Mechanism Of Polygonum Multiflorum Thunb And Its Main Ingredient 2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glycoside Improves Obesity-associated Endothelial Dysfunction And Hypertension In Obese Rats

BackgroundThe prevalence of obesity and hypertension has enhanced notably and the two often exist together.Obesity makes it more difficult for hypertensive patients to control their blood pressure,also promotes the accumulation of cardio-metabolic risk factors,thus aggravates cardiovascular and cerebrovascular injury.In China,the overweight/obesity rate of adult residents has been more than 50 %,and overweight/obesity has become an important driving factor for hypertension.Obesity-related cardiovascular diseases,especially hypertension,account for nearly 70% of the complications in obese patients.Given the high morbidity and mortality of obesity-related hypertension,there is an urgent need to find effective treatment strategies and drugs.However,weight loss alone has limited efficacy in obesity-related hypertension with target organ damage and there is limited number of effective drug interventions.Although there is no name of obesity-related hypertension in traditional Chinese medicine（TCM）,it could be classified as "vertigo","headache" and "stroke" according to TCM pathophysiology.This disease is the deficiency of Yin from liver and kidney deficiency and deficiency of Qi and blood.Therefore,for treatment of obesity-related hypertension by traditional Chinese medicine,doctors focus on reinforcing liver and kidney,clearing damp and turbidities.2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glycoside（TSG）is the main compound extracted from a traditional Chinese medicinal herb,Polygonum multiflorum Thunb,which is famous for its remarkable ability to reinforce liver and kidney.Modern pharmacologic researches have revealed that TSG could improve blood flow,treat hyperlipidemia and promote angiogenesis.However,the role of TSG in the physiological changes caused by obesity has been overlooked.Currently,several factors have been proposed for pathogenesis of obesity-associated hypertension,such as adipocytokines,renin‐an‐giotensin‐aldosterone system（RAAS）,oxidative stress and endothelial dysfunction.Adipokines participate in the regulation of multiple metabolic and biological processes related with obesity.Omentin-1,also known as intelectin-1（Itln-1）,is a novel adipokine.Clinical evidence has indicated that serum omentin-1 reduces notably in obese and diabetic objects.However,the mechanism of omentin-1 on hypertension in obesity and endothelial dysfunction has not been explored.ObjectivesThis study aims to examine（i）whether Polygonum multiflorum improves obesity-related hypertension and endothelial dysfunction;What are the effective components of Polygonum multiflorum in improving obesity-related hypertension and endothelial dysfunction;（ii）the mechanism of the functional component in Polygonum multiflorum which improves obesity-related endothelial dysfunction and hypertension.Methodsa)Analysis of main components of Polygonum multiflorum extract（PME）:HPLC method was established.The linear relationship,precision,stability,reproducibility and recovery rate were investigated.PME was compared with the mixture standards of TSG,emodin and physcion.And the average content of 3 main components in PME was calculated according to the peak area.b)Screening of functional components of PME in improving obesity-related endothelial dysfunction hypertension: According to the above proportion of PME,obese rats were given equal doses of PME（180 mg/kg）,TSG（100 mg/kg）,emodin（3 mg/kg）,physcion（1 mg/kg）or temmisartan（20 mg/kg）for 14 days,respectively.SBP was measured using a noninvasive tail-cuff system.Endothelial-dependent/independent mesenteric vasodilatation were determined.Serum of total cholesterol（TC）,triglyceride（TG）,low density lipoprotein cholesterol（LDL-C）and high density lipoprotein cholesterol（HDL-C）were detected.In addition,dose of TSG 50/100/200 mg/kg were set separately to determine the optimal dose.c)TSG activated Akt/eNOS/NO pathway and reduced oxidative/nitrative stress in mesenteric arterioles of obese rats: ZDF obese rats were given TSG（100 mg/kg）intragastrically for 14 days.The SBP and glucose tolerance were measured.The mesenteric arterioles were separated to assess the vasodialation function.Western blotting was used to detect the expression of（p-）Akt,（p-）e