Meirong Wu, Xiobin Wu, Jingxiong Zhu, Fngln Li, Xinlin Wei*, Yunfeng Wng,*

a Development Center of Plant Germplasm Resources, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China

b Department of Food Science & Technology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China

Keywords:

High-salt diet

Hypertension

Green tea

Selenium-enriched green tea

PI3K/Akt pathway

Microbial profile

A B S T R A C T

High-salt diet is well recognized as a risk factor for hypertension, and dietary intervention plays a critical role in the prevention of hypertension.The current study investigated the effects of selenium-enriched green tea (Se-GT) and ordinary green tea (GT) on prevention of hypertension of rats induced by high-salt diet, as well as their potential regulatory and mechanism.Our results showed that GT and Se-GT supplementations significantly prevented the increase of blood pressure (BP), activated the phosphoinosmde-3-kinase/protein kinase B (PI3K/Akt) signaling pathway, and regulated the gene expression related to BP, as well as improved the tissue damage like heart, liver, and kidneys.Besides, the key parameters associated with oxidative stress,inflammation and endothelial dysfunction were also altered by GT and Se-GT treatments.Importantly, GT or Se-GT administration adjusted the diversity and composition of the intestinal flora.Moreover, GT and Se-GT supplementations increased the abundance of beneficial bacteria and reduced the abundance of harmful or conditional pathogenic bacteria.More specifically, GT intake specifically and significantly enriched the relative abundance of Paraprevotella and Bacteroides, whereas Se-GT was characterized by specific and significant enrichment for Allobaculum and Bifidobacterium.Our results proved that dietary supplement of GT and Se-GT remarkably improved the vascular functions and effectively prevented tissue damage by regulation of intestinal flora, and thus preventing hypertension induced by high-salt diet.

1.Introduction

High-salt diet, one of the current unhealthy dietary ways that cannot be ignored, has caused a series of health issues.It is reported that high-salt intake causes damage to many target tissues and further lead to a series of cardiovascular diseases like hypertension,strong evidences indicate a causal link between salt intake and blood pressure (BP) [1].In addition to hypertension, high-salt diet can also disequilibrate intestinal microbial homeostasis and intestinal environment, and the unbalanced intestinal environment disrupt the intestinal barrier function, which results in systemic inflammation,insulin resistance and hypertension [2].

The intestinal microbiome is the largest and most complex microecosystem in the human body and is closely related to the host health.Studies have been reported that changes in the composition and function of the intestinal microbiome led to hypertension [3].It is estimated that only 1.9% of the gut microbiome is heritable,and more than 20% of the microbiome biodiversity can be inferred from diet-related environmental factors [4].Studies indicate that the homeostasis of the human intestinal ecosystem mainly depends on factors such as post-natal environment and diet [5].David et al.[6]proved that the gut microbiota can quickly respond to changes in diet.Many researchers have reported that tea intervention plays an important role in the prevention or control of hypertension [7,8].Additionally, many evidences indicate that tea can reduce the increased risk of cardiovascular diseases by regulating the intestinal flora [9].Therefore, tea intervention to reveal the relationship between the intestinal microbiota and the prevention of hypertension can better understand the potential function of tea in cardiovascular diseases.

Tea, as the second largest beverage in consumption after water, is mainly consisted of polyphenols, caffeine, minerals, amino acids and carbohydrates [10].Tea drinking has been reported to be beneficial for the prevention or reducing the risk of cardiovascular diseases [11,12],especially hypertension [13].It has been reported that black and green teas had a positive effect on prevention of hypertension [14].Negishi et al.[15]found that the polyphenols extracted from black and green teas could lower BP in spontaneously hypertensive rats.Yokogoshi et al.[16]demonstrated that tea rich in gamma-aminobutyric acid decreased BP and prevented the development of hypertension in rats fed with a high-salt diet.Selenium (Se) is an essential trace element closely related to human health and can prevent cardiovascular and cerebrovascular diseases [17].At present, PI3K/Akt signaling pathway has been widely studied in cardiovascular diseases,especially hypertension.Some studies further demonstrated that black tea polyphenol and (–)-epigallocatechin gallate (EGCG),depending upon the PI3K/Akt pathway, could induce nitric oxide synthase (eNOS) activation to promote the nitric oxide (NO) release,furthermore alleviate vessel pressure.Notably, PI3K/Akt pathway is a typical signal pathway in the cardiovascular physiology [18,19].Additionally, the tea rich in gamma-aminobutyric acid (GABA)attenuated cardiac apoptosis in spontaneously hypertensive rats by via activating PI3K/Akt pathway [20].All these reports revealed that tea or tea active ingredients could alleviate hypertension or metabolic disorders evoked by hypertension.Additionally, our previous studies have shown that Se-enriched green tea (Se-GT) contained highly active angiotensin-converting enzyme (ACE) inhibitory peptides that could effectively lower BP, and its BP-lowering activity was related to Se [21].Therefore, the combination of tea and Se can better play the anti-hypertension effect of tea.In addition to its positive role in BP regulation, tea can also effectively regulate the composition and structure of host intestinal flora, thus ameliorating various metabolic disorders, such as obesity and diabetes [22,23].These work prompted us to explore whether tea, especially Se-enriched tea, could prevent the increase in BP caused by high-salt diet by regulating the composition of intestinal microbes.

