阅读说明：本技术 胡椒碱在制备作为人的苦味受体14亚型的激动剂中的应用 (Use of piperine as an agonist of the human bitter taste receptor 14 subtype ) 是由 刘耀武 黄婷婷 于 2021-08-27 设计创作，主要内容包括：本发明涉及胡椒碱的新用途,胡椒碱在制备作为人的苦味受体14亚型(hTAS2R14)的激动剂中的应用,胡椒碱能够增加肠内分泌细胞Caco-2中hTAS2R14的mRNA水平和蛋白表达,以及增加其下游磷脂酶C β2和瞬时受体电位离子通道蛋白M5蛋白表达,增加胞内钙的动员,进而增加胰高血糖素样肽1 (GLP-1)前体胰高血糖素原的基因表达和GLP-1的分泌。hTAS2R14拮抗剂6-甲氧基黄酮没有减弱胡椒碱对胞内钙的动员的效应,反而促进了胡椒碱对胞内钙的动员。与hTAS2R14藕联的G蛋白的Gβγ亚基抑制剂明显减弱了胡椒碱对GLP-1的分泌,磷脂酶C β2抑制剂显著减弱了胡椒碱对GLP-1的分泌。该研究说明胡椒碱是hTAS2R14的高效激动剂。(The invention relates to a new application of piperine, in particular to an application of the piperine in preparing an agonist of human bitter taste receptor 14 subtype (hTAS2R14), wherein the piperine can increase the mRNA level and protein expression of hTAS2R14 in enteroendocrine cells Caco-2, increase the expression of downstream phospholipase C beta 2 and transient receptor potential ion channel protein M5 protein, increase the mobilization of intracellular calcium, and further increase the gene expression of glucagon precursor glucagon-like peptide 1 (GLP-1) and the secretion of GLP-1. The hTAS2R14 antagonist 6-methoxyflavone does not attenuate the mobilization effect of piperine on intracellular calcium, but rather promotes the mobilization of piperine on intracellular calcium. The G beta gamma subunit inhibitor of the G protein coupled with the hTAS2R14 obviously weakens the secretion of the piperine to the GLP-1, and the phospholipase C beta 2inhibitor obviously weakens the secretion of the piperine to the GLP-1. This study demonstrated that piperine is a highly potent agonist of hTAS2R 14.)

1. Use of piperine in the preparation of an agonist as bitter taste receptor 14, wherein said bitter taste receptor 14 subtypes comprise hTAS2R14, Tas2R140, Tas2R 113, Tas2R 116, and Tas2R 125; the molecular structure of the piperine is as follows:

Background

Taste strongly influences food preference and intake, and vertebrate receptors for food or compounds are distinct taste receptors (TASRs) that were originally found in the taste buds of the oral cavity. Human taste receptors include two types, sweet taste receptors (hTAS1Rs) and bitter taste receptors (hTAS2 Rs). hTAS2Rs is a class of G protein-coupled receptors (GPCRs) consisting of a hydrophobic seven-fold transmembrane domain, an extracellular short amino terminus, an intracellular carboxy terminus, 3 extracellular loop structures and 3 intracellular loop structures. The hTAS2Rs signaling pathway is highly conserved. After activation of hTAS2Rs by the ligand, hTAS2Rs separates from the G α β γ complex and functions in two ways: (1) the G.alpha.gust-PDE pathway, activation of TAS2Rs, results in the isolation of G protein a subunit gustducin (G.alpha.gust), leading to Phosphodiesterase (PDE) isomerization and reduced cAMP levels; the decrease in cAMP levels results in the release of cAMP inhibition of ion channels, triggering selective Ca²⁺Channels open, in turn causing intracellular Ca²⁺The increase in concentration depolarizes the cell membrane, releasing neurotransmitters or hormones. (2) The G beta gamma-PLC beta 2 pathway, hTAS2Rs, is activated to release G beta gamma subunit, further activates phospholipase beta 2(PLC beta 2) to generate inositol triphosphate (IP3) and diacylglycerol, IP3 activates its receptor 3 on the endoplasmic reticulum (IP3R3), causing Ca²⁺Mobilization, intracellular Ca²⁺Increased, but activation of PKC by DAG leads to intracellular Ca²⁺These, in turn, activate the transient receptor potential ion channel protein M5(TRPM5) on the cell membrane, leading to influx of cations such as Na + and the like and depolarization of the membrane, resulting in release of neurotransmitters or hormones.

