Application of Ackermann muciniphile and outer membrane protein Amuc1100 of Ackermann muciniphile in treatment of hypertension

Abstract

The invention discloses an application of Ackermann muciniphile and an outer membrane protein Amuc1100 of Ackermann muciniphile in treatment of hypertension. The outer membrane protein Amuc1100 is a protein of which the amino acid sequence is as shown in SEQ ID NO: 4. Experiments prove that the outer membrane protein Amuc1100 can enhance the intestinal barrier function of a mouse, blood pressure is reduced by remodeling the cardiovascular system and improving the intestinal microenvironment, and the blood pressure comprises systolic pressure and diastolic pressure. The invention has important application value in prevention and / or treatment of hypertension.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to application of Akkermansia muciniphila and its outer membrane protein Amuc_1100 in treating hypertension. Background Art

[0002] In recent years, the incidence of cardiovascular disease (CVD) has continued to rise, and the prevention of CVD has become an important public health strategy to improve the health level and life expectancy of the people. Since hypertension is one of the main causes of CVD in my country, the prevention and treatment of hypertension has become the primary task of CVD prevention and treatment. However, since hypertension is mostly primary, the cause is still unclear, and there is a lack of effective etiological prevention strategies, improving the cure rate and control rate of hypertensive patients has always been the main strategy to control the harm of hypertension.

[0003] In recent years, research on intestinal flora has continued to emerge, and the impact of intestinal flora on human health has received increasing attention. More and more scholars believe that intestinal flora imbalance is closely related to the risk factors of hypertension. Intestinal microorganisms refer to the microbial community existing in the human intestine, including bacteria, fungi, archaea, and protozoa (even viruses) and other microorganisms. Early studies have shown that the diversity of intestinal flora in patients with hypertension is reduced, and the number of beneficial bacteria such as Faecalibacterium prausnitzii, Roseburia, Coprococcus, Butyricimonas, Butyricimonas, and rectal true bacteria that produce short-chain fatty acids (SCFAs) is significantly reduced. At the same time, the number of harmful bacteria with increased inflammation and immune response, such as Prevotella, Klebsiella, and Fusobacterium, has increased, and these genera may be key factors in causing hypertension. In addition, intestinal flora imbalance is closely related to diet, and changing lifestyle and dietary structure plays an important role in intervening in hypertension. Therefore, readjusting the structure of intestinal flora by adjusting dietary structure, supplementing prebiotics, probiotics, and performing fecal microbiota transplantation may become a new way to treat hypertension.

[0004] As an emerging probiotic, Akkermansia muciniphila (Akk bacteria) has been widely studied for its use in major diseases such as diabetes, obesity, and immune system diseases, and its abundance level has an important impact on the body's physiological functions. Summary of the invention

[0005] The purpose of the present invention is to treat hypertension or improve blood pressure levels.

[0006] The present invention firstly protects the application of outer membrane protein Amuc_1100 and / or Akkermansia muciniphila ATCC BAA-835, which can be A1) or A2) or A3):

[0007] A1) Preparation of a medicament for the treatment of hypertension;

[0008] A1) preparing health products for improving blood pressure levels;

[0009] A3) Preparation of a medicament for lowering blood pressure.

[0010] In the above application, the outer membrane protein Amuc_1100 can be the following a1) or a2) or a3):

[0011] a1) the amino acid sequence is the protein shown in SEQ ID NO: 4;

[0012] a2) a fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of the protein shown in SEQ ID NO: 4;

[0013] a3) A protein associated with lowering blood pressure obtained by replacing and / or deleting and / or adding one or more amino acid residues of the protein shown in a1) or a2).

[0014] The specific labels of the proteins in a2) above are shown in Table 1.

[0015] Table 1. Sequences of tags

[0016] Label Residue sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHQ c-myc 10 EQKLISEEDL HA 9 YPYDVPDYA

[0017] In the protein described in a3) above, the substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0018] The protein in a3) above can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0019] The coding gene of the protein in a3) above can be obtained by deleting one or several codons of amino acid residues in the DNA sequence shown in SEQ ID No: 3, and / or performing missense mutation of one or several base pairs, and / or connecting the coding sequence of the tag shown in Table 1 to its 5′ end and / or 3′ end.

