formula groups significantly improved cardiomyocyte hypertrophy and disordered arrangement and inhibited myocardial fibrosis; the mRNA expression levels of Ang Ⅱ, AT1R, TGF-ß1, Smad2, Smad3, and MMP-9 in myocardial tissue of Compound Qidan Formula groups were significantly decreased, and the mRNA expression level of TIMP-1 was significantly increased. The protein expression levels of Ang Ⅱ, TGF-ß1, P-Smad2/3, and MMP-9 were significantly decreased. CONCLUSION: Compound Qidan Formula, composed of traditional Chinese herbs, can significantly improve cardiac function, improve atrial and ventricular remodeling, and prevent myocardial fibrosis and hypertrophy in rats with HFpEF induced by hypertension and diabetes mellitus. The mechanism may be related to regulating the Ang Ⅱ/TGF-ß1/Smad2/3 signaling pathway.

[Effect of electroacupuncture on transforming growth factor-ß1 and renal tubular epithelial mesenchymal transformation in renal tissue of spontaneously hypertensive rats].

OBJECTIVE: To observe the effects of electroacupuncture (EA) on renal fibrosis, the expression of transforming growth factor-ß1 (TGF-ß1) and epithelial mesenchymal transition (EMT) marker proteins in renal tissue of spontaneously hypertensive rats (SHR), so as to explore the underlying mechanism on EA alleviating hypertensive renal impairment. METHODS: Twenty-four male SHR were randomly divided into model group, losartan group and EA group, with 8 rats in each group, and eight male Wistar-Kyoto rats were taken as the normal group. Rats in the losartan group received gavage of losartan potassium solution (3 mg/mL, 30 mg·kg-1·d-1),once every other day for 12 weeks. Rats in the EA group received EA stimulation at bilateral "Shenshu" (BL23) and "Geshu" (BL17) (2 Hz/15 Hz, 1.0 mA), 15 min each time, once every other day for 12 weeks. The systolic blood pressure of caudal artery was measured before, and 4, 8 and 12 weeks after the intervention. The 24-hour urinary protein was measured before, and 6 and 12 weeks after the intervention. Histopathological changes of the left renal tissue were observed under light mircoscope after H.E. stain. Extracellular matrix (ECM) in renal tissues was observed after periodate Schiff staining. Basement membrane and collagen fibers were observed after Masson staining with collagen volume fraction (CVF) evaluated. The expression of TGF-ß1 mRNA in the right renal was detected by real-time fluorescence quantitative PCR. The expression of TGF-ß1 and EMT marker E-cadherin, α-SMA and fibronectin (FN) proteins in the left renal tissue was measured by immunohistochemistry. RESULTS: In the model group, irregular arrangement of nephrocytes, renal tubule atrophy, lumen stenosis, ECM hyperplasia and deposition, scar and sclerosis were observed, which were relatively milder in the EA and losartan groups. Compared with the normal group, tubulointerstitium CVF, systolic blood pressure of caudal artery before, and at 4, 8 and 12 weeks after the intervention, 24-hour urinary protein before, and at 6 and 12 weeks after the intervention, the expression of TGF-ß1 mRNA, area of TGF-ß1, α-SMA and FN positive staining in renal tissues were significantly increased (P<0.01), while the area of E-cadherin positive staining was significantly decreased (P<0.01) in the model group. Compared with the model group, tubulointerstitium CVF, systolic blood pressure of caudal artery at 4, 8 and 12 weeks after the intervention, 24-hour urinary protein at 6 and 12 weeks after the intervention, the expression of TGF-ß1 mRNA, area of TGF-ß1, α-SMA and FN positive staining in renal tissues were significantly decreased (P<0.01，P<0.05), while area of E-cadherin positive staining was significantly increased (P<0.01) in the losartan and EA groups. Compared with the losartan group, the area of E-cadherin was conside-rately increased (P<0.01), while the area of α-SMA protein decreased (P<0.01) in the EA group. CONCLUSION: EA could effectively alleviate hypertension and renal interstitial fibrosis in SHR, the mechanism of which may be related to its function in reducing the expression of TGF-ß1 and inhibiting EMT in renal tissue.