There are 25 currently known human TAS2Rs, including hTAS2R1, 3, 4, 5, 7, 8, 9, 10, 13, 14, 16, 19, 20, 30, 31, 38, 39, 40, 41, 42, 43, 45, 46, 50, 60, where the agonist profile of the three subtypes hTAS2R14, 10, 46 is much greater than the other subtypes. TAS2Rs is also present in extraoral tissues such as respiratory, gastrointestinal, brain, etc.

The intestine is the first extraoral tissue found to present taste sensory signals (Proc Natl Acad Sci USA 1996; 93(13): 6631-. Early reports found that activation of gastrointestinal hTAS2R14 promotes secretion of chymase peptide, thereby suppressing appetite. Recent reports have shown that respiratory tract hTAS2R14 can improve obstructive airway disease after activation. The biological function caused by the activation of hTAS2R14 is still to be studied.

Piperine (Pip) is the main irritant component and characteristic odorous compound of black pepper (pepper of the family Piperaceae) and is the highest natural alkaloid content in black pepper (2% -9%). The crude extract and active components (mainly alkaloids) of fructus Piperis have wide pharmacological activities, especially antioxidant, antidepressant, hepatoprotective, antimicrobial, anti-obesity, cognitive disorder treating, blood sugar lowering, hyperlipidemia resisting, immunity enhancing and antiinflammatory effects. However, no report of piperine as a bitter receptor 14 agonist was found.

Disclosure of Invention

1. Objects of the invention

The invention aims to find that the piperine is used as an agonist of a 14 subtype human bitter receptor, and the new application of the piperine is expanded.

2. Technical scheme

Use of piperine in the preparation of an agonist as rodent cognate receptors for hTAS2R14 and hTAS2R14, wherein the rodent cognate receptors for hTAS2R14 comprise Tas2R140, 113, 116, 125.

The research idea of the invention is as follows: the biological effect generated after the piperine activates the hTAS2R14 signal channel is researched by taking the fact that the hTAS2R14 in the endocrine cells of the intestinal tract can promote the secretion of incretin GLP-1 as an index. Piperine is experimentally shown to be an agonist of the 14 subtype of human bitter taste receptor.

The literature reports that human colorectal adenocarcinoma cell lines NCI-H716 and Caco-2 cells have the function of enteroendocrine cells (enteroendocrine) and can secrete certain intestinal peptide hormones. Caco-2 cells are anchorage-dependent growing cells, do not need differentiation and the like, and are easy to culture and observe administration. We studied the abundance of expression of hTAS2R4, 10 and 14 genes in Caco-2 cells and found that the abundance of expression of hTAS2R14, 4 and 10 genes was sequentially decreased, with the highest expression of hTAS2R14 gene being 9.46 and 28.84 times that of hTAS2R4 and hTAS2R10, respectively. Therefore, Caco-2 cells are taken as a research object, and the activation effect of piperine on the hTAS2R14 signal path and the influence of GLP-1 secretion are observed. Through a network platform Bitter X developed by a molecular design laboratory of Shanghai university of transportation, the hTAS2Rs capable of being combined with piperine is predicted, and 9 subtypes are found, wherein the possibility of hTAS2R4, 14 and 10 is high, and the maximum possibility is 75% -80%.

The invention discloses a function that piperine is a high-efficiency agonist of hTAS2R14, can activate an hTAS2R14 signal channel in an intestinal endocrine cell and further promote GLP-1 secretion, and researches show that the piperine can increase mobilization of calcium in a Caco-2 cell and secretion of GLP-1, increase protein expression and mRNA level of hTAS2R14 and increase PLC beta 2 protein expression and TRPM5 protein expression. On the other hand, the combination of the G beta gamma subunit inhibitor of the G protein coupled with the hTAS2R14 and the piperine obviously weakens the secretion of GLP-1 by the piperine, and the combination of the PLC beta 2inhibitor and the piperine obviously weakens the secretion of GLP-1 by the piperine. Most importantly, we established Caco-2 cells that knock down hTAS2R14 and found that the effect of piperine on increasing PLC β 2 and TRPM5 protein expression was counteracted following knock down of hTAS2R 14. In addition, the compound 6-Methoxyflavone (6-Methoxyflavone) is reported in the literature to be an hTAS2R14 antagonist and can antagonize the effect of the sensory complexing peptide 1 of the hTAS2R14 agonist (FASEB J.2021; 35(3): e 21375), however, we found that the intracellular calcium ion concentration did not decrease but significantly increased after different concentrations of 6-Methoxyflavone and piperine co-treated Caco-treated cells, indicating that the 6-Methoxyflavone is not antagonistic but agonistic to hTAS2R14 and has a synergistic effect with piperine.