[0020] In the above application, the Akkermansia muciniphila ATCC BAA-835 is a live bacterium, an inactivated bacterium, or a combination of live and inactivated bacteria.

[0021] The present invention also protects the use of a nucleic acid molecule encoding any of the outer membrane proteins Amuc_1100 described above, which may be A1) or A2) or A3):

[0022] A1) Preparation of a medicament for the treatment of hypertension;

[0023] A1) preparing health products for improving blood pressure levels;

[0024] A3) Preparation of a medicament for lowering blood pressure.

[0025] In the above application, the nucleic acid molecule encoding any of the above outer membrane proteins Amuc_1100 can be a DNA molecule as shown in b1) or b2) or b3) or b4) below:

[0026] b1) the coding region is the DNA molecule shown in SEQ ID NO: 3;

[0027] b2) the nucleotide sequence is a DNA molecule shown in SEQ ID NO: 3;

[0028] b3) a DNA molecule having 75% or more identity with the nucleotide sequence defined in b1) or b2), derived from Akkermansia muciniphila ATCC BAA-835 and encoding any of the outer membrane proteins Amuc_1100 described above;

[0029] b4) a DNA molecule which hybridizes with the nucleotide sequence defined in b1) or b2) under stringent conditions, is derived from Akkermansia muciniphila ATCC BAA-835 and encodes any of the outer membrane proteins Amuc_1100 described above.

[0030] The 75% or more identity may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity.

[0031] The stringent conditions are hybridization and membrane washing twice at 68°C in a 2×SSC, 0.1% SDS solution for 5 min each time, and hybridization and membrane washing twice at 68°C in a 0.5×SSC, 0.1% SDS solution for 15 min each time.

[0032] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0033] The present invention also protects the use of a composition containing any of the above outer membrane proteins Amuc_1100 and / or any of the above Akkermansia muciniphila ATCC BAA-835, which can be A1) or A2) or A3):

[0034] A1) Preparation of a medicament for the treatment of hypertension;

[0035] A1) preparing health products for improving blood pressure levels;

[0036] A3) Preparation of a medicament for lowering blood pressure.

[0037] In the above application, the composition containing any of the above outer membrane proteins Amuc_1100 and / or any of the above Akkermansia muciniphila ATCC BAA-835 can be a health product composition, a pharmaceutical composition or a probiotic composition.

[0038] Preferably, the composition containing any of the above outer membrane proteins Amuc_1100 and / or Akkermansia muciniphila ATCC BAA-835 may further include a second active ingredient. The second active ingredient may be a drug different from any of the above Akkermansia muciniphila ATCC BAA-835 and / or any of the above outer membrane proteins Amuc_1100.

[0039] Preferably, the probiotic composition may further contain probiotics different from any of the above-mentioned Akkermansia muciniphila ATCC BAA-835.

[0040] Preferably, the composition containing any of the above outer membrane proteins Amuc_1100 and / or Akkermansia muciniphila ATCC BAA-835 may also contain one or more of telmisartan, captopril, valsartan and amlodipine.

[0041] In the above application, the hypertension may be renal hypertension, spontaneous hypertension, metabolic hypertension caused by combined dietary factors, or isolated systolic hypertension.

[0042] In the above applications, the drug may be a medicine, and its dosage form may be tablets, capsules, granules, pills, ointments, solutions, suspensions, emulsions, creams, sprays, drops, patches or tube feeding preparations.

[0043] In the above application, the health care product may be a health food, and its type may be a dairy product or a fermented cereal food.

[0044] In any of the above-mentioned applications, the medicine or the health product may contain an effective amount of any of the above-mentioned outer membrane proteins Amuc_1100 and / or any of the above-mentioned Akkermansia muciniphila ATCC BAA-835.

[0045] In any of the above applications, the subject of the drug or the health product can be a mammal. Preferably, the mammal can be a human or a mouse.

[0046] In any of the above applications, lowering blood pressure may be lowering systolic blood pressure and / or diastolic blood pressure.

[0047] In any of the above applications, the blood pressure reduction may be to reduce the blood pressure level of angiotensin II-induced hypertension.

