CN116179667A - Kit and application for screening raw materials for skin pain relief based on TRPV1 signaling pathway in zebrafish model - Google Patents

Abstract

The invention provides a kit for screening raw materials for skin pain relief based on the TRPV1 signaling pathway in a zebrafish model. The kit includes RNA extraction reagents, RNA reverse transcription reagents, specific primers and qPCR reaction system reagents for detecting The expression of related genes, the related genes include at least one of TRPV1 gene, SP gene, NGF gene and TNF-α gene. The present invention also provides an application of the above-mentioned kit. By establishing a zebrafish model, the kit is used to screen out cosmetic raw materials that relieve skin pain. This greatly reduces the experimental cycle, which is of great significance to the development of cosmetic ingredients that relieve skin pain and itching.

Description

Kit and application for screening skin pain relief raw materials based on TRPV1 signaling pathway in zebrafish model

Technical Field

The present invention belongs to the technical field of cosmetic raw material screening, and specifically relates to a kit for screening skin pain relief raw materials based on the TRPV1 signaling pathway in a zebrafish model and its application.

Background Art

Transient receptor potential vanilloid 1 (TRPV1) is a target of primary sensory nerves that produce pain in response to physical and chemical stimuli. It is expressed in widely distributed skin cells and nociceptors such as human epidermal keratinocytes and human epidermal immortalized keratinocyte cell lines, and is closely related to the occurrence of local skin itching and pain. When the TRPV1 channel expressed at the end of the skin sensory afferent fiber is activated, it promotes the exocytosis of axonal vesicles to release a large number of neuropeptides, such as substance P (SP), calcitonin gene related peptide (CGRP) and other neurogenic inflammatory factors. These neuropeptides can activate a variety of skin cells (such as keratinocytes, antigen presenting cells, sebaceous gland cells, fibroblasts, etc.), ultimately leading to skin neurogenic inflammation. Therefore, it is of great significance to screen out highly effective and mild TRPV1 inhibitors for skin soothing.

TRPV1 can be directly activated by a variety of physical and chemical factors, such as capsaicin and other vanillic acid compounds, inflammatory mediators (such as arachidonic acid metabolites), heat (＞43℃), acid (pH＜5.3), and changes in extracellular osmotic pressure. It participates in physiological activities such as cell metabolism and signal transduction by mediating Ca2+ influx. Endogenous mediators such as bradykinin, prostaglandin E2, and nerve growth factor can phosphorylate specific structures of TRPV1 by mediating intracellular cascade signals such as protein kinase A (PKA), protein kinase C (PKC), and phospholipase C (PLC), sensitizing TRPV1 and thus lowering its activation threshold. Nerve Growth Factor (NGF) is widely present in the skin, immune cells, and other tissues. In the inflammatory response, NGF binds to tyrosine kinase receptor (TrkA) to activate PKA, PKC, ERK/MAPK, and PI3K pathways to sensitize TRPV1 channels, leading to thermal hyperalgesia; in addition, NGF can also enhance the expression level of TRPV1 protein by activating and phosphorylating the p38MAPK signaling pathway. Tumor Necrosis Factor-α (TNF-α) is an important inflammatory mediator. When the TRPV1 channel of the skin sensory afferent fibers is activated, the released neuropeptides such as SP and CGRP can induce mononuclear macrophages, NK cells and T lymphocytes to produce TNF-α, which can activate the mitogen-activated protein kinase (p38-MAPK) signaling pathway, change the cell membrane permeability, allow Na+ influx, reduce the excitation threshold, and aggravate the pain response.

Compared with other commonly used animal models such as mice and rabbits, the zebrafish model has the following unique advantages: (1) The zebrafish genome has been completely sequenced and has 70%–80% homology with the human genome; (2) The zebrafish species is stable and has small individual differences; (3) It is small in size, has strong reproductive capacity, is easy to culture, can obtain a large sample size, and has a short experimental cycle. Zebrafish can simulate neurogenic inflammation involving skin cells, nerve cells, and immune cells, making it an ideal experimental model.

In the prior art, skin cells such as HaCaT cells and 3D skin models are often used to evaluate the soothing effect of cosmetics. However, such methods are difficult to detect pain stimulation perceived by skin receptors, and the evaluation of the soothing effect of cosmetics is not comprehensive enough.