In this work, we studied the effects of GT and Se-GT on BP, fecal microbiota, BP-related serum biochemical and metabolic parameters and related key metabolic enzyme activities in high-salt-fed rats.In addition, we explored the potential mechanism of tea regulating BP.These studies will contribute to a further understanding of the health function of tea and its application in food or food additives.

2.Materials and methods

2.1 Materials and reagents

GT (Se content = (0.23 ± 0.05) mg/kg) and Se-GT (Se content =(2.09 ± 0.12) mg/kg) leaves were obtained from Enshi Selenium Impression Agricultural Technology Co., Ltd.(Hubei Province,China).Glutathione peroxidase (GPX), superoxide dismutase(SOD), mlondialdehyde (MDA), NO, eNOS, angiotensin-converting enzyme II (Ang II) detection kits were purchased from Nanjing Jiancheng Institute of Bioengineering Co.(Nanjing, China).The C-reactive protein (CRP) detection kit was purchased from Link Tech Co.(China).Lactate dehydrogenase (LDH) determination kit was purchased from Shenzhen Lei Du Life Technology Co.,Ltd.(Shenzhen, China).All catechin standards used in liquid chromatography were purchased from Chengdu RefMedic Biotech Co., Ltd.(Chengdu, China).All 21 amino acid standards were purchased from Sigma Co.(St.Louis, USA).Unless otherwise stated,all other chemicals were of analytical grade.

2.2 Preparation of tea extracts

The tea leaves were pulverized and then extracted with boiling water (1:10 and 1:9,m/V) twice, each for 2 h.After filtration, the extracts were combined and centrifuged, and the supernatant was concentrated, lyophilized, and then stored at –20 °C until use.

2.3 Active ingredient analysis

The Se content was determined by atomic fluorescence spectrometry regarding the National Standard of China (GB 5009.93-2010) [24].The content of tea polyphenols was determined by the Folin-Ciocalteu method regarding the National Standard of China(GB/T 8313-2018) [25].The content of tea polysaccharides was determined by the phenol-sulfuric acid method [26].The content of flavonoids was measured according to the description of the National Standard of China (SN/T 4592-2016) [27].The contents of catechin, alkaloid, and phenolic acid were evaluated according to the previous report by our laboratory [28].A high performance liquid chromatography LCC-AT20 system (Shimadzu, Tokyo, Japan) was used to analyze the amino acid content in the tea extracts.Various amino acid standards (dissolved in 0.1 mol/L HCl) were prepared into 1 mg/mL solutions, and then the standard solutions were mixed in equal amounts and diluted with 0.1 mol/L HCl to each concentration gradient.The samples were treated in the same way.A total of 200 μL of the mixed standard solution or sample was dissolved in the specific mixture (200 μL of OPA derivatization reagent + 600 μL of boric acid buffer), and then derivatized in the dark for 15 min.Next,HPLC analysis was performed.The HPLC system was as follows:C18column (250 mm × 4.6 mm, 5 μm), column temperature 35 °C,flow rate 1 mL/min, detection wavelength 338 nm, injection 20 μL,mobile phase A: 20 mmol/L sodium dihydrogen phosphate solution;mobile phase B: mixed solution (methanol:acetonitrile:distilled water = 45:45:10).

2.4 Animals and experimental design

Twenty-four 8-week-old clean Wistar male rats were obtained from Slaccas Laboratory Animal Co., Ltd.(Shanghai, China), they were divided into 4 groups: normal control (NC), model control(MC), GT and Se-GT.All animal experiments were carried out in strict accordance with the experimental animal ethics standards of the Shanghai Jiaotong University Laboratory Animal Ethics and Use Committee (approval number A2020080) to maximize animal welfare and reduce animal suffering in experiments.All experimental rats were raised in the Animal Experiment Center of Shanghai Jiaotong University, with free eating and drinking, and kept in a controlled animal room ((25 ± 1) °C, 70%-75% humidity, 12 h light-dark cycle).After 1 week of acclimatization, the NC group was given Soobree ordinary standard feed (NO.1010009, Jiangsu Xiehe Pharmaceutical Bioengineering Co., Ltd., Jiangsu, China) for 9 weeks of normal diet;MC, GT, and Se-GT groups received high-salt feed (92.45% ordinary standard feed + 7.55% sodium chloride, 20210308(x), Jiangsu Suzhou Hongxin Biotechnology Co., Ltd.) for 9 weeks to induce hypertension.The nutritional compositions of high-salt feed are shown in the Supporting information 1 (Table S1).Shoobree common standard feed ingredients were showed in our previous report [29].In addition, the rats in GT and Se-GT groups were given 500 mg/kg of GT and Se-GT aqueous extracts per day, respectively, added to drinking water, and the rats in the MC group and the NC group were given distilled water.In the first week, 5thweek, and 9thweek,the body weight and systolic pressure reflecting BP of the rats were measured.