[0048] In any of the above uses, the hypertension may also be induced by angiotensin II.

[0049] The inventors of the present application obtained the outer membrane protein Amuc_1100 with the amino acid sequence shown in SEQ ID NO: 4 from Akkermansia muciniphila (ATCC BAA-835). It was further proved through experiments that the outer membrane protein Amuc_1100 and Akkermansia muciniphila (ATCC BAA-835) can enhance the intestinal barrier function of mice, and reduce blood pressure by remodeling cardiovascular system and improving intestinal microenvironment, including systolic and diastolic blood pressure. The present invention has important application value in treating hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 These are the LEfSe analysis results of the 16S rDNA sequencing data and metagenomic data in Example 1.

[0051] Figure 2 These are the measurement results of systolic blood pressure (SBP) and diastolic blood pressure (DBP) of AngII hypertension group mice and control group mice in step 4 of Example 2.

[0052] Figure 3 The staining and statistical results of aortic tissue and colon tissue of AngII hypertension group mice and control group mice in step 5 of Example 2.

[0053] Figure 4 These are the Masson staining results of the aorta tissue and the PAS staining results of the colon tissue in step 3 of Example 3.

[0054] Figure 5 These are the statistical results of aortic wall thickness, aortic fibrosis area ratio, colon muscle layer thickness and goblet cell number under different treatments in step 3 of Example 3. DETAILED DESCRIPTION

[0055] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0056] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0057] The healthy subjects and hypertensive patients in the following examples all gave their informed consent to the experiment.

[0058] Example 1: Study on the flora of fecal samples from patients with hypertension

[0059] 1. In order to explore the differences in intestinal flora between hypertensive patients and healthy people, the inventors of the present application recruited 12 healthy subjects (systolic blood pressure (SBP) <120 mmHg and diastolic blood pressure (DBP) <80 mmHg) and 12 hypertensive patients (SBP>130 mmHg or DBP>80 mmHg and not receiving antihypertensive treatment). The 12 healthy subjects formed a healthy control group, i.e., the Con group. The 12 hypertensive patients formed a hypertension group, i.e., the HTN group. Except for the changes in blood pressure, the baseline data of the Con group and the HTN group (see Table 2) were basically consistent and there was no significant difference between the two groups.

[0060] Table 2

[0061] Health Group Hypertension group P-value Number of samples (pieces) 12 12 Female proportion (%) 66 42 Age, years (SEM) 47.83(2.32) 49.67(2.37) 0.587 SBP, mmHg (SEM) 115.33(1.19) 139.5(1.15) <0.001 DBP, mmHg (SEM) 73.83(1.27) 92.00(2.42) <0.001 <![CDATA[BMI,kg / m 2 (S.E.M)]]> 23.85(0.87) 26.04(0.60) 0.052 HBAC-1(SEM) 5.95(0.19) 5.72(0.14) 0.341 FBG, mmol / 1 (SEM) 6.35(0.92) 5.52(0.18) 0.393 TC, mmol / l (SEM) 5.37(0.26) 5.51(0.20) 0.677 TG, mmol / l (SEM) 1.21(0.12) 1.60(0.24) 0.178 HDL-C, mmol / l (SEM) 1.67(0.11) 1.40(0.07) 0.057 LDL-C, mmol / l (SEM) 3.14(0.17) 3.36(0.14) 0.353

[0062] Note: SEM: standard error; SBP: systolic blood pressure; DBP: diastolic blood pressure; BMI: body mass index; FBG: fasting blood glucose; TC: total cholesterol; TG: triglycerides; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; HBAC-1 is glycosylated hemoglobin.

[0063] 2. Extract genomic DNA from stool samples of 12 healthy subjects and 12 hypertensive patients in step 1 respectively.

[0064] 3. The genomic DNA obtained in step 2 was sequenced by 16S rDNA, and then the 16S rDNA sequencing data was subjected to linear discriminant effect size (LEfSe) analysis to obtain the communities and species that contributed significantly to the distinction between the hypertension group and the healthy group. The LEfSe analysis was as follows: the unpaired Kruskal-Wallis test was used to test the significant abundance differences at a specific taxonomic level, and then linear discriminant analysis (LDA) was used to evaluate the effect of the abundance of each species on the difference.