Various pharmacological tests of the piperine and the hTAS2R14 signal pathway and results thereof indicate that the piperine is a high-efficiency agonist of hTAS2R 14.

3. Advantageous effects

The pharmacological test results show that the invention has the following advantages:

(1) the invention discovers that piperine can activate an hTAS2R14 signal channel for the first time, and the piperine is a high-efficiency agonist of hTAS2R 14. hTAS2R14 is expressed in many systems of the body, and is particularly abundant in endocrine cells of the digestive tract. Furthermore, hTAS2R14 has a very broad agonist spectrum, belonging to the three bitter taste receptor subtypes with the broadest agonist spectrum as hTAS2R10 and hTAS2R 46.

The literature reports that steroid saponins extracted from native oroxylum indicum of south Africa can selectively activate hTAS2R7 and hTAS2R14, further promote the secretion of gastrointestinal tract intestinal tryptic peptide, and inhibit appetite, which suggests that the hTAS2R14 agonist can also play a role in improving metabolic diseases by influencing the secretion of other intestinal peptide hormones in the digestive tract. Because the function of hTAS2R14 is not completely clear, and the invention does not emphasize that piperine is used for treating metabolic diseases such as diabetes and the like, the invention emphasizes that piperine is used as an agonist of hTAS2R14, and the agonist of hTAS2R14 can start various signal channels and produce different functions, such as improving metabolic diseases, respiratory diseases and the like.

(2) The natural product piperine used in the invention is a high-efficiency hTAS2R14 agonist, the effective concentration for promoting intracellular calcium mobilization and GLP-1 secretion is lower, and is in the order of several micromoles per liter, and is lower than the effective concentration (in the order of dozens of micromoles per liter) of the synthetic hTAS2R14 agonist flufenamic acid (Cellular and Molecular Life Sciences 2020; 77: 531-542), and the effective concentration for promoting intracellular calcium mobilization of the hTAS2Rs agonist is usually in the order of hundreds of micromoles per liter and above. These show that the piperine agonizing hTAS2R14 in the invention causes higher potency of intestinal peptide hormone secretion, and is a good potential anti-metabolic disease drug.

(3) The two hTAS2R14 agonists piperine and flufenamic acid used in the invention can obviously promote GLP-1 secretion and activation of an hTAS2R14 signal channel, and the activation of the hTAS2R14 signal channel by piperine is obviously weakened by hTAS2R14 knockdown. However, the hTAS2R14 antagonist 6-methoxyflavone reported in the literature does not antagonize, but rather enhances the agonistic effect of piperine on hTAS2R 14. These suggest that hTAS2R14 is the target of piperine action and that the discovery of antagonists of hTAS2R14 is arbitrary and far away, thus illustrating the complexity of hTAS2R14 action.

Drawings

FIG. 1 Effect of piperine on PLC β 2 protein expression in Caco-2 cells. Ctrl is Control; pip-2, Pip-10 and Pip-50 respectively represent 2,10 and 50 mu M piperine. Scale bar:20 μm.

FIG. 2 Effect of hTAS2R14 knockdown on piperine to increase PLC β 2 protein expression in Caco-2 cells. Ctrl is Control; NC is negative control; pip-50: 50. mu.M piperine; shRNA sh-hTAS2R14RNA cellular immunofluorescence assay for protein expression levels. Scale bar:20 μm.

Detailed Description

Example 1: effect of piperine and Flufenamic acid on intracellular calcium mobilization and GLP-1 secretion in Caco-2 cells

1 materials and methods

1.1 cell lines

Caco-2 cells (FH0029) were purchased from Shanghai Rich Biotech, Inc.

1.2 drugs and Primary Agents

1.2.1 medicine: piperine (purity > 98%) was purchased from kyphos biotechnology limited; flufenamic acid (purity > 99%) was purchased from Shanghai-derived leaf Biotech, Inc.

1.2.2 reagents: DMEM high-glucose medium (4.5g/L glucose) was purchased from Beijing Solaibao Tech Co., Ltd; trizol reagent (15596-026) was purchased from Invitrogen, USA; PrimeScript RT reagent Kit (Perfect Real Time) (RR037A) and SYBR Premix Ex Taq II were purchased from TaKaRa, Dada, Inc.; DNase/RNase-FreeH₂O (T121-02) was purchased from Tiangen Biochemical technology (Beijing) Ltd; the CCK-8 kit is purchased from Shanghai Dongren chemical technology Co., Ltd; the Fura-2 calcium ion fluorescent probe was purchased from the Biotechnology institute of Jiangsu Bitian; human GLP-1(7-36) ELISA kits were purchased from Shanghai Bonemia Biotech, Inc.