2.5 Histology analysis

The submitted samples were fixed with 4% paraformaldehyde.After the fixation, the samples were trimmed, dehydrated, embedded,sectioned, stained, and mounted in strict accordance with the pathological test SOP procedures.Finally, the qualified samples were microscopically inspected.

2.6 Real-time reverse transcription quantitative PCR (qRTPCR) analysis

The total mRNA in heart tissue was extracted and reverse transcribed into cDNA according to the Servicebio?RT First Strand cDNA Synthesis Kit instructions (Service, Wuhan,China).The mRNA expression level was detected by SYBR qPCR Master Mix (High ROX, Wuhan, China) according to the light quantitative PCR kit instructions.The specific primers used were as follows: ACE, 5’-TCATCCAGTTCCAGTTCCACG-3’(F), 5’-C G T G T T T G G T G T C C A G G-3’ (R); E T-1, 5’-T T G C T C C T G C T C C T C C T T G A T-3’ (F),5’-CTGTTCCCTTGGTCTGTGGTC-3’ (R); eNOS,5’-G G T A T T T G A T G C T C G G G A C T G C-3’ (F),5’-GTGATGGCTGAACGAAGATTGC-3’ (R); HO-1,5’-A T C G T G C T C G C A T G A A C A C T-3’ (F),5’-C A G C T C C T C A A A C A G C T C A A T G-3’ (R);S O D, 5’-A G C A T T C C A T C A T T G G C C G T A-3’ (F),5’-GCAATCCCAATCACACCACAA-3’ (R); iNOS,5’-C T T G G A G C G A G T T G T G G A T T G T-3’ (F),5’-GGTAGTGATGTCCAGGAAGTAGGTG-3’ (R);GAPDH, 5’-CCTCGTCCCGTAGACAAAATG-3’ (F),5’-TGAGGTCAATGAAGGGGTCGT-3’ (R).TheGAPDHgene was employed as an internal reference and the relative mRNA level of target genes was calculated by using the 2?ΔΔCtmethod.

2.7 Biochemical analysis

The serum levels of GPX, SOD, MDA, NO, eNOS, LDH, Ang II and CRP were all determined by using commercially available kits.

2.8 Immunohistochemical analysis of protein expression

The heart tissue was prepared into approximately 4 μm lateral tissue sections for immunohistochemical analysis PI3K, pAkt, Akt,vascular endothelial growth factor receptor2 (VEFGR2) expression.After dewaxing, rehydrating, and blocking of endogenous enzymes, sections were blocked with bovine serum albumin (BSA)(Servicebio, Wuhan, China) for 30 min and then rewarmed at room temperature.The corresponding polyclonal antibody (1:100)was added and incubated overnight at 4 °C.After washing with PBS, a biotin-labeled secondary antibody and avidin-labeled horseradish peroxidase were sequentially added and incubated at room temperature for 50 min.After adding DAB staining solution (Servicebio, Wuhan, China), it was counter-stained with hematoxylin for 3 min, dehydrated with gradient ethanol and dried naturally, and then sealed with neutral glue to observe the protein expression in each tissue.The relative expression of the section was calculated by the ratio of positive areas.

2.9 Microbiome profiling of fecal samples

The fresh feces of rats were collected aseptically and stored at–80 °C until detection.16S rRNA sequencing was used to analyze the composition of intestinal micro flora.Detailed analysis and sequencing steps were summarized in Supporting Information 2.

2.10 Statistical analysis

The data were expressed as the arithmetic mean ± standard deviation.Comparisons between groups were assessed by student’st-test for two groups and one-way ANOVA for multiple groups using the Tukey test.A level ofP＜ 0.05 was considered statistically significant.All statistical analysis was performed by SPSS 20.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA).