[0065] The results showed that the abundance of Akkermansia was significantly reduced in the hypertensive group compared with the healthy group ( Figure 1 (A).

[0066] 4. Since the sequencing depth of 16S rDNA in the V3-V4 region was not enough to cover the species level of the intestinal flora, the genomic DNA obtained in step 2 was subjected to metagenomic sequencing after excluding abnormal samples (16S rRNA depth not exceeding 60,000 and / or samples with possible partial degradation of feces), and then the metagenomic data was subjected to LEfSe analysis to obtain the communities and species that contributed significantly to the distinction between the hypertension group and the healthy group. The LEfSe analysis was as follows: the unpaired Kruskal-Wallis test was used to test the significant abundance differences at a specific taxonomic level, and then the linear discriminant analysis (LDA) was used to evaluate the influence of the abundance of each species on the difference.

[0067] The results showed that compared with the healthy group, the number of Akkermansia muciniphila (Akk bacteria) in the hypertension group was significantly reduced ( Figure 1 (middle B).

[0068] Example 2. Preparation and detection of angiotensin II (AngII) hypertensive mouse model

[0069] 1. Take an implantable osmotic pressure capsule (product of Alzet, model 1004, delivery rate 0.11 μl / hr) and prepare an AngII capsule pump and a NaCl capsule pump according to the instructions.

[0070] 2. Take 10 SPF-grade C57BL / 6J mice of 7-8 weeks old and weighing 20±5g (hereinafter referred to as mice, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), anesthetize them with 0.7% pentobarbital solution (solvent is normal saline), then make a small incision of about 5mm in length on the back of the mouse's neck, insert the AngII capsule pump, and finally suture the back of the mouse. The AngII capsule pump injects AngII through an osmotic micropump for 4 weeks, and the AngII injection dose is 800ng / (kg·min). After the mouse wakes up, the AngII hypertensive mouse model is prepared.

[0071] The AngII hypertension group (referred to as the AngII group) consisted of 10 AngII hypertension mouse models.

[0072] 3. Take 10 SPF C57BL / 6J mice aged 7-8 weeks and weighing 20±5g, anesthetize them with 0.7% pentobarbital solution (solvent is normal saline), then make a small incision about 5mm long on the back of the mouse's neck, insert the NaCl capsule pump, and finally suture the back of the mouse. The NaCl capsule pump injects 0.9% NaCl through an osmotic micropump for 4 weeks. After the mouse wakes up, the control mouse is prepared.

[0073] The control group consisted of 10 control mice.

[0074] 4. Non-invasive blood pressure measurement in mice (tail-cuff pressure method)

[0075] After completing steps 2 and 3, mice in the AngII group and the control group were taken, and the systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured using the BP-300A fully automatic non-invasive BP measurement system (Chengdu Taimeng Software Co., Ltd.). Each mouse was measured three times with an interval of more than 30 seconds. The average of the three measurements was taken as the measurement result, that is, the measurement result of week 0. The measurement was carried out every week for 4 consecutive weeks.

[0076] Due to the influence of environmental factors, the blood pressure of mice generally fluctuates within 10 mmHg. Therefore, it is necessary to measure under the same objective conditions as much as possible to reduce measurement errors and ensure the stability of measurement results.

[0077] The measurement results are shown in Figure 2(A is the statistical result of systolic blood pressure, B is the statistical result of diastolic blood pressure, # represents P value <0.01). The results showed that the blood pressure of mice in the AngII hypertension group gradually increased, and the systolic blood pressure reached 154.6±2.2 mmHg and the diastolic blood pressure reached 132.8±2.5 mmHg at the 4th week, which were significantly higher than the systolic blood pressure (108.4±3.4 mmHg) and diastolic blood pressure (94.85±3.9 mmHg) of the control group at the same time point, respectively. This proves that AngII can induce an increase in blood pressure in mice and prepare a mouse hypertension model.