Summary of the invention

In view of the above problems, the first purpose of the present invention is to design a kit for screening skin pain relieving raw materials based on the TRPV1 signaling pathway in the zebrafish model to detect the mRNA expression of related genes such as TRPV1, SP, NGF and TNF-α; the second purpose is to use the above kit to realize the rapid screening of cosmetic raw materials for TRPV1 signaling pathway inhibitors, and to screen out cosmetic raw materials for skin pain relief.

In order to achieve the above-mentioned object, the present invention provides a kit for screening skin pain relieving raw materials based on the TRPV1 signaling pathway in a zebrafish model, comprising an RNA extraction reagent, an RNA reverse transcription reagent, specific primers and a qPCR reaction system reagent, for detecting the expression of related genes, wherein the related genes include at least one of the TRPV1 gene, the SP gene, the NGF gene and the TNF-α gene.

Furthermore, the specific primer sequences for detecting TRPV1 gene expression are shown in SEQ ID NO: 1-2:

Forward: 5’-ATGCAGGGTTTACATGAGTATCTC-3’;

Reverse: 5’-GTCGGTGTAAGCAGCATTGATA-3’.

Furthermore, the specific primer sequences for detecting the expression of SP gene are shown in SEQ ID NO: 3-4:

Forward: 5’-CAAGAAGAGCCAGAGTCGTATG-3’;

Reverse: 5’-CTTTTGGGAGCGAATGTGAA-3’.

Furthermore, the specific primer sequences for detecting NGF gene expression are shown in SEQ ID NO: 5-6:

Forward: 5’-CGGCTGACTTCATTCAAGAACC-3’;

Reverse: 5’-GGAAAACATCCACCATCACATAC-3’.

Furthermore, the specific primer sequences for detecting TNF-α gene expression are shown in SEQ ID NOs: 7-8:

Forward: 5’-ATCTTCAAAGTCGGGTGTATGG-3’;

Reverse: 5’-GATTGCCCTGGGTCTTATGG-3’.

The present invention also provides an application of the above-mentioned kit, by establishing a zebrafish model, and using the kit to screen cosmetic raw materials for relieving skin pain.

Furthermore, by establishing a zebrafish model, inhibitors of the TRPV1 signaling pathway were screened out to screen cosmetic raw materials for relieving skin pain.

Furthermore, the zebrafish model uses the zebrafish AB strain and is established by LPS induction.

Furthermore, a method for using the kit to screen cosmetic raw materials for relieving skin pain comprises the following steps:

(1) Cultivating zebrafish and setting up a model group, a control group and a sample treatment group, wherein the model group and the sample treatment group are fertilized zebrafish embryos at 24 hpf transferred to an embryo culture medium containing LPS for induction, and the control group is 24 hpf zebrafish embryos transferred to an embryo culture medium without LPS for culture.

(2) adding the cosmetic raw materials to be screened to the culture medium of the sample treatment group, respectively, at a safe concentration of the cosmetic raw materials, and continuing the culture;

(3) Using the kit, RNA was extracted and purified, reverse transcribed, and PCR amplified from zebrafish embryos in the model group, control group, and sample treatment group, and the effects of cosmetic raw materials on the mRNA expression of related genes were detected and analyzed to screen cosmetic raw materials.

The beneficial effects achieved by the present invention are as follows: (1) Based on the zebrafish model, the cosmetic raw materials that relieve skin inflammation by inhibiting the TRPV1 signaling pathway are quickly screened, which greatly reduces the experimental cycle; (2) The gene expression of TRPV1 and its related pain and itch mediators is quantitatively detected, and the effects of the cosmetic raw materials to be screened are sensitively and conveniently detected; (3) The kit designed by the present invention can be used to quickly screen out cosmetic raw materials that inhibit the TRPV1 signaling pathway, which is of great significance for the development of cosmetic functional ingredients that relieve skin pain and itching.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the purity and integrity detection of RNA extracted in Example 1.

FIG. 2 is a schematic diagram of the detection results of TRPV1 mRNA after treatment in Example 1.

FIG. 3 is a schematic diagram of the SP mRNA detection results after treatment in Example 1.

FIG. 4 is a schematic diagram of the NGF mRNA detection results after treatment in Example 1. FIG.

FIG. 5 is a schematic diagram of the TNF-α mRNA detection results after treatment in Example 1.