3.Results

3.1 Chemical composition in GT and Se-GT extracts

We determined the content of main components in GT and Se-GT extracts, as displayed in Tables 1 and 2, GT and Se-GT extracts contained appreciable amounts of tea polyphenols, soluble sugar,total flavonoids, caffeine, (–)-epigallocatechin (EGC), EGCG andL-theanine.The higher contents of tea polysaccharides, tea polyphenols, EGCG, EGC andL-theanine were found in Se-GT extracts, and the higher contents of total flavonoids were found in GT extracts.However, there were significant differences in most components between these two tea extracts.Our results indicated that GT and Se-GT extracts might contain beneficial functional ingredients that were associated with regulation of hypertension.

Table 1Comparison of the main components of GT and Se-GT (mg/g).

Table 2Comparison of 21 kinds of amino acids in GT and Se-GT (mg/g).

3.2 Effects of GT and Se-GT on body weight, BP and the serum biochemical parameters related to metabolic disorders caused by hypertension

After 9 weeks observation, GT and Se-GT showed a limit effect on body weight (Fig.1A).However, the BP of GT and Se-GT groups were significantly decreased compared with MC group (Fig.1B).Furthermore, the BP-regulating effect of Se-GT group was better than that of the GT group (Fig.1B).Tea and its bioactive ingredients can regulate BP by reducing oxidative stress, inflammation and improving endothelial function [30].In addition to observing systolic blood pressure (SBP) levels, we also analyzed the levels of serum factors,including oxidative stress, inflammation, endothelial function, lipid metabolism and lactate dehydrogenase.As could be seen, GT and Se-GT treatments significantly increased the levels of SOD, GPX,NO, and eNOS (Figs.1C, 1D, 1G, 1H).Moreover, GT and Se-GT treatments significantly decreased the levels of MDA, Ang II and LDH, Se-GT treatments significantly lowered the levels of CRP, but GT had a limit (Figs.1E, 1F, 1I, 1J).

Fig.1 The effects of GT and Se-GT on the body weight, blood pressure and serum biochemical parameters of hypertensive rats induced by high salt diet.(A) Body weight; (B) SBP; (C) SOD; (D) GPX; (E) MDA; (F) Ang II; (G) NO; (H) eNOS; (I) CRP; (J) LDH.Different letters represent a significant difference among multiple groups.*P ＜ 0.05, **P ＜ 0.01, ns represents no significance.One-way analysis of variance and Tukey’s test were used to estimate statistical significance.

3.3 Effects of GT and Se-GT on the heart, liver and kidney tissues, as well as the expression of BP-related genes

Histopathological analysis showed that both GT and Se-GT reversed the structural damage of the heart, liver, and kidney tissues caused by high-salt diet (Fig.2A).GT and Se-GT ameliorated the cytoplasmic porosity of individual cardiomyocytes, hepatic vacuolar degeneration, and edema of renal tubular epithelial cells.Studies have shown that long-term high-salt diets led to increased BP [31], thus we investigated the effects of GT and Se-GT on the gene expressions closely related to blood pressure regulation, includingACE,ET-1,eNOS,HO-1,SODandiNOs.From the data in Figs.2B–2H, it could be seen that the mRNA expressions ofACE,ET-1andiNOsin the heart tissue of the MC group was significantly increased compared with the NC group, while theeNOS,HO-1andSODexpression was significantly decreased.However, compared with the MC group, GT and Se-GT treatment significantly down-regulated the expression ofACEandiNOs, and the expression ofeNOS,HO-1andSODwere significantly up-regulated.Besides, theET-1expression of Se-GT group was significantly down-regulated, but no significant difference was found in the GT group.In particular, the expressions ofET-1,eNOS,HO-1andSODwere significantly different between Se-GT and GT groups.

Fig.2 GT and Se-GT on the histology of heart, liver and kidney and the expression of blood pressure-related genes.(A) Histological analysis of heart, liver and kidney; (B) ACE expression; (C) ET-1 expression; (D) eNOS expression; (E) HO-1 expression; (F) SOD expression; (D) iNOS expression.*P < 0.05, **P < 0.01,ns represents no significance.One-way analysis of variance and Tukey’s test were used to estimate statistical significance.

Fig.2 (Continued)

3.4 GT and Se-GT activated cardiac PI3K/Akt signaling pathway

The PI3K/Akt signaling pathway is one of the most studied signal transduction pathways in oxidative stress, inflammation and endothelial function [32].Based on this, we measured the expression of some key proteins in signal transduction including PI3K p85, pAkt,Akt and VEFGR2.The immunohistochemistry result was displayed in Fig.3.Compared with MC group, Se-GT significantly up-regulated the protein expressions of PI3K p85, pAkt, Akt and VEFGR2, GT had no significant effect on the pAkt protein expression (Fig.3).