[0078] 5. After completing steps 2 and 3, the mice were killed and the aortic and colonic tissues were obtained. The aortic tissue was subjected to Masson staining (for specific steps, please refer to the following literature: Liu Yang, Chen Guihai. Effects and mechanisms of Hanjing Decoction on phenotypic transformation of vascular smooth muscle cells, inflammation, and oxidative stress in rats with cold-coagulation type hypertension [J]. Journal of Jinan University (Natural Science and Medicine Edition), 2023, 44(04): 347-357.). The colonic tissue was subjected to PAS staining (for specific steps, please refer to the following literature: Guo Weiling. Effect and mechanism of inosine-producing intestinal bacteria on the alleviation of colitis [D]. Jiangnan University, 2023.).

[0079] Results Figure 3 (A is the result of Masson staining of the aorta and PAS staining of the colon, B is the statistical result, the data in the figure are P values, and P values ​​less than 0.05 indicate significant differences). The results showed that the aorta and colon tissues of the mice in the AngII hypertension group showed signs of hypertrophy, while the aorta and colon tissues of the mice in the control group showed no significant changes; compared with the mice in the control group, the aortic wall of the mice in the AngII group was significantly thickened, and the proportion of aortic fibrosis area was significantly increased; compared with the mice in the control group, the thickness of the colon muscle layer of the mice in the AngII group was significantly increased, and the proportion of goblet cells was significantly reduced.

[0080] These results indicate that AngII-induced aortic and colon tissue remodeling in mice is affected by elevated blood pressure and altered intestinal permeability.

[0081] Example 3: Application of Akkermansia muciniphila and its outer membrane protein Amuc_1100 in reducing hypertension

[0082] The strain of Akkermansia muciniphila in this example is specifically Akkermansia muciniphila (ATCC BAA-835) (product of ATCC). Hereinafter, Akkermansia muciniphila (ATCC BAA-835) is referred to as Akk bacteria.

[0083] 1. Preparation of Akkermansia muciniphila culture fluid

[0084] Akk bacteria were inoculated into Brian Heart Infusion (BHI) medium containing 10 mg / L resazurin and cultured anaerobically (during the culture period, plate counts were performed using mucin medium containing 1% agarose under anaerobic conditions). The final Akk bacteria concentration was 1×10 9 CFU / 200 μl of Akkermansia muciniphila bacterial liquid.

[0085] 2. Obtaining the outer membrane protein Amuc_1100

[0086] 1. Using the DNA molecule with nucleotide sequence as shown in SEQ ID NO: 3 as template, primer F: 5'-GGGAATTC CATATG AGCAATTGGATTACAGAC-3' (underlined is the recognition site of restriction endonuclease NdeI, shown in SEQ ID No: 1) and primer R: 5'-C CTCGAG PCR amplification was performed using a primer pair consisting of TTAATCTTCAGACGGTTCCTG-3' (the underline is the recognition site of restriction endonuclease Xhol, shown in SEQ ID No: 2) to obtain a PCR amplification product of about 970 bp.

[0087] 2. Plasmid pet-22b(+) (GenScript, Nanjing) was double-digested with restriction endonucleases NdeI and Xhol to recover a vector backbone of about 5 kb.

[0088] 3. Double digest the PCR amplification product obtained in step 1 with restriction endonucleases NdeI and Xhol, and recover a digestion product of about 950 bp.

[0089] 4. The enzyme digestion product recovered in step 3 and the vector backbone recovered in step 2 are connected to obtain the recombinant plasmid pET-22b-1100.

[0090] The recombinant plasmid pET-22b-1100 was sequenced. The sequencing results showed that the recombinant plasmid pET-22b-1100 was obtained by replacing the small DNA fragment between the restriction endonucleases NdeI and Xhol of the plasmid pet-22b(+) with the DNA molecule shown in SEQ ID NO:3 from the 4th to the 954th position from the 5' end.