Dietary intake of table olives exerts antihypertensive effects in association with changes in gut microbiota in spontaneously hypertensive rats.

Arbequina table olive (AO) consumption lowers blood pressure (BP) in spontaneously hypertensive rats (SHR). This study evaluates whether dietary supplementation with AO induced changes in the gut microbiota that are consistent with the purported antihypertensive effects. Wistar-Kyoto rats (WKY-c) and SHR-c received water, while SHR-o were supplemented by gavage with AO (3.85 g kg-1) for 7 weeks. Faecal microbiota was analysed by 16S rRNA gene sequencing. SHR-c showed increased Firmicutes and decreased Bacteroidetes compared to WKY-c. AO supplementation in SHR-o decreased BP by approximately 19 mmHg, and reduced plasmatic concentrations of malondialdehyde and angiotensin II. Moreover, reshaped faecal microbiota associated with antihypertensive activity by lowering Peptoniphilus and increasing Akkermansia, Sutterella, Allobaculum, Ruminococcus, and Oscillospira. Also promoted the growth of probiotic strains of Lactobacillus and Bifidobacterium and modified the relationship of Lactobacillus with other microorganisms, from competitive to symbiotic. In SHR, AO promotes a microbiota profile compatible with the antihypertensive effects of this food.

Corticotropin-releasing factor potentiates glutamatergic input and excitability of presympathetic neurons in the hypothalamus in spontaneously hypertensive rats.

Hyperactivity of presympathetic neurons in the hypothalamic paraventricular nucleus (PVN) plays a key role in generating excess sympathetic output in hypertension. However, the mechanisms driving hyperactivity of PVN presympathetic neurons in hypertension are unclear. In this study, we determined the role of corticotropin-releasing factor (CRF) in the PVN in augmented glutamatergic input, neuronal excitability and sympathetic outflow in hypertension. The number of CRF or c-Fos immunoreactive neurons and CRF/c-Fos double-labeled neurons in the PVN was significantly greater in spontaneously hypertensive rats (SHRs) than in normotensive Wistar-Kyoto (WKY) rats. Blocking glutamatergic input reduced the CRF-potentiated excitability of spinally projecting PVN neurons. Furthermore, CRF knockdown via Crispr/Cas9 in the PVN decreased the frequencies of spontaneous firing and miniature excitatory postsynaptic currents (mEPSCs) in spinally projecting PVN neurons in SHRs. In addition, the mRNA and protein levels of CRFR1, but not CRFR2, in the PVN were significantly higher in SHRs than in WKY rats. Blocking CRFR1 with NBI-35965, but not blocking CRFR2 with Antisauvagine-30, reduced the frequencies of spontaneous firing and mEPSCs of spinally projecting PVN neurons in SHRs. Also, microinjection of NBI-35965 into the PVN significantly reduced arterial blood pressure (ABP) and renal sympathetic nerve activity (RSNA) in anesthetized SHRs, but not in WKY rats. However, microinjection of Antisauvagine-30 into the PVN had no effect on ABP or RSNA in WKY rats and SHRs. Our findings suggest that endogenous CRF in the PVN potentiates glutamatergic input and firing activity of PVN presympathetic neurons via CRFR1, resulting in augmented sympathetic outflow in hypertension.

Beneficial effects of the phytoestrogen genistein on hippocampal impairments of spontaneously hypertensive rats (SHR).