1.3 methods

1.3.1 grouping and administration: caco-2 is an adherently growing cell, conventionalCulturing in DMEM high-glucose medium containing 10% FBS and 1% double antibody (streptomycin qing) and 5% CO₂And culturing in a constant temperature incubator at 37 ℃. When the growth state is good, replacing serum-free culture medium to synchronize the cell cycle for 12h, dividing the cell cycle into a control group (Ctrl), a Pip treatment group with different concentrations and an FA treatment group, culturing for 30min, collecting cells, and determining the concentration of calcium ions in cells; culturing for 1h, collecting the culture medium, and measuring the GLP-1 level; and measuring other relevant indexes after culturing for 24 h.

1.3.2 cell viability assay: the cell viability was determined by the CCK-8 method. Cells were cultured according to 1.3.1, Pip was set at five concentrations of 0.1,1,10,50, 100. mu. mol/L and Ctrl group at the same time, and cells were collected after 24 hours of culture and assayed according to the kit procedures.

1.3.3mRNA level determination: the mRNA level was determined by RT-qPCR. Extracting total RNA by using Trizol, determining the concentration of the total RNA, and operating according to the instruction of a reagent. Determination of its purity, OD_260/280Samples between 1.8 and 2.0 are qualified samples. 0.5. mu.g of total RNA was taken from each sample for reverse transcription into cDNA, and the detailed procedure was performed according to the instructions of the cDNA reverse transcription synthesis kit. The synthesized cDNA was amplified using Roche 480 fluorescent quantitative PCR instrument. After the amplification was completed, the lysis curve was amplified to check the purity of the amplified fragment. The smaller the Cp value, the higher the content of the objective gene. By 2^-⊿CpValue ([ delta ] Cp ═ delta ] Cp_β-actin﹣⊿Cp_{Purpose(s) to}) As a relative value of gene expression for the sample.

1.3.4 determination of calcium ion concentration: the intracellular calcium ion concentration is determined by a fluorescent probe method. The cells are inoculated in a 96-well plate for pre-culture for 24h, washed by PBS for three times, added with the work solution of the Fura-2 calcium ion fluorescent probe and incubated for 45min in the dark, washed by PBS for three times, and added with a culture medium containing two drugs with different concentrations and incubated for 30min in the dark. And (3) respectively detecting the fluorescence intensity at the excitation wavelength of 340nm and the emission wavelength of 380nm by using a fluorescence microplate reader, wherein the calcium ion concentration is reflected by the fluorescence intensity ratio of 340nm to 380nm, and the larger the ratio is, the higher the calcium ion concentration is.

1.3.5GLP-1 concentration determination: GLP-1 concentration was determined by ELISA. Cells were cultured according to 1.3.1 and cell culture medium was collected and assayed exactly as per the kit instructions.

2 results

2.1 comparison of expression abundance of hTAS2R14, 4, 10 in Caco-2 cells

After the Caco-2 cells are cultured for 24h, the expression of three human bitter taste receptors is measured by a real-time quantitative PCR method, and the hTAS2R14, 4 and 10 are expressed, while the hTAS2R14 has the highest expression level, and the mRNA levels are reduced in sequence according to the sequence of hTAS2R14, 4 and 10 (see Table 1). Therefore, Caco-2 cells can be used for functional studies of hTAS2R14 agonists.

TABLE 1 expression abundance of hTAS2R14, 4, 10 in Caco-2 cells

Note RT-qPCR method to determine hTAS2Rs levels. Mean ± SD.

2.2 screening of piperine concentration in Caco-2 cells

The cell activity is used as an index to screen the concentration of the piperine medicine which has no damage to Caco-2 cells. As a result, it was found that piperine concentrations of less than 100. mu. mol/L had no significant effect on the cell viability of Caco-2 cells compared to the drug-untreated group (see Table 2). Thus, three concentrations of piperine, 50,10, 2 μmol/L, were initially set up for their study of their effect on intracellular calcium mobilization.

TABLE 2 Effect of piperine on Caco-2 cell viability

Note Ctrl Control; pip-0.1, Pip-1, Pip-10, Pip-50, Pip-100 represent 0.1,1,10,50,100 μ M piperine. mean ± SD, respectively, n is complex number ## P <0.01, compared to Ctrl group.

