| Objective: To explore the medication rule of coincidence of hypertension and functional gastrointestinal diseases in elderly patients by collecting and analyzing the effective cases of hypertension and functional gastrointestinal diseases.In the part of experimental study in vivo,we observed the changes of intestinal microorganisms in spontaneously hypertensive rats(SHRs)and evaluated the effects and explored the mechanism of Eucommia ulmoides & Tribulus terrestris on intestinal microbiota in aged SHRs.Through the fecal bacteria transplantation,the role of intestinal microbiota in the process of hypertension was further studied.We hope to provide experimental support for Eucommia ulmoides & Tribulus terrestris on antihypertension from the perspective of regulating intestinal flora in future.Methods:1.Study on the medication rule of coincidence of hypertension and functional gastrointestinal diseases in the elderly: 157 cases of coincidence of senile hypertension and functional gastrointestinal disease were collected.Based on the traditional Chinese Medicine Inheritance Assistant Platform,the medication rule and prescription regularity of Professor Yang Chuanhua in treating coincidence of hypertension and functional gastrointestinal disease was analyzed by using modern data mining concepts and methods.2.Changes of intestinal microbiota in SHRs and the effect of valsartan on intestinal microorganisms in aged SHRs: Fourteen 18-month-old male SHRs and seven18-month-old male WKY rats were selected.SHRs were randomly divided into valsartan administration group and disease model group.The above three groups were gavaged for 8weeks and the body weight and blood pressure were measured weekly.Serum levels of N-terminal pro brain natriuretic peptide(NT-pro BNP),neuropeptide Y(NPY),C reactiveprotein(CRP),angiotensin II(Ang II)and norepinephrine(NE)were detected by ELISA,and the expression levels of IKK-a,IKK-beta,IKBalpha and NF-kappa B p65 in thoracic aorta were detected by real-time fluorescence quantitative PCR(q RT-PCR).The stool samples were collected by aseptic principle and the amplicon information of 16 S V4 region was collected and analyzed.3.The effect of Eucommia ulmoides & Tribulus terrestris on intestinal microbiota in aged SHRs: A total of twenty-eight 18-month-old male SHRs were randomly divided into control group and Eucommia ulmoides & Tribulus terrestris high,medium and low dose group.Four groups were gavaged for 8 weeks.Blood pressure and body weight were measured weekly.Eight weeks later,serum level of NT-pro BNP,NPY,CRP,Ang II and NE were measured through inferior vena cava blood sampling,and the expression levels of IKK-?,IKK-?,IKB? and NF-?B p65 were determined by real-time fluorescence quantitative PCR(q RT-PCR).Stool samples were taken from rats under the aseptic operation principle,and the amplicon information of 16 S V4 region was collected and analyzed.The content of short chain fatty acids(SCFAs)in stool samples was detected by gas chromatography.4.Fecal bacteria transplantation experiment: Fourteen 18-month-old male SHRs were randomly divided into two groups: ET?SHR(fecal bacteria from middle dose group of Eucommia ulmoides & Tribulus terrestris were transplanted to SHR rats)group and N?SHR(given with isodose saline)group;Fourteen 18-month-old male WKY rats were randomly divided into two groups :SHR?WKY(fecal bacteria from SHR rats)group and N?WKY(given with isodose saline)group.After 8 weeks of oral-upper gavage administration,the levels of NT-pro BNP,NPY,CRP,Ang II and NE were detected by ELISA,blood routine,urine routine,liver and kidney function were detected by chemical method,stool samples were taken by aseptic principle,and the amplicon information of16 S V4 region was collected and analyzed.Results:1.Study on the medication rule of coincidence of hypertension and functional gastrointestinal diseases in the elderly: Through analysis of the data of 157 cases,Angelica sinensis,Astragalus membranaceus,Ligusticum chuanxiong,Coptis chinensis,Alisma orientalis,Tribulus terrestris,Scutellaria baicalensis,Poria cocos,Achyranthes bidentata and Eucommia ulmoides were commonly used in the first ten of all drugs.According to the analysis of using frequency,drugs play the role of invigorating deficiency,clearing awayheat,calming liver and quenching wind,activating blood circulation and removing blood stasis in the forefront.The most frequently used herb couples were Tribulus terrestris-Eucommia ulmoides.2.Changes of intestinal microbiota in SHRs and the effect of valsartan on intestinal microorganisms in aged SHRs: The body weight of rats in valsartan group were significantly lower than that of SHR control group(253.21 +21.65 vs 298.69±17.02 g,P <0.05).Valsartan decreased the blood pressure of SHRs(systolic pressure / diastolic pressure: 170.33±10.74/113.22±7.39 vs 203.75±11.42/136.77±8.01 mm Hg,P<0.05)as same as serum NT-pro BNP,NPY,NP-pro BNP and NPY(NT-pro BNP: 304.11±23.62 vs471.67±61.87ng/L;NPY:1896.39±14.44 vs 3826.72±68.35ng/L;CRP:37.73±16.27 vs68.51 ± 9.47?g/ml,P < 0.05).However,valsartan did not recover all the changes in intestinal microbiota caused by hypertension.The bacteria biomarker at genus level was Lactobacillus.3.The effect of Tribulus terrestris-Eucommia ulmoides on intestinal microbiota in aged SHRs: The blood pressure of rats in high and medium dose group were significantly lower than that of SHR control group(systolic pressure / diastolic pressure:170.33 ±10.74/114.03±6.62 mm Hg,171.57±10.35/112.36±7.32 mm Hg vs 203.75±11.42/136.77±8.01 mm Hg,P<0.05).There was no significant change in blood pressure of rats in low dose group,and no difference between high and medium dose group in blood pressure.Medium dose Tribulus terrestris-Eucommia ulmoides significant decreased NT-pro BNP,NPY,CRP and Ang II levels(NT-pro BNP: 321.47±31.45 vs 471.67±61.87ng/L;NPY:2001.87±26.65 vs 3826.72±68.35 ng/L;CRP: 42.37±12.97 vs 68.51±9.47?g/ml;Ang II: 481.92 ± 21.45 vs 591.61 ± 88.72 ng/L,P < 0.05),while high dose Tribulus terrestris-Eucommia ulmoides significant decreased NPY level(NPY:2977.61±64.51 vs3826.72 ± 68.35 ng/L,P < 0.05).High and medium dose Tribulus terrestris-Eucommia ulmoides significantly changed the expression of key genes of NF-?B pathway in thoracic aorta,while low dose had no significant effect on it.The composition,both alpha diversity and beta diversity of intestinal microbiota were significantly different.Anaeroplasma was microbiota biomarker of medium dose group at genus level.High dose Tribulus terrestris-Eucommia ulmoides significantly increased the levels of acetic acid and propionic acid(acetic acid:1976.32±42.36 vs 1349.67±74.31?g/g,propionic acid:755.26±31.21 vs 358.76±40.83?g/g,P<0.05),the level of acetic acid,propionic acid and butyric acid was significantly increased by medium dose(acetic acid: 2346.78±37.91 vs 1349.67±74.31?g/g,propionic acid: 759.37±52.89 vs 358.76±40.83?g/g,butyric acid:1783.36±46.52 vs 389.21±48.34?g/g,P<0.05),low dose significantly increased the levels of butyric acid,isobutyric acid,valeric acid and isovaleric acid(butyric acid: 862.37±60.18 vs 389.21±48.34?g/g,isobutyric acid: 112.37±9.87 vs 52.17±9.16?g/g,valeric acid:193.27±11.36 vs 70.31±9.67?g/g,isovaleric acid:150.78 ±10.36 vs 51.52±9.37?g/g,P< 0.05).The abundance of Anaeroplasma had negative linear correlation with CRP concentration and NF-?B p65 level.4.Fecal microbiota transplantation(FMT)experiment: After fecal bacteria transplantation,compared with N?WKY group,the blood pressure level of SHR?WKY group was increased(systolic/diastolic blood pressure before and after transplantation:144.73±11.34/83.36±9.73 mm Hg vs 164.83±12.98/102.24±9.47 mm Hg,P<0.05)as well as NT-pro BNP and CRP levels(NT-pro BNP:461.42±58.32 vs 229.70±21.45ng/L,CRP:53.15±8.37 vs 26.12±3.92?g/ml,P<0.05).Compared with N?SHR group,the blood pressure level of ET?SHR group was decreased(systolic/diastolic blood pressure before and after transplantation: 198.36±10.21/131.90±7.83 mm Hg vs 172.23±11.84/117.03±6.62 mm Hg,P < 0.05),as well as NT-pro BNP and CRP levels(NT-pro BNP:331.27±57.83 vs 460.55±63.96ng/L,CRP: 41.37±8.27 vs 67.10±9.30?g/ml,P<0.05).Both Prevotellaceae and Prevotella which were considered to be closely related to hypertension were found in SHR?WKY group.Mollicutes,which is the bacteria biomarker of medium dose group of Tribulus terrestris-Eucommia ulmoides was found in ET?SHR group.Conclusion: Both senile hypertension and functional gastrointestinal diseases have the pathological state of Qi deficiency,blood stasis and phlegm turbidity from the viewpoint of traditional Chinese medicine.In the treatment,Professor Yang focused on invigorating kidney and spleen,promoting blood circulation and regulating qi,in which Tribulus terrestris-Eucommia ulmoides is the most frequently used herb couple according to the principle.In the experiments in vivo,Tribulus terrestris-Eucommia ulmoides effectively decreased blood pressure and inflammation in old SHRs,and that Tribulus terrestris-Eucommia ulmoides demonstrated better advantages in maintaining intestinal flora diversity than valsartan.Optimization of intestinal flora composition and diversitywas hypothesized as one of the mechanism of Tribulus terrestris-Eucommia ulmoides.The strains related to hypertension and the antihypertensive effect of Tribulus terrestrisEucommia ulmoides could be transplanted through FMT. |
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