Dietary supplementation of Huangshan Maofeng green tea preventing hypertension of older C57BL/6 mice induced by desoxycorticosterone acetate and salt

 Senile hypertension affects the life quality of aged population

• Dietary intervention plays a pivotal role in the prevention of hypertension
• There are few reports concerning the effects and mechanisms of green tea supplementation preventing age related hypertension.
• In this study, Huangshan Maofeng green tea (HSMF) dose-dependently prevented the increase of systolic and diastolic blood pressure and increased blood pressure induced by DOCA plus salt in 51-week old mice.

Hypertension is one of the chronic cardiovascular diseases, and plays an important role in development of aortic aneurysm, atherosclerosis, and cerebral stroke

• It has been estimated that hypertension affects one billion people and causes 9.4 million deaths every year globally.
• Dietary intervention for preventing senile hypertension might be an excellent strategy for aging people.

Tea sample and analysis

• HSMF purchased from Huangshan Maofeng Tea Group Company (Huangshan, Anhui, China).
• The main catechins and caffeine were analyzed following our previous protocol [34] on a Waters High Performance Liquid Chromatography (HPLC) system supported with a Waters 600 controller and Waters 2489 UV/Visible Detector (280 nm).

Animals experiment

• Male C57BL/6 mice, aged 28 weeks, were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China).
• The mice were housed in standard mouse cages in the specific pathogen free laboratory animal facility center, which was controlled with a constant temperature (22 ± 1°C) and humidity (50 ± 5%) under a 12:12 h light-dark cycle falls on 8:00 AM to 8:30 PM at Anhui Agricultural University.
• They were fed with normal chow and water ad libitum until to age of 36-week-old.

DOCA salt induced hypertension

• After 6 weeks dietary intervention, the mice were anesthetized by intraperitoneal injection of pentobarbital (100 mg/kg).

The measurement of mesenteric artery contraction

• Potassium chloride (KCl), angiotensin Ⅱ, 5-hydroxytryptamine (5-HT), U46619 and norepinephrine (NE) were used to stimulate the contraction of MA.

Quantitative real time PCR analysis

• A small piece of right kidney and aorta were fixed in RNA stabilization solution (Thermo Fisher Scientific, Waltham, MA, USA) and total RNA was extracted using RNA isolator (Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China).
• Reverse transcription was performed using HiScript II 1st Strand cDNA Synthesis kit.
• Quantitative Real Time PCR (qPCR) was performed with AceQ qPCR SYBR Green Master Mix kit by a Bio-Rad CFX System.

Histological staining and immunohistochemical analysis

• Kidney tissues for both experiments were fixed in 10% neutrally buffered formalin solution at room temperature, and then dehydrated, and embedded in paraffin (Paraplast Tissue Embedding Medium, LEICA, Buffalo Grove, IL, USA) using a modular tissue embedding system.
• Hematoxylin-eosin staining was carried out on 5 μm sections, which were incisized by a fully automated rotary microtome and mounted onto positively charged slides (Adhesion Microscope Slides, CITOGLAS, Shanghai, China).
• He was staining using an HE staining kit (Boster Biological Technology Company, Pleasanton, CA, USA).

Western blot analysis

• Total protein was extracted from the aorta tissues using 2 × SDS buffer, and was quantified using BCA kit (Vazyme Biotech Co., Ltd., Nanjing, China).
• The primary antibodies used are as following: M130 from R&D Systems (Minneapolis, MN, USA), p-MYTP1 and p-MLC20 from Cell Signaling Technology (MA), PKCα, p-CPI-17, etc.
• Second antibodies are from Santa Cruz Biotechnology (CA, USA)
• Protein binding was detected using an ECL reagent, and densitometry analysis was performed using Image J software

Statistics

• Data were analyzed using Graphpad Prism Software v5.0.10
• One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test
• Comparisons between two groups were performed with an unpaired two-tailed Student's t-test
• P <.05 was considered statistical significance.

HSMF preventing DS induced hypertension in old mice

• Over activation of MR disequilibrates the balance of the renin-aniotension-aldosterone system and leads to hypertension
• In this study, induction of 43-week-old C57BL/6 mice by DS for 2 weeks remarkably increased SBP (137.8 ± 2.18 mm Hg) and DBP (81.5±2.75 mm HG), respectively, by ~18mm Hg and ~11 mm HGs, compared with control group (SBP, 119.7 ± 0.55 mm, DBP, 69.8± 1.58 mm).
• This increase of SBP induced by DS was prevented by low dosage, middle dosage, and high dosage tea dietary intervention at the 45 week in mice.
• These preventive effects showed dose-dependence manner.

Ameliorated MA hypercontractility in old mice

• High concentration of potassium chloride (KCl 143 mM) and angiotensinⅡ(100 nM) significantly induced the contraction of MA from DS group mice compared to control group mice.
• Decreased contraction by tea intervention showed dose-dependently.

HSMF down-regulated the contractile genes and proteins expression in aorta of old mice

• Contractile proteins in blood vessels play pivotal roles in regulating smooth muscle contraction
• Myosin phosphatase targeting subunit 1 (MYPT1), CPI-17, MLC20, and 20 kDa regulatory light chain (MLC20) of myosin-II are key events in this pathway
• The expression of contractile proteins such as RhoA, Rock1, Rock2, and protein kinase C (PKC)-potentiated inhibitory protein of 17 kDa (CPI-17) were significantly up-regulated in DS group mice compared to that of control group mice.
• Tea intervention inhibited all of those genes expression level with dose-dependent effects.

HSMF inhibited inflammation-related genes expression in the aorta of old mice

• The expression of IL-6, IL-1β, TNF-ɑ, and MCP-1 increased significantly in DS group mice compared to that in control group mice, but were suppressed by tea intervention with dose-dependent (Fig. 5).
• IL-11β expression was significantly inhibited only in DS+H intervention group mice.

HSMF protected kidney injury induced by DOCA plus salt in old mice

• Kidney is a key organ for water retention and fluid homeostasis, and plays an important role in BP regulation
• Renal injury induces renal hypertension, which is one of hypertensive complications
• In our study, DS treatment caused severe kidney injury in older C57BL/6 mice.
• DS treatment significantly increased the NGAL level in plasma of older mice compared to control mice.

HSMF-reduced macrophage infiltration and inflammation-related genes expression in kidney

• Chronic inflammation in kidney leads to renal injury and may accelerate the development of hypertension.
• Here are the main findings:
• Dietary tea supplement decreased these genes expression with dose-dependent.
• The expression of MCP-1 and CRP was significantly suppressed in the kidney of all three tea treated group mice compared to DS group mice (Fig. 8D and E).

Discussion Hypertension is chronic age-related cardiovascular disease, which seriously threats the health of aged people

• It is always observed that the total peripheral resistance increases in patients and experimental animal models with hypertension
• The excessive contraction of small vessels induced by DS leads to peripheral resistance and consequently elevates BP.
• Several papers have reported that tea secondary metabolites suppressed the MA contraction and suppressed the peripheral resistance to decrease BP.

Reference

10.1016/j.jnutbio.2020.108530