2.3 Effect of Flufenamic acid and piperine on calcium mobilization in Caco-2 cells

Flufenamic acid was used as a positive control for the agonism of hTAS2R14 by piperine in this study. Although the literature reports that flufenamic acid increases the EC of intracellular calcium levels₅₀The value was 238nmol/L, but the EC for calcium mobilization, determined by studies performed after overexpression of hTAS2R14 by the tool cell HEK293, was used₅₀The value is obtained. Depending on the cell type, EC₅₀The values differed, so this study screened for their concentrations. It was found that flufenamic acid significantly increased intracellular calcium levels at 20 and 50 μmol/L (see table 3). Since the percentage increase in intracellular calcium levels was more pronounced at 50. mu. mol/L, 50. mu. mol/L of flufenamic acid was selected for subsequent studies.

TABLE 3 Effect of hTAS2R14 agonist flufenamic acid on intracellular calcium mobilization in Caco-2 cells

Note Ctrl Control; FA-2, FA-5, FA-10, FA-20 and FA-50 represent 2,5,10,20 and 50 mu M flufenamic acid. mean + -SD, n is the number of complex pores, # P <0.05, # P <0.01, compared with Ctrl group.

We treated Caco-2 cells with three concentrations of piperine, low, medium and high, 2,10, 50. mu. mol/L, and found that 10 and 50. mu. mol/L piperine significantly increased intracellular calcium levels, while 2. mu. mol/L piperine showed only an increasing trend (see Table 4). In addition, the effect of 10. mu. mol/L piperine was slightly stronger than that of 20. mu. mol/L flufenamic acid (see tables 3, 4).

TABLE 4 Effect of hTAS2R14 agonist piperine on intracellular calcium mobilization in Caco-2 cells

Note Ctrl Control; pip-2, Pip-10, Pip-50 represent 2,10,50 μ M piperine. mean ± SD, respectively, n is the number of replicate wells ## P <0.01, compared to Ctrl group.

2.4 Effect of Piperine and Flufenamic acid on GLP-1 secretion by Caco-2 cells

Compared with Ctrl group (25mmol/L glucose culture), the secretion of GLP-1 by Caco-2 cells was significantly stimulated after 10 and 50. mu. mol/L piperine treatment, resulting in a significant increase in GLP-1 concentration in the culture medium, while 2. mu. mol/L piperine showed only an increasing trend (see Table 5). Furthermore, the extracellular GLP-1 concentration was also significantly increased after 50. mu. mol/L flufenamic acid treatment, but much less than 10. mu. mol/L piperine (see Table 5).

TABLE 5 Effect of piperine and flufenamic acid on GLP-1 secretion by Caco-2 cells

Note Ctrl Control; pip-2, Pip-10, Pip-50 represent 2,10, 50. mu.M piperine, respectively;

FA represents 50 μ M flufenamic acid. mean. + -. SD, n is cell batch number, # P <0.05,

# P <0.01, compared to Ctrl group.

2.5 Effect of piperine on Caco-2 cell GLP-1 precursor Proglucagon mRNA levels

High concentrations of piperine significantly increased GCG levels in Caco-2 cells compared to the Ctrl group, whereas low and medium concentrations of piperine showed only an increasing trend (see table 6). The result shows that piperine can promote the expression of GLP-1 precursor glucagon, thereby increasing the synthesis of GLP-1.

TABLE 6 Effect of piperine on Caco-2 cell GLP-1 precursor glucagon mRNA levels

Note Ctrl Control; pip-2, Pip-10 and Pip-50 respectively represent gene symbols of 2,10 and 50 mu M piperine. GCG which is glucagon (proglucagon). beta-actin is used as an internal reference, and the level of hTAS2Rs is measured by an RT-qPCR method. Mean ± SD, n is the number of cell batches ## P <0.01, compared to Ctrl group.

Example 2: effect of piperine on the hTAS2R14 signaling pathway in Caco-2 cells

1 materials and methods

1.1 cell line example 1.

1.2 pharmaceutical products and Main reagent BCA protein assay kit (23225) purchased from Thermo-Scientific (Rockford, IL, USA); rabbit hTAS2R14 antibody (DF5159) was purchased from Affinity antibodies; rabbit PLC β 2 antibody (a8141) was purchased from ABclonal antibodies; rabbit TRPM5 antibody (18027-1-AP) was purchased from Proteitech antibody; rabbit β -actin (# AP0060) antibody was purchased from Bioworld technology antibody; goat anti-rabbit Dylight 594 affinipuregg (H + L) secondary antibody (# V926-32211) was purchased from Li-Cor, Inc (Lincoln, NE); the rest is the same as example 1.