[0091] SEQ ID No:3:

[0092] ATGAGCAATTGGATTACAGACAACAAGCCCGCCGCCATGGTCGCGGGCGTGGGACTTCTCTTATTCCTGGGGTTATCCGCGACAGGGTACATCGTCAATTCCAAACGCAGTGAACTGGACAAAAAAATCAGCATCGCCGCCAAGGAAATCAAGTCCGCCAATGCTGCGGAAATCACTCCGAGCCGATCATCCAACGAAGAGCTGGAAAAAGAACTGAACCGCTATGCCAAGGCCGTGGGCAGCCTGGAAACGGCCTACAAGCCCTTCCTTGCCTCCTCCGCGCTGGTCCCCACCACGCCCACGGCATTCCAGAATGAACTGAAAACATTCAGGGATTCCCTGATCTCCTCCTGCAAGAAAAAGAACATTCTCATAACGGACACATCCTCCTGGCTCGGTTTCCAGGTTTACAGCACCCAGGCTCCCTCTGTTCAGGCGGCCTCCACGCTGGGTTTTGAATTGAAAGCCATCAACAGCCTGGTCAACAAACTGGCGGAATGCGGCCTGTCCAAATTCATCAAGGTGTACCGCCCCCAGCTCCCCATTGAAACCCCGGCGAACAATCCGGAAGAATCGGACGAAGCCGACCAGGCCCCATGGACTCCCATGCCTCTGGAAATAGCCTTCCAGGGCGACCGGGAAAGTGTATTGAAAGCCATGAACGCCATAACCGGCATGCAGGACTATCTGTTCACGGTCAACTCCATCCGTATCCGCAACGAACGGATGATGCCCCCTCCCATCGCCAATCCGGCAGCCGCCAAACCTGCCGCGGCCCAACCCGCCACGGGTGCGGCTTCCCTGACTCCGGCGGATGAGGCGGCTGCACCTGCAGCCCCGGCCATCCAGCAAGTCATCAAGCCTTACATGGGCAAGGAGCAGGTCTTTGTCCAGGTCTCCCTGAATCTGGTCCACTTCAACCAGCCCAAGGCTCAGGAACCGTCTGAAGATTAA

[0093] The recombinant plasmid pET-22b-1100 expresses the outer membrane protein Amuc_1100 with the amino acid sequence shown in SEQ ID NO:4.

[0094] SEQ ID NO: 4 is:

[0095] MSNWITDNKPAAMVAGVGLLLFLGLSATGYIVNSKRSELDKKISIAAKEIKSANAAEITPSRSSNEELEKELNRYAKAVGSLETAYKPFLASSALVPTTPTAFQNELKTFRDSLISSCKKKNILITDTSSWLGFQVYSTQAPSVQAASTLGFELKAIN SLVNKLAECGLSKFIKVYRPQLPIETPANNPEESDEADQAPWTPMPLEIAFQGDRESVLKAMNAITGMQDYLFTVNSIRIRNERMMPPPIANPAAAKPAAAQPATGAASLTPADEAAAPAAPAIQQVIKPYMGKEQVFVQVSLNLVHFNQPKAQEPSED

[0096] 5. Transform the recombinant plasmid pET-22b-1100 into E. coli BL21 (DE3) to obtain recombinant E. coli.

[0097] 6. Inoculate a single colony of the recombinant E. coli into 5 mL of LB liquid medium containing kanamycin (dissolve 10 g of tryptone, 5 g of yeast extract and 10 g of NaCl in 1 L of distilled water), and culture at 37°C and 180 rpm for 8 h to obtain culture solution 1. Inoculate culture solution 1 into 500 mL of LB liquid medium containing kanamycin, and culture at 37°C and 180 rpm for 4 h to obtain culture solution 2. Add IPTG to culture solution 2 to make the concentration of IPTG in the system 1 mM, then culture at 37°C and 180 rpm for 3 h, centrifuge at 5000 g for 10 min, discard the supernatant, and collect the bacteria.

[0098] 7. Take the bacteria collected in step 6, resuspend them in PBS buffer with pH 7.2 and 0.01 mol / L containing lysozyme, and then ultrasonically disrupt them to obtain a bacterial cell disruption solution. Centrifuge the bacterial cell disruption solution at 8000 rpm for 10 min and collect the supernatant.

[0099] 8. Take the supernatant collected in step 7 and use Ni-NTA HIS*BIND RESIN His fusion tag protein purification resin for metal affinity purification under natural conditions to obtain the outer membrane protein Amuc_1100 solution.