Fig.3 GT and Se-GT activated the PI3K/Akt signaling pathway in heart tissue, including protein expressions of PI3K p85, pAkt, Akt and VEFGR2.*P < 0.05,**P ＜ 0.01, ns represents no significance.One-way analysis of variance and Tukey’s test were used to estimate statistical significance.

Fig.3 (Continued)

3.5 Effect of GT and Se-GT on gut microbial diversity

We used 16S rRNA high-throughput sequencing technology to evaluate the implication of intestinal microbiota with GT and Se-GT extracts.A total of 2 402 679 high-quality sequences were collected from 23 samples (n= 6 in each group, except for NCn= 5), and 4 527 distinct operational taxonomic units (OTUs) were observed.Bacterial richness, quantified by Chao index, was significantly lower in MC group compared with the NC or GT and Se-GT groups (Fig.4A),which suggests that GT and Se-GT mitigate the reductions in bacteria richness due to high-salt diet.Species richness and community diversity, quantified by Shannon index, were lower in the high-salt diet group than GT and Se-GT groups (Fig.4B).

Fig.4 GT and Se-GT influenced the diversity of intestinal flora in fecal samples.(A) Chao index; (B) Shannon index; (C) Venn diagram; (D) PCA analysis diagram;(E) B/F ratio.*P < 0.05, **P ＜ 0.01, ns represents no significance.One-way analysis of variance and Tukey’s test were used to estimate statistical significance.

The OTU richness in each group was compared.The intervention of GT and Se-GT alleviated the decline in the OTU richness.The Venn diagram of OTUs (Fig.4C) showed 485 mutual OTUs of all groups.The total OTUs in each group of NC, MC, GT and Se-GT were 2 039, 1 721, 1 739, and 1 774, respectively.In the PCoA analysis, the samples in the MC group showed a distinct change compared with those in the NC or GT and Se-GT-treated groups.The GT and Se-GT treatments reversed the gut microbiota composition and made it close to the NC group (Fig.4D).Moreover, Se-GT showed a better alteration effect.

3.6 Composition and abundance analysis at the phylum and genus level

To further assess the effect of GT and Se-GT on the composition and structure of intestinal flora, the community abundance analysis on phylum and genus levels was carried out.At the phylum level(Fig.5A), the intestinal flora of rats was mainly dominated by Firmicutes and Bacteroidetes.The high-salt diet caused the Bacteroides/Firmicutes (B/F) ratio imbalance (Fig.4F), and GT and Se-GT reversed this trend and normalized it.At the genus level, the top 10 genera wereLactobacillus, Unspecified_S24_, Unspecified_Clostridiales,Turicibacte,Allobaculum,Unspecified_Clostridiaceae,Unspecified_Lachnospiraceae,Bifidobacterium,Unspecified_Ruminococcaceae, and Unspecified_Peptostreptococcaceae (Fig.5B).The cladogram in (Fig.4C) and linear discriminant analysis (LDA)in (Fig.4D) indicate that GT intake specifically and significantly enriched the relative abundance ofParaprevotellaandBacteroides,whereas Se-GT was characterized by specific and significant enrichment forAllobaculumandBifidobacterium.Likewise, the significant selective enhancement forTuricibacter,Ralstonia,andEnterococcusgenera in NC group was found.Unexpectedly,Lactobacilluswas specifically enriched by high-salt diet.

Fig.5 GT and Se-GT changed gut microbiota structure at phylum and genus levels in rats in response to high-salt diet.(A) Percent community abundance diagram on phylum level; (B) Percent community abundance diagram on genus level; (C) LEfSe multi-level classification tree diagram; (D) LDA discriminant histogram.

Fig.5 (Continued)

3.7 Gut microbial OTU composition and its correlation with hypertension related indexes

Among the top 20 OTUs with the highest abundance, 16 and 18 different OTUs were greatly altered by GT and Se-GT respectively(Fig.6A).Accordingly, 12 and 13 of the OTUs regulated by the highsalt diet were reversed in response to GT and Se-GT treatments,respectively.Also, Spearman correlation analysis (Fig.6B) revealed that 9 OTUs were identified to have significant correlations with one or more parameters.Among the OTUs identified,Christensenella,Clostridium,Coprobacillus,Elusimicrobium,Dorea,PeptoniphilusandAgrobacteriumwere significantly and negatively correlated with hypertension.The OTU belonging toAdlercreutziashowed significantly and positively correlation with hypertension-related disorders.These genera may participate in the occurrence and development of hypertension induced by a high-salt diet.Notably,Lactobacilluswas significantly associated with hypertension and showed controversial effect on regulation of BP.