DETAILED DESCRIPTION

The present invention is further described in detail by way of examples. The following examples are only used to further illustrate the present invention and should not be construed as limiting the present invention.

The present invention provides a kit for screening skin pain relief raw materials based on the TRPV1 signaling pathway in a zebrafish model, comprising an RNA extraction reagent, an RNA reverse transcription reagent, a specific primer and a qPCR reaction system reagent, for detecting the expression of related genes, wherein the related genes include at least one of a TRPV1 gene, a SP gene, a NGF gene and a TNF-α gene; wherein:

The specific primer sequences used to detect TRPV1 gene expression are shown in SEQ ID NO: 1-2:

Forward: 5’-ATGCAGGGTTTACATGAGTATCTC-3’;

Reverse: 5’-GTCGGTGTAAGCAGCATTGATA-3’.

The specific primer sequences used to detect SP gene expression are shown in SEQ ID NO: 3-4:

Forward: 5’-CAAGAAGAGCCAGAGTCGTATG-3’;

Reverse: 5’-CTTTTGGGAGCGAATGTGAA-3’.

The specific primer sequences used to detect NGF gene expression are shown in SEQ ID NO: 5-6:

Forward: 5’-CGGCTGACTTCATTCAAGAACC-3’;

Reverse: 5’-GGAAAACATCCACCATCACATAC-3’.

The specific primer sequences used to detect TNF-α gene expression are shown in SEQ ID NOs: 7-8:

Forward: 5’-ATCTTCAAAGTCGGGTGTATGG-3’;

Reverse: 5’-GATTGCCCTGGGTCTTATGG-3’.

The present invention also provides an application of the above kit, which is to use the kit to screen cosmetic raw materials for relieving skin pain by establishing a zebrafish model, and the specific operation steps include:

(1) Experimental fish culture: AB strain zebrafish purchased from the Chinese Zebrafish Resource Bank were used as experimental fish. Fertilized zebrafish eggs and embryos were cultured in an environment with a temperature of 28.5° C., a pH of 7.2-7.6, and a light/darkness ratio of 14 h:10 h for 24 h.

(2) Zebrafish grouping: 24 hpf fertilized zebrafish embryos were transferred to embryo culture medium containing 20 μg/ml LPS for induction to set up the model group. At the same time, another 30 24 hpf fertilized zebrafish embryos were transferred to embryo culture medium without LPS to set up the control group. The remaining sample treatment groups were treated in the same way as the model group.

(3) After 4 hours, the raw materials to be screened were added to the sample treatment group of zebrafish culture medium containing LPS. The raw material concentration was within the safe concentration (mortality ≤ 95%). The number of experimental fish in each treatment experiment was 30, and each experiment was repeated three times. The culture was continued for 24 hours.

(4) RNA extraction and purification: Take 30 zebrafish embryos from each group and homogenize them, add 1 ml of Trizol, and let it stand for 3 minutes; add 300 μL of chloroform and vortex to mix, and let it stand for 3 minutes; adjust the centrifuge speed to 12000 rpm and centrifuge at 4°C for 15 minutes. After completion, pipette the supernatant containing RNA into a new enzyme-free 1.5 mL centrifuge tube. Add an equal amount of isopropanol to the supernatant to precipitate RNA, and let it stand in a -20°C refrigerator for 20 minutes; after checking the RNA purity and integrity, the extracted and separated RNA will be immediately reverse transcribed.

(5) Reverse transcription: The isolated and purified RNA is reverse transcribed.

(6) Primer design and sequence.

(7) PCR amplification: The synthesized cDNA was amplified by PCR and analyzed by fluorescence using the SYBR GREENI dye method.

(8) Result determination: Based on the obtained CT value, the expression level was calculated using the 2- ^ΔΔCT method with GAPDH as the internal reference.

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

Example

By establishing a zebrafish model, the above-mentioned kit for screening skin pain relieving raw materials based on the TRPV1 signaling pathway was used to screen out cosmetic raw materials for skin pain relief. The cosmetic raw materials selected in the embodiment of the present invention are matrine, berberine and baicalin, and these three cosmetic raw materials are tested for skin pain relief by the following steps.