1.3 methods

1.3.1 grouping and administration: the same as in example 1.

1.3.2 protein expression assay (Westernblot method): the protein concentration of the cell homogenate supernatant was measured by BCA method and the protein solution was diluted with SDS loading buffer. The conventional Westernblot method is used for measuring protein expression, namely electrophoresis, membrane conversion, sealing, primary antibody incubation, secondary antibody incubation, development and scanning are sequentially carried out, and the change of the expression quantity of the target protein is analyzed by taking the relative gray scale of the target protein and the internal reference protein as indexes.

1.3.3 protein expression assay (Immunofluorescence method): placing a sterile circular cover glass into a 12-hole plate, inoculating cells into the hole plate, after the cells stably grow on the cover glass, giving a serum-free culture medium for cell cycle synchronization treatment for 12 hours, replacing a complete culture medium during administration, respectively giving medicine treatment, discarding the culture medium after 24 hours, and performing subsequent experiments.

1) Washing cell slide with PBS for 5 min/times and multiplying by 3 times;

2) adding 4% paraformaldehyde or methanol to cover the slide, and fixing at-20 deg.C for 15 min;

3) PBS washing, 5 min/times multiplied by 3 times;

4) adding 0.1% TritonX-100, and perforating for 10 min;

5) PBS washing, 5 min/times multiplied by 3 times;

6) adding 1% BSA (PBS) to block for 30 min;

7) the primary antibody was diluted as required by the instructions, covered the entire slide, and placed in a wet box overnight at 4 ℃.

8) PBS washing, 5 min/times multiplied by 3 times;

9) dylight 594 (anti-rabbit) labeled secondary antibody was formulated as per the instructions, covering the entire slide, and incubated at 37 ℃ for 1 h;

10) PBS washing, 5 min/times multiplied by 3 times;

11) DAPI staining for 2 min;

12) PBS washing, 5 min/times multiplied by 3 times;

13) residual PBS was blotted dry with filter paper, a drop of anti-fluorescence quencher was added, and the slide was transferred to a slide with the front side down.

14) And (4) taking a picture by a fluorescence microscope, and observing the cell location of the target protein in the cell.

As a result, the target protein fluoresces red and the nucleus fluoresces blue.

1.3.4mRNA level determination (RT-qPCR method): the same as in example 1.

2 results

2.1 Effect of piperine on Caco-2 cell hTAS2R14mRNA levels and protein expression

Compared with Ctrl group, high concentration piperine can significantly increase the level of hTAS2R14mRNA in Caco-2 cells, and low and medium concentrations piperine can also increase the level of hTAS2R14mRNA, but the difference is not significant (see table 7). Meanwhile, the expression of the hTAS2R14 protein in Caco-2 cells can be obviously increased by medium and high concentrations of piperine, and the expression of the hTAS2R14 protein can also be increased by low concentrations of piperine, but the difference is not obvious (see Table 7). The results show that piperine can up-regulate hTAS2R14 in Caco-2 cells.

TABLE 7 Effect of piperine on Caco-2 cell hTAS2R14mRNA levels and protein expression

Note Ctrl Control; pip-2, Pip-10 and Pip-50 respectively represent 2,10 and 50 mu M piperine, beta-actin is used as an internal reference, and the level of hTAS2Rs is measured by an RT-qPCR method. The protein expression level is determined by a Westernblot method by taking beta-actin as an internal reference. Mean ± SD, N ═ 4(protein) cell batches, N ═ 5(mRNA)

Cell batch # P <0.05, # P <0.01, compared to Ctrl group.

2.2 Effect of piperine on PLC beta 2 protein expression in Caco-2 cells

Compared with the Ctrl group, the expression of the PLC beta 2 protein in Caco-2 cells can be obviously increased by the piperine with medium and high concentrations, and the expression of the piperine with low concentrations is not obviously increased (see figure 1). The results show that piperine can activate hTAS2R14 in Caco-2 cells, thereby separating G alpha and G beta gamma subunits of G protein and increasing the expression of PLC beta 2.

2.3 Effect of piperine on TRPM5 protein expression in Caco-2 cells

Compared with the Ctrl group, the medium and high concentrations of piperine can significantly increase the expression of TRPM5 protein in Caco-2 cells, and the low concentration of piperine does not significantly increase (see Table 8). The result shows that the piperine can activate the hTAS2R14/G beta gamma pathway in Caco-2 cells, promote the mobilization of intracellular calcium and further increase the expression of TRPM 5.