Fig.6 GT and Se-GT treatments reversed the imbalance of intestinal flora abundance of rats caused by high-salt diet.(A) Heatmap in the abundance of the top 20 OTUs of different treatment groups.The & and # indicate the less and more relative abundances of OTUs in GT or Se-GT groups in comparison with MC group,respectively.The * indicates that the OTU of the NC group that was changed by hypertension was reversed by GT or Se-GT treatment.(B) Spearman correlation analysis between the microbial genera in the intestinal flora and related parameters of hypertension.The parameters related to blood pressure were on the X-axis,and the bacteria genera were on the Y-axis.The R value was displayed in different colors, and the * sign indicates that there was a significant correlation between them.*P ＜ 0.05, **P < 0.01, ***P < 0.001.

4.Discussion

Many studies have shown that long-term consumption of highsalt diet could cause hypertension and intestinal flora disturbance [33].It is reported that dietary adjustment could significantly prevent and control the elevated BP [34].The researchers have found that tea exhibited good antihypertensive effect in clinical research [30].However, there were few reports on the regulating effect of Seenriched tea on hypertension induced by high-salt diet.This study aims to investigate the effect of Se-GT on BP and intestinal flora of rats fed a high-salt diet and its potential regulatory mechanism.

The current study showed that supplement with GT and Se-GT both had a remarkable inhibitory effect on BP and a protective effect on heart, liver and kidney tissues, but the regulatory effect on body weight was limited.Similar results were also found by Liang et al.[35],which suggested that the anti-hypertensive effect of tea was not conducted by regulation of body weight.However, our results also revealed the antihypertensive activity of Se-GT was better than that of GT, which could be explained in part by their different chemical compositions (Tables 1 and 2).The higher contents of tea polysaccharides (TPS), tea polyphenols, EGCG, EGC andL-theanine were found in Se-GT, while the higher contents of total flavonoids were found in GT.As a potential treatment option for metabolic diseases, many studies had shown that TPS had a good effect on antioxidative stress and anti-inflammatory, but there were few studies on TPS directly regulating hypertension [36].Relevant studies have shown that the hypertension could be controlled and improved to a certain extent when the oxidative stress and inflammation of hypertensive rats were further improved [37,38].It could be seen that the higher content of polysaccharides in GT extracts may indirectly relieve high blood pressure.Previous research revealed that GT polyphenols attenuated the increased BP in spontaneously hypertensive rats [39], and EGCG significantly decreased SBP in the spontaneously hypertensive rats (SHR) [40].In addition,Yokogoshi et al.[16]founded thatL-theanine administration significantly reduced the BP in SHR rats.The regular consumption of flavonoids could play a cardiovascular protective role and reduced the occurrence or progression of many cardiovascular diseases, especially hypertension [41].Thus, these active ingredients had a positive effect on alleviating hypertension, and the distribution of active compounds lowering BP was significantly different the distinction in the category and source of tea.

Variousin vivohypertensive models show the association of vascular inflammation and endothelial dysfunction with hypertension,whereas oxidative stress could result in inflammation, endothelial dysfunction and vascular dystonia.Additionally, pro-oxidation and pro-inflammation may also mediate the influx of VSMCs Ca2+and vasoconstriction, whereas the endothelial dysfunction could inhibit eNOS activity and reduce NO level, thereby inducing hypertension [42].Our results showed that the high-salt intake decreased the levels of SOD and GPX and increased the levels of MDA and CRP in serum,indicating an increasing state of oxidative stress and inflammation.Meanwhile, the high-salt diet led to a decrease in NO and eNOS levels and an increase in Ang II level, indicating more serious endothelial dysfunction.ACE is an important member of the reninangiotensin system (RAS), it catalyzes Ang I into Ang II with high vasoconstrictor activity and promotes the proliferation of vascular smooth muscle cells, thereby inducing hypertension [43].Vascular superoxide anion can be enhanced by Ang II-induced hypertension,and this is mainly attributed to the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase by Ang II, the activated NADPH oxidase can further influence vasomotor tone [44].It has been reported that ET-1 and eNOS were important members in the endothelial function system.Accurately, ET-1 is a potent vasoconstrictor peptide with pro-oxidation and pro-inflammation and can participate in the development of endothelial dysfunction [45],whereas eNOS can oxidizeL-arginine to produceL-citrulline and NO.eNOS can also participate in the development of vascular endothelial dysfunction related to hypertension [46].In addition, the abnormal iNOS (the same family as eNOS) activity can also up-regulate the arginase activity, and arginase can reduce the NO formation by competing witheNOS forL-arginine and promote superoxide formation by uncoupling eNOS[47].Furthermore, the HO-1 with anti-oxidation was reported to improve endothelium-dependent by augmenting the activation of the Ca2+-activated K+channel [48].More importantly, ET-1 also induces the redistribution of eNOSfrom the plasma membrane to the mitochondria through the phosphorylation of eNOS, thereby indirectly decreasing the endothelial vasodilation and the NO bioavailability [49].Likewise, our results showed that excessive salt intake enhanced the endothelial dysfunction state of rats and the elevated mRNA expression ofACE,ET-1andiNOSand the decreased mRNA expression ofeNOS,SODandHO-1.Therefore,oxidative stress leads to a decrease in NO bioavailability causing endothelial dysfunction and vasoconstrictive effect.