NOS,NOX-2 and p22 phox in mesenteric arterioles.NO/ O₂^·-/ ONOO-production in rat mesenteric arterioles were detected by fluorescent probes.d)TSG improved obesity-related hypertension by upregulating OMT-1: Serum OMT-1 and total TG concentrations of overweight/obese patients were detected by ELISA kits,and the brachial artery FMD was detected by color Doppler ultrasonography.Mature HPA-v and HUVECs were co-cultivated in a 6-well transwell system.Mature adipocytes were cultured in upper chamber and HUVECs were cultivated in lower chamber.TSG（100 μM,24 h）was added to upper chamber.Then,global omentin-1 gene knockout（OMT-/-）mice were generated under CRISPR/Cas9.The target sites were designed in exon 3-7 of the mouse Itln-1 gene.Twelve-week-old male OMT-/-mice and wild-type（WT） littermates were randomized into two groups with saline or TSG（100 mg/kg/day）gavage for a fortnight.e)TSG upregulates OMT-1 level by activating PPAR-γ in adipose tissues from obese rats: Total RNA was extracted from OMT-/-mice,high-throughput sequencing was performed on Illumina Hi Seq 2500 platform and transcriptome microarray information was analyzed.Ch IP assay was performed with a Ch IP-IT high-sensitivity kit.The expression of PPAR-γ and OMT-1 in adipocytes were detected by PCR and Western blotting after transfection with Itln-1 si RNA or treatment with TSG（100 μM）or PPAR-γ inhibitor T0070907（10 μM）.Resultsa)The standard curves were: TSG y=24206x-20.34 r2=0.99996,emodin y=26273x+0.665 r2=0.99998,physcion: y=26445x-0.547 r2=1,respectively.Precision RSDs were 0.11%,0.37% and 0.36%.Stability RSDs were 0.29%,1.35% and 0.88%.Reproducibility RSDs were 2.73%,2.00% and 2.18%.Recoveries were 112.08%,100.47%,92.86%,and the RSDs were 3.676%,3.275%,0.671%.According to the peak area,the average contents of 3 main components in PME were as follows: TSG 563.75 mg/g,emodin 15.89 mg/g,physcion 5.26 mg/g.b)Obese rats showed a significant increase in systolic blood pressure（SBP）and impaired endothelial vasorelaxation.PME（180 mg/kg）and TSG（100 mg/kg）,but not emodin（3 mg/kg）or physcion（1 mg/kg）notably reduced SBP and improved mesenteric endothelial vasorelaxation in ZDF rats.TSG also caused body weight loss and decreased TG,TC and LDL-C levels in obese rats,but failed to improve glucose tolerance.c)Vasorelaxation was abolished by pre-incubated with L-NAME（a specific NO synthase inhibitor,100 μM,30 min）in mesenteric arterioles from obese rats.TSG treatment enhanced phosphorylations of Akt and e NOS,reduced NOX-2 and p22 phox expression,and O₂^·-and ONOO-concentrations in mesenteric arteries from obese rats.d)Obese rats exhibited low concentration of circulating and visceral adipose omentin-1,and TSG administration promoted the expression of omentin-1 in obese rats.Additionally,rh-omentin significantly up-regulated the phosphorylations of Akt/e NOS and was accompanied by increase NO concentration,down-regulated the expressions of NOX-2 and p22 phox,as well as decreased the productions of O₂^·-and ONOO-in HUVECs under HF condition.Furtherly,Itln-1 depletion induced elevated arterial blood pressure and endothelial dysfunction in vivo,whereas TSG treatment failed to reverse this.e)TSG treatment increased the combination between transcript factor PPAR-γ and promoter of Itln-1 gene,and finally resulted in up-regulation of omentin-1 m RNA/protein expression in adipocytes.T0070907 effectively decreased the m RNA level of Itln-1,as well as PPAR-γ and omentin-1 expression in adipocytes,and further inhibited the activation of Akt/e NOS signaling in HUVECs from the co-cultured system.ConclusionsIn this study,1)a HPLC method for simultaneous determination of TSG,emodin and physcion in PME was developed.PME significantly improved microvascular endothelial dysfunction and decreases arterial blood pressure in obese rats.TSG was the key component of PME to protect vascular endothelium and postpone the process of obesity-related hypertension.2)We demonstrated for the first time that reduced PPAR-γ expression in adipocytes led to downregulation of OMT-1,thereby induced obesity-related hypertension and endothelial dysfunction.TSG enhanced OMT-1 level（at least partially）by promoting binding of PPAR-γ and Itln-1 promoter in adipose tissues from obese rats,activated Akt/e NOS/NO signal,alleviated oxidative/nitrative stress,improved endothelial dysfunction in mesenteric arteries and thus improved obesity-related hypertension.