The specific steps are as follows:

1. RNA Extraction

(1) Experimental fish culture: AB strain zebrafish purchased from the Chinese Zebrafish Resource Bank were used as experimental fish. Fertilized zebrafish eggs and embryos were cultured in an environment with a temperature of 28.5° C., a pH of 7.2-7.6, and a light/darkness ratio of 14 h:10 h for 24 h.

(2) Zebrafish grouping: Fertilized zebrafish embryos at 24 hpf were transferred to embryo culture medium containing 20 μg/ml LPS for induction to set up the model group. At the same time, another 30 fertilized zebrafish embryos at 24 hpf were transferred to embryo culture medium without LPS to set up the control group. The three sample treatment groups of matrine, berberine and baicalin were treated in the same way as the model group.

(3) After 4 hours, the raw materials to be screened were added to the zebrafish culture medium containing LPS in the sample treatment group. The sample concentration was set to 5 mg/mL. The number of experimental fish in each treatment experiment was 30, and each experiment was repeated three times. The culture was continued for 24 hours. The three sample treatment groups were model + matrine, model + berberine, and model + baicalin.

(4) RNA extraction and purification: Take 30 zebrafish embryos from each group and homogenize them. Add 1 ml of Trizol and let stand for 3 minutes. Add 300 μL of chloroform and vortex to mix. Let stand for 3 minutes. Adjust the centrifuge speed to 12,000 rpm and centrifuge at 4°C for 15 minutes. After completion, aspirate the supernatant containing RNA into a new enzyme-free 1.5 mL centrifuge tube. Add an equal amount of isopropanol to the supernatant to precipitate the RNA. Invert and mix. Place in a -20°C refrigerator and let stand for 20 minutes. Set the speed to 12,000 rpm and centrifuge at 4°C for 15 minutes. After completion, discard the supernatant. Add 1 mL of 75% ethanol (DEPC-prepared), wash the precipitate, and centrifuge. Set the speed to 12,000 rpm and centrifuge at 4°C for 5 minutes to remove the liquid. Finally, dry the precipitated RNA in a fume hood. After the RNA is slightly dry, add an appropriate volume of DEPC water to dissolve the RNA.

(5) RNA purity and integrity detection: Take 8 μL of total RNA and use TGem microspectrophotometer for detection. Load 1.5 μL each time and detect 5 times. Record the total RNA concentration and OD _260nm /OD _280nm and calculate the average value. If the OD _260nm /OD _280nm ratio is between 1.8 and 2.1, it means that the extracted RNA has a high purity. Take 3 μL of total RNA sample and detect it by 1.5% agarose gel electrophoresis at a constant voltage of 150V for 15 minutes. Use a gel imaging system to observe RNA bands and take pictures. The detection results of the above model group, control group and sample treatment group are shown in Table 1 and Figure 1.

Table 1 RNA purity test

As shown in the results of RNA purity test in Table 1, the ratio of OD _260nm / OD _280nm of RNA extracted from the above groups was between 1.8 and 2.1, which indicated that the extracted RNA had high purity. As shown in Figure 1, from left to right, the groups were control group, model group, model + matrine, model + berberine, model + baicalin, among which the 18S and 28S bands were clear and complete, indicating that the extracted RNA had good integrity.

2. qPCR detection

The extracted RNA that has passed the purity and integrity test is reverse transcribed to synthesize cDNA. The specific steps are as follows:

(1) Reverse transcription: The separated and purified RNA was subjected to the reaction system shown in Table 2 and the reaction conditions shown in Table 3. In this embodiment, the reverse transcriptase in the reaction system was MMLV reverse transcriptase, and the reverse transcription buffer was MMLV reverse transcription buffer.

Table 2 Reverse transcription reaction system

Table 3 Reverse transcription reaction conditions

(2) The primer sequences designed for qPCR are shown in Table 4.

Table 4 Specific primers

(3) After reverse transcription, qPCR quantitative analysis

The synthesized cDNA was amplified by PCR according to the reaction system shown in Table 5 and the reaction conditions shown in Table 6 below, and fluorescence analysis was performed using the SYBR GREENI dye method.

Table 5 PCR amplification reaction system

Table 6 PCR amplification reaction conditions

(4) Result determination: Based on the obtained CT value, GAPDH was used as the internal reference and the 2 ^-ΔΔCT method was used for calculation.