This finding supports previous research which links tea and its active compounds and hypertension.Furthermore, our research also showed that GT and Se-GT significantly activated the PI3K-Akt signaling pathway, enhanced eNOS activity, and promoted NO production,which was consistent with the previous report that tea extract could activate the Akt pathway to improve endothelial function [30].Tea and its secondary metabolites may regulate BP through the following mechanisms: relaxation of smooth muscle contraction,enhancement of endothelial nitric oxide synthase activity, reduction of vascular inflammation, and inhibition of renin activity and antivascular oxidative stress [30].The PI3K-Akt pathway is a significant signal pathway in the activation of eNOS.Studies have shown that black tea polyphenol-induced eNOS activation was dependent upon the PI3K/Akt pathway [50].Specifically, catechin stimulates NO synthesis via the formation of an active complex between eNOS, AKT,and HSP90.Moreover, Fyn (a member of the Src family), mediates the EGCG-induced PI3K/Akt-mediated activation of eNOS [19].In addition, vascular endothelial growth factor A (VEGFA) can activate the PI3K/Akt signaling pathway and increase NO production by activation of eNOS through calmodulin (CaM) binding [51].Interestingly, we found that the regulation effect of Se-GT was better than that of GT.A possible explanation for this might be that the enrichment of Se in Se-GT changes or enhances the activity of tea polyphenols, tea polysaccharides, EGCG and theanine in Se-GT [52].

Several studies have proposed a relationship between the gut microbiome and hypertension.Specifically, alterations to the composition of gut microbiota, as well as a reduction of bacterial richness and diversity, have been correlated with hypertension [53].We also observed that high-salt diet had a certain effect on the composition of intestinal microbiota.Recent studies have shown that high-salt diet alone caused a change in microbiota profile,characterized by the loss of their diversity [54].The supplement of GT polyphenols ameliorated the disorder of gut microbiota and mitigated intestinal inflammation in canines with high-fat-diet-induced obesity [55].Consistent with these reports, our results (Figs.4A-4D)show the richness and diversity (includingαandβ) of the intestine flora of hypertensive rats induced by high salt diet decreased significantly, while the supplementation of GT and Se-GT significantly reversed the dysbiosis of microbial richness and diversity, especially Se-GT.In addition to GT and Se GT, our lab also indicated that Liubao tea could increase the richness and diversity of intestinal microbiota [23].All these proved a regulation effect of tea on intestinal flora, which might be attributed to the rich active components in tea [56].

Studies at the phylum level have shown that a high-salt diet would increase the colonization of Firmicutes, thereby increasing the F/B ratio of [57].Moreover, it was reported that the F/B ratio was significantly decreased in spontaneously hypertensive rats [58].In this work, the significant increased F/B ratio, which was reported to be correlated with the improvement of metabolic disorder [59], was observed in the gut microbiota of GT and Se-GT-treated groups.High-salt diet could reduce the abundances ofBifidobacteriumandLactobacillus[57].Unexpectedly, our study showed thatLactobacilluswas specifically enriched by high-salt diet.It is known thatLactobacillushas been associated with a wide range of purported health benefits, such as antimicrobial, anti-oxidative, and anti-diabetic effects [60].Most notably, studies have shown that there were higher levels ofLactobacillusin hypertensive rats [61].Furthermore, theLactobacillusis a taxonomically complex bacteria and is composed of over 170 species.Their antimicrobial susceptibilities are poorly defined in part because of their taxonomic complexity [62].It was reported that treatment withLactobacillusincreased body weight in diabetic rats [63].Consequently, it is possible thatLactobacillusenhanced the prevalence of hypertension induced by a high-salt diet.The relative abundance ofPrevotellawas negatively related with the insulin-resistance and blood glucose level [64].Our data also showed that GT significantly enriched the abundance of theParaprevotellaandBacteroides.Bacteroideswas recently shown to prevent obesity and improve insulin sensitivity in mice [65].In addition, the abundance ofAllobaculumandBifidobacteriumwas specifically and greatly enriched by Se-GT.Allobaculumwas positively related to the anti-inflammatory effect and the production of short-chain fatty acids (SCFAs), whereas SCFAs have been reported to exhibit many beneficial functions [66].Li et al.[67]reported the decreasedBifidobacteriumin the guts of hypertensive patients [68].Clinical trials conducted by Li et al.[67]showed that patients with hypetension had a reduced intestinalBifidobacterium.Our study showed that supplement of Se-GT could significantly increase the intestinalBifidobacteria[69].The changes in the abundance of intestinal flora might be related to the active ingredients in GT and Se-GT.It was reported that a diet rich in tea polyphenols led to a decrease in Firmicutes and an increase in Bacteroides[70].EGCG treatment resulted in stimulation of the beneficial bacteria likeBacteroides,Christensenellaceae, andBifidobacterium.Additionally, the abundance of pathogenic bacteria includingFusobacteriumvarium,Bilophila, and Enterobacteriaceae were inhibited [71].Furthermore,studies have shown thatL-theanine increased the proportion ofPrevotella,Lachnospira,andRuminococcuswhile increasing the total SCFAs in the feces [72].Flavonoids in tea could regulate intestinal immunity, which included regulating T cell differentiation, changing the intestinal microbiota and regulating cytokines [73].Thus, the antihypertension effects of GT and Se-GT might be attributed to the upregulation of beneficial bacteria in the gut microbiota.