[0100] The concentration of the outer membrane protein Amuc_1100 solution was determined by the Coomassie Brilliant Blue method (i.e., Bradford method, specific references: L. Wang, et al., A purified membrane protein from Akkermansia muciniphila or the pasteurized bacterium blunts colitis associated tumourigenesis by modulation of CD8(+)T cells inmice, Gut 69(2020)1988–1997,); H. Plovier, et al., A purified membrane proteinfrom Akkermansia muciniphila or the pasteurized bacterium improves metabolismin obese and diabetic mice, Nat. Med. 23(2017)107–113), and then the outer membrane protein Amuc_1100 solution was stored at -20°C.

[0101] The results showed that the concentration of the outer membrane protein Amuc_1100 solution was 30 μg / 200 μl.

[0102] 3. Application of Akk bacteria and its outer membrane protein Amuc_1100 in reducing hypertension

[0103] 1. Randomly take 18 AngII hypertensive mouse models prepared in step 2 of Example 2 and divide them into 3 groups, namely AngII group, AngII+Akk group and Amuc_1100 group. Randomly take 6 control mice prepared in step 3 of Example 2 as the control group. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured using BP-300A fully automatic non-invasive BP measurement system (Chengdu Taimeng Software Co., Ltd.). Each mouse was measured three times and the interval time was required to be more than 30 seconds. The average value of the three measurements was taken as the measurement result, i.e., the blood pressure result of week 0 (0W).

[0104] 2. After completing step 1, each group is processed as follows:

[0105] AngII group: Each mouse was gavaged with 200 μl of normal saline once a day for 4 weeks.

[0106] AngII+Akk group: Each mouse was intragastrically administered with 200 μl of the Akkermansia muciniphila liquid prepared in step 1, once a day for 4 consecutive weeks.

[0107] Amuc_1100 group: Each mouse was intragastrically administered with 200 μl of the outer membrane protein Amuc_1100 solution prepared in step 2, once a day for 4 consecutive weeks.

[0108] Control group: Each mouse was gavaged with 200 μl of normal saline once a day for 4 consecutive weeks.

[0109] During step 2, the systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured weekly using the BP-300A fully automatic non-invasive BP measurement system (Chengdu Taimeng Software Co., Ltd.). Each mouse was measured three times and the interval time was required to be more than 30 seconds. The average of the three measurements was taken as the measurement result, that is, the blood pressure results of the first week (1W), the second week (2W), the third week (3W), and the fourth week (4W) were obtained.

[0110] Some of the test results are shown in Table 3. The results showed that compared with the control group, the blood pressure (including systolic and diastolic pressure) of mice in the AngII group, AngII+Akk group and Amuc_1100 group were significantly reduced.

[0111] Table 3

[0112]

[0113] Note: $ means P < 0.01, the difference is significant; # means P < 0.0001, the difference is extremely significant.

[0114] 3. After completing step 2, kill each group of mice and take the aortic tissue and colon tissue of the mice. Perform Masson staining on the aortic tissue (see step 5 in Example 2 for the specific staining method). Perform AB-PAS staining on the colon tissue (refer to the following literature for specific steps: Zhao Mingjiao. Effects of different processing methods on the relief of ulcerative colitis in mice by carrots and carotenoids [D]. Nanchang University, 2023.). The results of Masson staining and AB-PAS staining are shown in Figure 4 The statistical results are shown in Figure 5 (The data in the figure are P values, and P values ​​less than 0.05 indicate significant differences). The results showed that compared with the AngII group, the aortic wall thickness of mice in the AngII+Akk group and the Amuc_1100 group was significantly reduced, and the proportion of aortic fibrosis area was significantly reduced; compared with the AngII group, the colon muscle layer thickness of mice in the AngII+Akk group and the Amuc_1100 group was significantly reduced, and the number of goblet cells was significantly increased. This shows that Akk bacteria and the outer membrane protein Amuc_1100 solution can enhance the intestinal barrier function of the AngII hypertension mouse model.

[0115] The above results indicate that Akk bacteria and outer membrane protein Amuc_1100 solution lower blood pressure by remodeling cardiovascular system and improving intestinal microenvironment.