TABLE 8 Effect of piperine on TRPM5 protein expression in Caco-2 cells

Note Ctrl Control; pip-2, Pip-10 and Pip-50 respectively represent 2,10 and 50 mu M piperine, beta-actin is used as an internal reference, and the protein expression level is determined by a Westernblot method. Mean ± SD, n is the number of cell batches, # P <0.05, # P <0.01, compared to Ctrl group.

Example 3: effect of Caco-2 cell hTAS2R14 pathway inhibition on its agonist piperine effect

1 materials and methods

1.1 cell line example 1.

1.2 drug and main reagents: gallein (GC13945, purity > 98%) was purchased from Glpbio, USA; u73122(T6243, purity 97.98%) was purchased from TargetMol corporation, usa; 6-Methoxyflavone (6-methoxyflavone, 6-MF, purity > 98%) was purchased from Shanghai-derived leaf Biotech, Inc. The other steps are the same as those in examples 1 and 2.

1.3 methods

1.3.1 grouping and administration: caco-2 was routinely cultured in DMEM high-glucose medium containing 10% FBS and 1% double antibody (streptomycin qing) with 5% CO₂And culturing in a constant temperature incubator at 37 ℃. When the growth state is good, the cell cycle is synchronized for 12h by changing into a serum-free culture medium, the medium is divided into a Pip high-concentration group (Pip-50) and a Pip-50 and different intervention preparations co-processing group, and the intracellular calcium ion concentration is measured after the medium is cultured for 30 min; GLP-1 levels in the medium were determined after 1h of culture.

1.3.2 measurement of calcium ion concentration (fluorescent Probe method): the same as in example 1.

1.3.3GLP-1 level assay (ELISA method): the same as in example 1.

2 results

2.Effect of 16-Methoxyflavone on the promotion of intracellular calcium mobilization in Caco-2 cells by Piperine

The compound 6-methoxyflavone is reported in the literature to be an hTAS2R14 antagonist, and 6-methoxyflavone (30. mu. mol/L) can antagonize the effect of hTAS2R14 agonist sensing comparative peptide 1 (FASEBJ.2021; 35(3): e21375.), however, in the research we found that after Caco-treating Caco-2 cells with 6-methoxyflavone at a concentration of 25, 50, 100. mu. mol/L and piperine at a high concentration (50. mu. mol/L), the intracellular calcium ion concentration did not decrease but increased significantly (see Table 9), indicating that 6-methoxyflavone does not antagonize hTAS2R14, and has a synergistic effect with piperine.

TABLE Effect of 96-Methoxyflavone on piperine promotion of intracellular calcium mobilization in Caco-2 cells

Pip-50 for 50. mu.M piperine; 6-MF-25, 6-MF-50, 6-MF-100 represent 25, 50, 100. mu.M 6-methoxyflavone 6-methoxyflavone. mean. + -. SD, n is the number of multiple wells, # # P <0.01, respectively, compared to Pip-50 group.

2.2 Effect of hTAS2R14 pathway inhibition on piperine-stimulated Caco-2 cell GLP-1 secretion

Compared with the high-concentration piperine group, GLP-1 secretion is remarkably reduced after the high-concentration piperine and a G beta gamma inhibitor Gallein are co-treated on Caco-2 cells (see Table 10); meanwhile, after the Caco-2 cells are co-treated by the high-concentration piperine and the PLC beta 2inhibitor U73122, GLP-1 secretion is also obviously reduced (see Table 10). These results indicate that inhibition of the hTAS2R14/G β γ pathway attenuates the effect of piperine in promoting GLP-1 secretion.

TABLE 10 Effect of hTAS2R14 pathway inhibition on piperine-stimulated GLP-1 secretion from Caco-2 cells

Pip-50 for 50. mu.M piperine; galelin aG beta gamma inhibitor; u73122 ac β 2inhibitor. mean ± SD, n is cell index ## P <0.01, compared to Pip-50 group.

Example 4: effect of Caco-2 cell hTAS2R14 knockdown on its agonist piperine Effect

1 materials and methods

1.1 cell line example 1.

1.2 drug and main reagents: three LV-sh-hTAS2R14RNA (shRNA) products and Negative Control (NC) thereof were purchased from Shanghai Jikai Gene science and technology Co., Ltd; the other steps are the same as those in examples 1 and 2.