Hippocampal neuropathology is a recognized feature of the spontaneously hypertensive rat (SHR). The hippocampal alterations associate with cognitive impairment. We have shown that hippocampal abnormalities are reversed by 17ß-estradiol, a steroid binding to intracellular receptors (estrogen receptor α and ß subtypes) or the membrane-located G-protein coupled estradiol receptor. Genistein (GEN) is a neuroprotective phytoestrogen which binds to estrogen receptor ß and G-protein coupled estradiol receptor. Here, we investigated whether GEN neuroprotection extends to SHR. For this purpose, we treated 5-month-old SHR for 2 weeks with 10 mg kg-1 daily s.c injections of GEN. We analyzed the expression of doublecortin+ neuronal progenitors, glial fibrillary acidic protein+ astrocytes and ionized calcium-binding adapter molecule 1+ microglia in the CA1 region and dentate gyrus of the hippocampus using immunocytochemistry, whereas a quantitative real-time polymerase chain reaction was used to measure the expression of pro- and anti-inflammatory factors tumor necrosis factor α, cyclooxygenase-2 and transforming growth factor ß. We also evaluated hippocampal dependent memory using the novel object recognition test. The results showed a decreased number of doublecortin+ neural progenitors in the dentate gyrus of SHR that was reversed with GEN. The number of glial fibrillary acidic protein+ astrocytes in the dentate gyrus and CA1 was increased in SHR but significantly decreased by GEN treatment. Additionally, GEN shifted microglial morphology from the predominantly activated phenotype present in SHR, to the more surveillance phenotype found in normotensive rats. Furthermore, treatment with GEN decreased the mRNA of the pro-inflammatory factors tumor necrosis factor α and cyclooxygenase-2 and increased the mRNA of the anti-inflammatory factor transforming growth factor ß. Discrimination index in the novel object recognition test was decreased in SHR and treatment with GEN increased this parameter. Our results indicate important neuroprotective effects of GEN at the neurochemical and behavioral level in SHR. Our data open an interesting possibility for proposing this phytoestrogen as an alternative therapy in hypertensive encephalopathy.

Involvement of Interleukin-1 ß/Insulin-Like Growth Factor 1 in Ameliorating Effects of Electroacupuncture on Myocardial Fibrosis Induced by Essential Hypertension.

OBJECTIVE: To investigate the effect of electroacupuncture (EA) at Neiguan (PC 6) on myocardial fibrosis in spontaneously hypertensive rats (SHRs), and to explore the contribution of interleukin-1 ß (IL-1 ß), insulin-like growth factor 1 (IGF-1), and transforming growth factor ß 1 (TGF- ß 1) to the effects. METHODS: Nine 12-weeks-old Wistar Kyoto (WKY) male rats were employed as the normal group. Twenty-seven SHRs were equally randomized into SHR, SHR+EA, and SHR + sham groups. EA was applied at bilateral PC 6 once a day 30 min per day in 8 consecutive weeks. After 8-weeks EA treatment at PC 6, histopathologic changes of collagen type I (Col I), collagen type 1 (Col 1) and the levels of IGF-1, 1L-1 ß, TGF- ß 1, matrix metalloproteinase (MMP)-2 and MMP-9 were examined in myocardial tissure respectively. RESULTS: After 8-weeks EA treatment at PC 6, the enhanced myocardial fibrosis in SHRs were characterized by the increased mean fluorescence intensity of Col I and Col 1 in myocardium tissue (P<0.01). All these abnormal alterations above in SHR + EA group was significantly lower compared with the SHR group (P<0.01). Meanwhile, the increased levels of IL-1 ß, IGF-1, TGF-ß 1 in serum or myocardial tissue of SHRs, diminished MMP 9 mRNA expression in SHRs were also markedly inhibited after 8 weeks of EA treatment (P<0.05 or P<0.01). Furthermore, the contents of IL-1 ß, IGF-1, TGF-ß 1 in myocardial tissue were positively correlated with the systolic blood pressure and hydroxyproline respectively (P<0.01). CONCLUSION: EA at bilateral PC 6 could ameliorate cardiac fibrosis in SHRs, which might be mediated by regulation of 1L-1 ß/IGF-1-TGF- ß 1-MMP9 pathway.