The final results of each group are shown in Figures 2 to 5. In the model group induced by LPS, the expression level of zebrafish TRPV1 mRNA increased significantly, and the mRNA expression of pain-related genes such as SP, NGF and TNF-α was significantly increased. As shown in Figure 2, after adding appropriate concentrations of matrine, berberine and baicalin, the mRNA expression of TRPV1 was higher than that of the control group, but it was significantly decreased compared with the model group; as shown in Figure 3, after adding appropriate concentrations of matrine, berberine and baicalin, the mRNA expression of SP was higher than that of the control group, but it was significantly decreased compared with the model group; as shown in Figure 4, after adding appropriate concentrations of matrine, berberine and baicalin, the mRNA expression of NGF was higher than that of the control group, but it was significantly decreased compared with the model group; as shown in Figure 5, after adding appropriate concentrations of matrine, berberine and baicalin, the mRNA expression of TNF-α was higher than that of the control group, but it was significantly decreased compared with the model group. The above results also show that the cosmetic raw materials matrine, berberine and baicalin selected in the examples of the present invention can be used as skin pain relief raw materials.

In summary, the present invention uses the kit for screening skin pain relieving raw materials based on the TRPV1 signaling pathway of the zebrafish model, which can accurately and quickly detect the TRPV1 gene and its related pain mediators SP, NGF and TNF-α genes, and is suitable for effective and efficient screening of skin pain relieving cosmetic functional ingredients.

The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims (9)

1. A kit for screening raw materials for skin pain relief based on the TRPV1 signaling pathway in a zebrafish model, characterized in that it includes RNA extraction reagents, RNA reverse transcription reagents, specific primers and qPCR reaction system reagents for detecting related genes expression, the related genes include at least one of TRPV1 gene, SP gene, NGF gene and TNF-α gene. 2. The kit according to claim 1, wherein the specific primer sequence for detecting TRPV1 gene expression is as shown in SEQ ID NO: 1-2: Forward: 5'-ATGCAGGGTTTACATGAGTATCTC-3'; Reverse: 5'-GTCGGTGTAAGCAGCATTGATA-3'. 3. The kit according to claim 1, wherein the specific primer sequence for detecting SP gene expression is as shown in SEQ ID NO: 3-4: Forward: 5'-CAAGAAGAGCCAGAGTCGTATG-3'; Reverse: 5'-CTTTTGGGAGCGAATGTGAA-3'. 4. The kit according to claim 1, wherein the specific primer sequence for detecting NGF gene expression is as shown in SEQ ID NO: 5-6: Forward: 5'-CGCTGACTTCATTCAAGAACC-3'; Reverse: 5'-GGAAAACATCCACCATCACATAC-3'. 5. The kit according to claim 1, wherein the specific primer sequence for detecting TNF-α gene expression is as shown in SEQ ID NO: 7-8: Forward: 5'-ATCTTCAAAGTCGGGTGTATGG-3'; Reverse: 5'-GATTGCCCTGGGTCTTATGG-3'. 6. An application of the kit according to any one of claims 1-5, characterized in that, by establishing a zebrafish model, the kit is used to screen skin pain-relieving cosmetic raw materials. 7. The application according to claim 6, wherein the inhibitor of the TRPV1 signaling pathway is screened by establishing a zebrafish model to screen cosmetic raw materials for skin pain relief. 8. The application according to claim 6, characterized in that the zebrafish model uses the zebrafish AB strain, and establishes a related model through LPS induction. 9. The application according to claim 6, characterized in that, the method of using the kit to screen the skin pain-relieving cosmetic raw materials comprises the following steps: (1) Cultivate zebrafish, and set up model group, control group and sample treatment group, wherein, model group and sample treatment group are the zebrafish embryos that fertilize 24hpf are transferred to containing LPS embryo culture medium to induce, and the control group is that Zebrafish embryos at 24hpf were transferred to LPS-free embryo culture medium; (2) Add the cosmetic raw materials to be screened respectively to the culture solution of the sample treatment group, the concentration of the cosmetic raw materials is a safe concentration, and continue to cultivate; (3) Using the kit, carry out RNA extraction and purification, reverse transcription, and PCR amplification on the zebrafish embryos of the model group, the control group, and the sample treatment group, and detect and analyze the influence of cosmetic raw materials on the mRNA expression of related genes , to screen cosmetic raw materials.

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