1.3 methods

1.3.1 establishment of Caco-2 cells with knockdown of hTAS2R 14: cell count was 1X 10⁵Cells/ml were seeded into 6-well plates, grown to about 60-80%, changed to serum-free or 1% serum in DMEM medium, and transfection was initiated by addition of Lentivirus (LV). LV carries hTAS2R14 and empty vector (NC) shRNA, after transfection for 72h, GFP fluorescence intensity is observed, and when fluorescence abundance is about 90%, transfection is completed. Meanwhile, 2 mu g/ml puromycin is added for cell screening and killing, after 24 hours, the concentration is reduced to 1 mu g/ml for maintaining and culturing for 72 hours, and after the normal cells are all dead, namely the transfection is finished, a stable transfectant cell strain is collected for verification of the transfection effect or subculture preservation.

1.3.2 grouping and administration: transfected Caco-2 cells of LV-sh-hTAS2R14 and NC were routinely cultured in DMEM high-glucose medium containing 10% FBS and 1% double antibody (streptomycin qinghao), 5% CO₂And culturing in a constant temperature incubator at 37 ℃. When the growth state is good, the cell cycle is synchronized for 12h by changing into a serum-free culture medium, and the cells are divided into an NC group, an hTAS2R14 knock-down group and two groups which are treated by Pip high concentration (Pip-50) and cultured for 24h for carrying out related index determination.

1.3.3mRNA level determination (RT-qPCR method): the same as in example 1.

1.3.4 protein expression assay (Westernblot method): the same as in example 2.

1.3.5 protein expression assay (Immunofluorescence method): the same as in example 2.

2 results

2.1 validation of Caco-2 cells knocking down hTAS2R14

After Caco-2 cells were transfected with three LV-sh-hTAS2R14 cells and negative controls thereof, the hTAS2R14mRNA level of the third sh-hTAS2R14 group was found to be extremely significantly reduced, whereas the first sh-hTAS2R14 and negative controls showed an increasing trend, and the second sh-hTAS2R14 showed a decreasing trend without significant difference (see Table 11). These results indicate that the third sh-hTAS2R14 was effective and that hTAS2R14 protein expression was verified after its transfection. As a result, after a third sh-hTAS2R14 transfected Caco-2 cells, the expression of hTAS2R14 protein was significantly reduced (see Table 12).

TABLE 11 Change in hTAS2R14mRNA levels following transfection of Caco-2 cells with three LV-sh-hTAS2R14

Note Ctrl: control; NC is negativecontrol; shRNA-1, 2 and 3 are three LV-sh-hTAAS2R14RNA. mean. + -. SD, respectively, and n is cell batch number ## P <0.01, compared with Ctrl group.

TABLE 12 Change in hTAS2R14 protein expression following transfection of Caco-2 cells with three LV-sh-hTAS2R14 cells

Injecting NC, negative control; shRNA-3 is expressed as the third LV-sh-hTAS2R14RNA. mean. + -. SD, n is the number of cell batches ## P <0.01, compared to the NC group.

2.2 Effect of knockdown of hTAS2R14 on the expression of PLC β 2 protein

Compared with Ctrl group, there was no significant change in PLC beta 2 protein expression in NC group. Compared with the NC group, the high-concentration piperine group PLC β 2 protein expression was significantly increased, while the hTAS2R14 knock-down group was significantly decreased. Compared with the NC group cultured by high-concentration piperine, the hTAS2R14 knocked-down group cultured by the high-concentration piperine has obviously reduced PLC beta 2 protein expression. These results indicate that knock-down of hTAS2R14 attenuated the effect of piperine on increased expression of PLC β 2 protein (see figure 2).

2.3 Effect of knockdown of hTAS2R14 on the expression of TRPM5 protein by Piperine

Compared with the NC group, the high-concentration piperine group TRPM5 protein expression was significantly increased, while the hTAS2R14 knock-down group was significantly decreased. Compared with the NC group cultured by high-concentration piperine, the hTAS2R14 knock-down group cultured by high-concentration piperine has obviously reduced TRPM5 protein expression. These results indicate that knock-down of hTAS2R14 attenuated the effect of piperine on the increase in TRPM5 protein expression (see table 13).

TABLE 13 variation of knock-down sh-hTAS2R14 on TRPM5 protein expression

Injecting NC, negative control; shRNA: LV-sh-hTAS2R14 RNA; pip-50:50 μ M piperine mean ± SD, n is the number of cell batches, # P <0.05, # P <0.01, compared to NC group; p <0.01, compared to NC + Pip-50 group.