Involvement of Interleukin-1 β/Insulin-Like Growth Factor 1 in Ameliorating Effects of Electroacupuncture on Myocardial Fibrosis Induced by Essential Hypertension / 中国结合医学杂志

OBJECTIVE@#To investigate the effect of electroacupuncture (EA) at Neiguan (PC 6) on myocardial fibrosis in spontaneously hypertensive rats (SHRs), and to explore the contribution of interleukin-1 β (IL-1 β), insulin-like growth factor 1 (IGF-1), and transforming growth factor β 1 (TGF- β 1) to the effects.@*METHODS@#Nine 12-weeks-old Wistar Kyoto (WKY) male rats were employed as the normal group. Twenty-seven SHRs were equally randomized into SHR, SHR+EA, and SHR + sham groups. EA was applied at bilateral PC 6 once a day 30 min per day in 8 consecutive weeks. After 8-weeks EA treatment at PC 6, histopathologic changes of collagen type I (Col I), collagen type 1 (Col 1) and the levels of IGF-1, 1L-1 β, TGF- β 1, matrix metalloproteinase (MMP)-2 and MMP-9 were examined in myocardial tissure respectively.@*RESULTS@#After 8-weeks EA treatment at PC 6, the enhanced myocardial fibrosis in SHRs were characterized by the increased mean fluorescence intensity of Col I and Col 1 in myocardium tissue (P<0.01). All these abnormal alterations above in SHR + EA group was significantly lower compared with the SHR group (P<0.01). Meanwhile, the increased levels of IL-1 β, IGF-1, TGF-β 1 in serum or myocardial tissue of SHRs, diminished MMP 9 mRNA expression in SHRs were also markedly inhibited after 8 weeks of EA treatment (P<0.05 or P<0.01). Furthermore, the contents of IL-1 β, IGF-1, TGF-β 1 in myocardial tissue were positively correlated with the systolic blood pressure and hydroxyproline respectively (P<0.01).@*CONCLUSION@#EA at bilateral PC 6 could ameliorate cardiac fibrosis in SHRs, which might be mediated by regulation of 1L-1 β/IGF-1-TGF- β 1-MMP9 pathway.

Choline induced cardiac dysfunction by inhibiting the production of endogenous hydrogen sulfide in spontaneously hypertensive rats.

To investigate the exact effects of dietary choline on hypertensive heart disease (HHD) and explore the potential mechanisms, male spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) were randomly divided into five groups as follows: WKY group, WKY + Choline group, SHR group, SHR + Choline group, and SHR + Choline + NaHS group. In choline treatment groups, rats were fed with 1.3% (w/v) choline in the drinking water for 3 months. The rats in the SHR + Choline + NaHS group were intraperitoneally injected with NaHS (100 micromol/kg/day, a hydrogen sulfide (H2S) donor) for 3 months. After 3 months, left ventricular ejection fraction (LVEF) and fractional shortening (LVFS), the indicators of cardiac function measured by echocardiography, were increased significantly in SHR as compared to WKY, although there was no significant difference in collagen volumes and Bax/Bcl-2 ratio between the two groups, indicating the early stage of cardiac hypertrophy. There was a significant decrease in LVEF and LVFS and an increase in collagen volumes and Bax/Bcl-2 ratio in SHR fed with choline, meanwhile, plasma H2S levels were significantly decreased significantly in SHR fed with choline accompanying by the decrease of cystathionine-gamma-lyase (CSE) activity. Three months of NaHS significantly increased plasma H2S levels, ameliorated cardiac dysfunction and inhibited cardiac fibrosis and apoptosis in SHR fed with choline. In conclusion, choline aggravated cardiac dysfunction in HHD through inhibiting the production of endogenous H2S, which was reversed by supplementation of exogenous H2S donor.

TangNaiKang, herbal formulation, alleviates obesity in diabetic SHR/cp rats through modulation of gut microbiota and related metabolic functions.