The abundance alteration of the top 20 OUTs was in line with the above results.The GT and Se-GT treatment significantly changed 16 and 18 different OTUs, respectively.More accurately,compared with MC group,Allobaculum,Turicibacter,Bacteroidesand Lachnospiraceae were significantly increased in the GT and Se-GT groups.AllobaculumandTuricibacterwere reported to show good anti-inflammatory effect, which were related to the production of the SCFA [66].Lachnospiraceae is an abundant obligate anaerobic member of the healthy human microbiota and can affect the host by producing SCFA, converting primary bile acids into secondary bile acids, and promoting colonization resistance to intestinal pathogens [74].Compared with the MC group,BifidobacteriumandRuminococcuswere increased in rats of GT and Se-GT groups.BifidobacteriumandRuminococcusare the main microbial members negatively associated with the severity of fibrosis in non-obese subjects [75].More notably, Se-GT treatment significantly increased the abundance of Peptostreptococcacea andEnterococcus, but their abundance decreased in GT treatment group.Peptostreptococcacea is a microbial re-colonization bacteria of mice characterized by the oxygen-tolerant function [76].Previous report has shown thatEnterococcuswas a probiotic which had anti-inflammatory activity and hypocholesterolemic effect [77].Simultaneously, the abundance ofCoprococcuswas greatly decreased in the rats of GT and Se-GT groups compared with MC group, its relative abundance was negatively correlated with the content of SCFAs [78].Spearman correlation analysis indicated that most of the microbes belonging toChristensenella,Clostridium,Coprobacillus,Elusimicrobium,Dorea,PeptoniphilusandAgrobacteriumshowed a significantly negative correlation with BP.Among the phylotypes mentioned above,Christensenellawas reported to be enriched in healthy host and could ameliorate insulin resistance and protect mice from intestinal diseases [79].Besides, a previous study showed that the relative abundance ofCoprobacillus,DoreaandElusimicrobiumwere negatively correlated with inflammation [80].Specifically,Coprobacillusreduced re-activation of the immune system and inflammatory events.Doreawas a beneficial bacteria associated with anti-inflammation [81].Elusimicrobiumis a strong opposite candidate for inflammatory cytokine regulation and is highly negatively correlated with IL-6 and IL-17 [82].Clostridiumhas been reported to show good anti-glycopeptide effect [83].Therefore, our results indicated that these genera associated with hypertensionrelated metabolic disorders might participate in the composition in the intestinal microbiota, thereby exerting its role in regulating BP.

5.Conclusion

In this study, we found that GT and Se-GT could significantly activated the PI3K/Akt signaling pathway, regulated the mRNA expression related to BP, and reduced the levels of BP, oxidative stress, inflammation and endothelial dysfunction, as well as protected the heart, liver, kidney tissues.However, Se-GT showed a better effect than GT on preventing hypertension and metabolic disorders induced by high-salt diet.Additionally, GT and Se-GT could regulate the composition and diversity of the intestinal flora.Specifically, GT and Se-GT could increase the abundance of beneficial bacteria and decrease the harmful and conditional pathogenic bacteria to prevent hypertension.

Conflict of interest

The authors declared no conflict of interest.

Acknowledgement

The authors are grateful for financial sponsored by the National Key R&D Program of China (No.2018YFC1604405), Fund of Shanghai Engineering Research Center of Plant Germplasm Resources (No.17DZ2252700), and Research on the health function of tea and deep-processed products in preventing metabolic diseases(No.C-6105-20-074).

Appendix A.Supplementary data

Supplementary data associated with this article can be found in the online version, at http://doi.org/10.1016/j.fshw.2021.12.031.