[0116] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. The use of outer membrane protein Amuc_1100 and / or Akkermansia muciniphila ATCC BAA-835 is A1) or A2) or A3): A1) Preparation of a medicament for the treatment of hypertension; A1) preparing health products for improving blood pressure levels; A3) preparing a medicament for lowering blood pressure; The outer membrane protein Amuc_1100 is as follows a1) or a2) or a3): a1) the amino acid sequence is the protein shown in SEQ ID NO: 4; a2) a fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of the protein shown in SEQ ID NO: 4; a3) A protein associated with lowering blood pressure obtained by replacing and / or deleting and / or adding one or more amino acid residues of the protein shown in a1) or a2).

2. The use according to claim 1, characterized in that: The Akkermansia muciniphila ATCC BAA-835 is a live bacterium, an inactivated bacterium, or a combination of live and inactivated bacteria.

3. The use of the nucleic acid molecule encoding the outer membrane protein Amuc_1100 described in claim 1 is A1) or A2) or A3): A1) Preparation of a medicament for the treatment of hypertension; A1) preparing health products for improving blood pressure levels; A3) Preparation of a medicament for lowering blood pressure.

4. The use according to claim 3, characterized in that: The nucleic acid molecule encoding the outer membrane protein Amuc_1100 in claim 1 is a DNA molecule as shown in the following b1) or b2) or b3) or b4): b1) the coding region is the DNA molecule shown in SEQ ID NO: 3; b2) the nucleotide sequence is a DNA molecule shown in SEQ ID NO: 3; b3) a DNA molecule having 75% or more identity with the nucleotide sequence defined in b1) or b2), derived from Akkermansia muciniphila ATCC BAA-835 and encoding the outer membrane protein Amuc_1100 as claimed in claim 1; b4) a DNA molecule which hybridizes with the nucleotide sequence defined in b1) or b2) under stringent conditions, is derived from Akkermansia muciniphila ATCC BAA-835 and encodes the outer membrane protein Amuc_1100 described in claim 1.

5. Use of the composition containing the outer membrane protein Amuc-1100 and / or Akkermansia muciniphila ATCC BAA-835 as claimed in claim 1, which is A1) or A2) or A3): A1) Preparation of a medicament for the treatment of hypertension; A1) preparing health products for improving blood pressure levels; A3) Preparation of a medicament for lowering blood pressure.

6. The use according to claim 5, characterized in that: The composition containing the outer membrane protein Amuc_1100 and / or Akkermansia muciniphila ATCC BAA-835 of claim 1 is a health product composition, a pharmaceutical composition or a probiotic composition; Preferably, the composition containing the outer membrane protein Amuc_1100 and / or Akkermansia muciniphila ATCC BAA-835 of claim 1 further comprises a second active ingredient; the second active ingredient is a drug different from the outer membrane protein Amuc_1100 and / or Akkermansia muciniphila ATCC BAA-835 of claim 1; Preferably, the probiotic composition further contains probiotics different from the Akkermansia muciniphila ATCC BAA-835 described in claim 1; Preferably, the composition containing the outer membrane protein Amuc_1100 and / or Akkermansia muciniphila ATCC BAA-835 as claimed in claim 1 further comprises one or more of telmisartan, captopril, valsartan and amlodipine.

7. The use according to any one of claims 1 to 6, characterized in that: The hypertension is renal hypertension, spontaneous hypertension, metabolic hypertension caused by combined dietary factors or isolated systolic hypertension.

8. The use according to any one of claims 1 to 7, characterized in that: The drug is a medicine, and its dosage form is tablets, capsules, granules, pills, ointments, solutions, suspensions, emulsions, creams, sprays, drops, patches or tube feeding preparations; and / or, The health care products are health care foods, and the types thereof are dairy products or fermented cereal foods.

9. The use according to any one of claims 1 to 8, characterized in that: The medicine or the health product contains an effective amount of the outer membrane protein Amuc_1100 and / or Akkermansia muciniphila ATCC BAA-835 described in claim 1.

10. The use according to any one of claims 1 to 9, characterized in that: The subject of the medicine or the health product is a mammal; Preferably, the mammal is a human or a mouse.

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