CONTEXT: Tangnaikang (TNK) is a Chinese herbal formulation that has lipid-lowering effects, but its effect on reducing obesity has not been studied. OBJECTIVE: To observe the effect of TNK on obesity and explore its effect on gut microbiota of obese rats. MATERIALS AND METHODS: The SHR/NDmcr-cp rats were divided into three groups: (1) 3.24 g/kg TNK (High TNK), (2) 1.62 g/kg TNK (Low TNK), and (3) an untreated control (CON). Wistar-Kyoto rats were used as normal controls (WKY). After 8 weeks of TNK oral administration, body weight, abdominal circumference, triglycerides (TC) and total cholesterol (CHO) were measured. Gut microbiota diversity was studied by 16S rDNA sequencing, and metagenomes analysis was conducted to determine alteration in functional gene expression. RESULTS: The body weight (496.60 ± 6.0 g vs. 523.40 ± 5.6 g), abdomen circumference (24.00 ± 0.11 cm vs. 24.87 ± 0.25 cm), TC (3.04 ± 0.16 mmol/L vs. 4.97 ± 0.21 mmol/L), CHO (2.42 ± 0.15 mmol/L vs. 2.84 ± 0.09 mmol/L) of rats in the High TNK group were decreased significantly (all p < 0.05). TNK administration regulates intestinal flora, up-regulates Eisenbergiella and down-regulates Clostridium_sensu_stricto_1, which is beneficial to the production of short-chain fatty acids (SCFAs). Metagenomes analysis shows that TNK is closely related to the fatty acid synthesis pathway. DISCUSSION AND CONCLUSIONS: TNK can regulate gut microbiota to reduce obesity, which may be related to fatty acid metabolism. Our research supports the clinical application of TNK preparation and provides a new perspective for the treatment of obesity.

GJD Modulates Cardiac/Vascular Inflammation and Decreases Blood Pressure in Hypertensive Rats.

Gedan Jiangya decoction (GJD) (aqueous ethanol extract), a traditional Chinese medicine formula which contain six botanical drugs (Uncaria rhynchophylla (Miq.) Miq., Salvia miltiorrhiza Bunge, Pueraria lobata (Willd.) Ohwi, Eucommia ulmoides Oliv., Prunella vulgaris L., and Achyranthes bidentata Blume) was designed to treat hypertension; however, the underlying mechanism of action is unclear. This study aimed to determine the mechanisms of action of GJD in the treatment of hypertension in spontaneously hypertensive rats (SHR). Male SHRs were randomly divided into five groups: GJD doses were low (1.36 g/kg/d), medium (2.72 g/kg/d), and high (5.44 g/kg/d), captopril (13.5 mg/kg/d), and SHR groups, with Wistar-Kyoto rats (WKY) serving as the control. Every rat was gavaged once a day. The ALC-NIBP, a noninvasive blood pressure device, measured systolic (SBP) and diastolic (DBP) blood pressures. Six weeks following treatment, all rats were anesthetized. The blood samples were obtained from the abdominal aorta and then serum isolated to assess endothelin-1 and angiotensin II, interleukin-1beta, interleukin-6, and TNF-alpha. The left ventricular and thoracic aortas were taken for HE staining, immunohistochemistry, RT-qPCR, and western blot examination. Following GJD therapy, SBP and DBP were significantly lowered, as were serum levels of endothelin-1 and angiotensin II. The thickness of the left ventricular and thoracic aorta walls reduced, as did type I collagen, type III collagen, and alpha-SMA expression in the left ventricular and aortic tissues. The GJD treatment significantly reduced serum levels of the inflammatory markers interleukin-1beta, interleukin-6, and TNF-alpha. Furthermore, interleukin-1 beta, interleukin-6, TNF-alpha, TAK1, and NF-κB/p65 levels were significantly reduced in left ventricular and aortic tissues, whereas IkB-alpha levels were significantly elevated. GJD has a dose-dependent effect on all parameters. In conclusion, GJD has been shown to lower blood pressure, improve cardiovascular remodeling, and reduce inflammation via regulating NF-κB in SHRs.

[Effect of Rehmanniae Radix Praeparata on energy metabolism in prefrontal cortex of rats with attention deficit hyperactivity disorder based on "static Yin and dynamic Yang" theory].

The present study investigated the effect of Rehmanniae Radix Praeparata(RRP) on the energy metabolism of prefrontal cortex(PFC) of spontaneously hypertensive rats with attention deficit hyperactivity disorder(ADHD) based on the "static Yin and dynamic Yang" theory.Thirty spontaneously hypertensive male rats aged 3 weeks were randomly divided into a model group, a methylphenidate(MPH) group(2 mg·kg~(-1)), and an RRP group(2.4 g·kg~(-1)).Wistar-Kyoto(WKY) male rats of the same age were assigned to the normal group.Rats were treated with corresponding drugs twice per day, and those in the model group and the normal group received the same volume of 0.9% sodium carboxymethyl cellulose(CMC-Na) solution by gavage.The open-field test was performed to evaluate the spontaneous and impulsive behaviors of rats before treatment and on the 4~(th) week after treatment.Four weeks after treatment, PFC was isolated and mitochondria were prepared.The content of adenosine triphosphate(ATP), adenosine diphosphate(ADP), and adenosine monophosphate(AMP) in the PFC was determined by high-performance liquid chromatography(HPLC), and energy charge(EC) was calculated.The parameters related to mitochondrial respiratory function were measured by the Clark oxygen electrode, specifically, state 3 respiration(ST3), state 4 respiration(ST4), and respiratory control rate(RCR).Enzymatic activities of succinate dehydrogenase(SDH), cytochrome C oxidase(COX), Na~+-K~+-ATPase, and Ca~(2+)-Mg~(2+)-ATPase were measured by chemical colorimetry.Mitochondrial permeability transition pore(mPTP) opening was measured by spectrophotometry.Protein expression of glucose transporter 1(GLUT1) and GLUT3 in PFC was tested by Western blot.Compared with the results in the model group, RRP could significantly reduce the total distance of movement, vertical times, and distance in the central area in the open field test(P<0.05 or P<0.01), increase the content of ATP and EC, decrease the content of AMP(P<0.05), elevate ST3 and RCR(P<0.05), potentiate activities of SDH, COX, Na~+-K~+-ATPase, and Ca~(2+)-Mg~(2+)-ATPase(P<0.05 or P<0.01), inhibit the opening of mPTP, and increase the expression levels of GLUT1 and GLUT3 proteins(P<0.05).It was inferred that RRP could inhibit hyperacti-vity and impulsivity by improving the energy metabolism disorder in PFC of ADHD rats, and its mechanism may be related to the improvement of mitochondrial respiratory function, potentiation of Na~+-K~+-ATPase, Ca~(2+)-Mg~(2+)-ATPase, and mitochondrial respiratory enzymes, inhibition of the opening of mPTP, and up-regulation of the expression of glucose transporter proteins.This study initially reveals the biological connotation of the "static Yin and dynamic Yang" theory in ADHD.

[Tianma Gouteng Decoction regulates MFN2 expression to delay vascular aging in spontaneously hypertensive rats].

We studied the effect of Tianma Gouteng Decoction on vascular aging in spontaneously hypertensive rats(SHRs) to explore the molecular mechanism of the decoction in improving arterial vascular aging by regulating the expression of mitofusin 2(MFN2).Twenty 64-weeks-old SHRs were randomly assigned into the aging group and the treatment group(Tianma Gouteng Decoction 5.48 mg·kg~(-1)).Wistar-Kyoto(WKY) rats of 64 weeks old were taken as the normal group and SHR rats of 14 weeks old as the young group.The intervention with Tianma Gouteng Decoction lasted for 12 weeks.We then employed HE staining and Masson staining to observe the morphology of thoracic aorta under an electron microscope and measured the malondialdehyde(MDA) content, superoxide dismutase(SOD) activity, respiratory chain complex Ⅲ level, and thioredoxin peroxidase(TPX) activity.The vascular aging was detected via the biomarker senescence-associated beta-galactosidase(SA-ß-Gal).The expression levels of MFN2 and aging marker proteins silent information regulator 1(SIRT1), Klotho, p21, and p53 in thoracic aorta were detected by immunohistochemistry/fluorescence, qRT-PCR, and Western blot.Compared with the young group and the normal group, the aging group had risen blood pressure, lumen stenosis caused by thickened intima of blood vessels, decreased SOD and TPX activities, increased MDA and mitochondrial respiratory chain complex Ⅲ levels, down-regulated expression of MFN2, SIRT1, and Klotho, and up-regulated expression of p21 and p53(P<0.01 or P<0.05).The treatment with Tianma Gouteng Decoction significantly lowered blood pressure, mitigated vascular intimal thickening, increased SOD and TPX activities, and decreased MDA and mitochondrial respiratory chain complex Ⅲ levels, thus alleviating vascular aging.At the same time, the decoction up-regulated the expression of MFN2, SIRT1, and Klotho, while down-regulated that of p21 and p53(P<0.01 or P<0.05).In summary, Tianma Gouteng Decoction can significantly delay the vascular aging in hypertension.Specifically, it may up-regulate the expression of MFN2 and regulate the expression of aging-related proteins to alleviate oxidative stress.

Influence of electroacupuncture on ghrelin and the phosphoinositide 3-kinase/protein kinase B/endothelial nitric oxide synthase signaling pathway in spontaneously hypertensive rats.

OBJECTIVE: To investigate the influence of electroacupuncture (EA) on ghrelin and the phosphoinositide 3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3K/Akt/eNOS) signaling pathway in spontaneously hypertensive rats (SHRs). METHODS: Eight Wistar-Kyoto rats were used as the healthy blood pressure (BP) control (normal group), and 32 SHRs were randomized into model group, EA group, EA plus ghrelin group (EA + G group), and EA plus PF04628935 group (a potent ghrelin receptor blocker; EA + P group) using a random number table. Rats in the normal group and model group did not receive treatment, but were immobilized for 20 min per day, 5 times a week, for 4 continuous weeks. SHRs in the EA group, EA + G group and EA + P group were immobilized and given EA treatment in 20 min sessions, 5 times per week, for 4 weeks. Additionally, 1 h before EA, SHRs in the EA + G group and EA + P group were intraperitoneally injected with ghrelin or PF04628935, respectively, for 4 weeks. The tail-cuff method was used to measure BP. After the 4-week intervention, the rats were sacrificed by cervical dislocation, and pathological morphology of the abdominal aorta was observed using hematoxylin-eosin (HE) staining. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of ghrelin, nitric oxide (NO), endothelin-1 (ET-1) and thromboxane A2 (TXA2) in the serum. Isolated thoracic aortic ring experiment was performed to evaluate vasorelaxation. Western blot was used to measure the expression of PI3K, Akt, phosphorylated Akt (p-Akt) and eNOS proteins in the abdominal aorta. Further, quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to measure the relative levels of mRNA expression for PI3K, Akt and eNOS in the abdominal aorta. RESULTS: EA significantly reduced the systolic BP (SBP) and diastolic BP (DBP) (P < 0.05). HE staining showed that EA improved the morphology of the vascular endothelium to some extent. Results of ELISA indicated that higher concentrations of ghrelin and NO, and lower concentrations of ET-1 and TXA2 were presented in the EA group (P < 0.05). The isolated thoracic aortic ring experiment demonstrated that the vasodilation capacity of the thoracic aorta increased in the EA group. Results of Western blot and qRT-PCR showed that EA increased the abundance of PI3K, p-Akt/Akt and eNOS proteins, as well as expression levels of PI3K, Akt and eNOS mRNAs (P < 0.05). In the EA + G group, SBP and DBP decreased (P < 0.05), ghrelin concentrations increased (P < 0.05), and the concentrations of ET-1 and TXA2 decreased (P < 0.05), relative to the EA group. In addition, the levels of PI3K and eNOS proteins, the p-Akt/Akt ratio, and the expression of PI3K, Akt and eNOS mRNAs increased significantly in the EA + G group (P < 0.05), while PF04628935 reversed these effects. CONCLUSION: EA effectively reduced BP and protected the vascular endothelium, and these effects may be linked to promoting the release of ghrelin and activation of the PI3K/Akt/eNOS signaling pathway.