Application of folium cortex eucommiae in preparation of product for regulating gonadotropin-releasing hormone receptor pathway

Abstract

The invention relates to the technical field of biological medicines, and discloses application of folium cortex eucommiae in preparation of a product for regulating a gonadotropin-releasing hormone receptor pathway. The invention provides application of folium cortex eucommiae in preparation of a product for regulating a gonadotropin-releasing hormone receptor pathway, and provides a novel method for regulating gonadotropin-releasing hormone, particularly regulating the expression level of related genes of the gonadotropin-releasing hormone receptor pathway.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to use of eucommia leaves in preparing products for regulating a gonadotropin-releasing hormone receptor pathway. Background Art

[0002] Eucommia ulmoides Oliver is a plant of the genus Eucommia in the family Eucommia, and is a national second-level precious protected tree species. Eucommia is a Chinese medicinal material, and the bark of Eucommia has a long history of medicinal use and is widely used in clinical practice. Eucommia was first recorded in Shennong's Herbal Classic, which describes it as "mainly treating pain in the waist and knees, tonifying the middle, replenishing the essence and qi, strengthening the tendons and bones, removing itching and dampness under the genitals, and residual urine. Long-term use can make the body light and resist aging." Traditional Chinese medicine believes that Eucommia is sweet in taste, warm in nature, and belongs to the liver and kidney meridians. It has the effects of tonifying the liver and kidneys, strengthening the tendons and bones, and calming the fetus. It is used to treat liver and kidney deficiency, pain in the waist and knees, weakness of the tendons and bones, dizziness, and discomfort during pregnancy. Modern pharmacological research has also proved that Eucommia ulmoides (bark, leaves and male flowers) has significant effects in "three reductions" (lowering blood pressure, blood sugar and blood lipids), "four protections" (protecting the cardiovascular system, liver, kidneys and eyesight), and "six resistances" (anti-tumor, anti-infarction, anti-osteoporosis and aging, anti-virus and bacteria, anti-allergy and anti-oxidation), with no toxic side effects.

[0003] However, the regulatory effect of Eucommia ulmoides on the gonadotropin-releasing hormone receptor pathway has not been reported. Summary of the invention

[0004] In view of this, the present invention provides use of eucommia leaves in preparing products for regulating the gonadotropin-releasing hormone receptor pathway.

[0005] In a first aspect, the present invention provides a use of Eucommia ulmoides leaves in preparing a product for regulating a gonadotropin-releasing hormone receptor pathway.

[0006] In an optional embodiment, the regulating the gonadotropin-releasing hormone receptor pathway comprises:

[0007] Regulates the expression of genes involved in the gonadotropin-releasing hormone receptor pathway.

[0008] In an optional embodiment, the gonadotropin-releasing hormone receptor pathway-related genes include:

[0009] RAC1 gene, ID3 gene, PTK2 gene, MAP3K4 gene, EGR1 gene, JUNB gene, DUSP1 gene, TCF7 gene, SLC2A1 gene, PLA2G6 gene, MAP2K3 gene, BMP4 gene, MIF gene, MAPK3 gene, FST gene.

[0010] In an optional embodiment, the regulation of the expression of gonadotropin-releasing hormone receptor pathway-related genes comprises:

[0011] Down-regulate the expression of RAC1, ID3, PTK2, MAP3K4 and EGR1 genes;

[0012] and / or, up-regulating the expression of JUNB gene, DUSP1 gene, TCF7 gene, SLC2A1 gene, PLA2G6 gene, MAP2K3 gene, BMP4 gene, MIF gene, MAPK3 gene and FST gene.

[0013] In an optional embodiment, the product is prepared from a raw material comprising Eucommia ulmoides leaves and / or Eucommia ulmoides leaf extract.

[0014] In an optional embodiment, the product contains Eucommia ulmoides leaves and / or Eucommia ulmoides leaf extract as the only active ingredient.

[0015] In an optional embodiment, the product includes at least one of medicine, food or health care product.

[0016] The technical solution provided by the present invention has at least the following beneficial effects:

[0017] The present invention provides the use of eucommia leaves in preparing products for regulating the gonadotropin-releasing hormone receptor pathway, and provides a new method for regulating gonadotropin-releasing hormone, especially regulating the expression level of genes related to the gonadotropin-releasing hormone receptor pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is a diagram showing the gene expression sequencing results of the gonadotropin-releasing hormone receptor pathway in the group treated with the eucommia leaf extract in an example of the present invention. DETAILED DESCRIPTION

[0020] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0021] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0022] The present invention is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed by the present invention.

[0023] Example

[0024] 1. Experimental Medicinal Materials

[0025] Eucommia leaves were collected from Beijing Medicinal Botanical Garden. The dried Eucommia leaves were ground and extracted with distilled water at 100°C for 2 hours. The supernatant was sieved, concentrated under reduced pressure at below 40°C using a rotary evaporator, and freeze-dried to obtain Eucommia leaf extract. 1 kg of dry extract powder (Eucommia leaf extract) was produced for every 10 kg of Eucommia dry leaves.

[0026] 2. Cell Culture and Drug Treatment

[0027] HepG2 cells were placed in 37°C, CO 2 The cells were cultured in an incubator with a volume fraction of 5% and a humidity of 97%. The experiment was divided into: a blank control group, DMEM medium containing 10% fetal bovine serum; a drug treatment group, 100 μg / mL of Eucommia ulmoides leaf extract was added to the complete medium.

[0028] 3. Total RNA extraction and detection

[0029] According to the instructions of the RNA extraction kit, the blank control group and the drug treatment group (treated for 24 hours) were operated respectively. The main process is as follows:

[0030] (1) All consumables used in the experiment were treated with 0.1% DEPC (diethylpyrocarbonate) aqueous solution at 37°C for 12 h, and then autoclaved at 120°C for 30 min to remove residual DEPC;

[0031] (2) Add appropriate amount of RNAiso Plus to cells in each group and fully homogenize;

[0032] (3) Transfer the homogenate to a centrifuge tube and let it stand at room temperature for 5 min;

[0033] (4) Centrifuge at 12,000 x g at 4°C for 5 min, then carefully aspirate the supernatant and transfer it to a new centrifuge tube (do not aspirate the precipitate);

[0034] (5) Add chloroform equivalent to 1 / 5 of the volume of RNAiso Plus to the supernatant, shake and mix until the solution is emulsified and milky white, and let stand at room temperature for 5 min;

[0035] (6) Centrifuge at 12,000 x g for 15 min at 4°C. The homogenate will separate into three layers: a colorless supernatant (containing RNA), a white protein layer in the middle (mostly DNA), and a colored lower organic phase. Carefully transfer the supernatant to a new centrifuge tube (do not aspirate the white middle layer).

[0036] (7) Add 0.5-1 times the volume of RNAiso Plus isopropanol to the supernatant, invert the tube to mix thoroughly, and let stand at room temperature for 10 min;

[0037] (8) Centrifuge at 12,000 x g for 10 min at 4°C. RNA precipitate will appear at the bottom of the tube. Carefully discard the supernatant and wash the precipitate with an equal amount of 75% ethanol as RNAiso Plus.

[0038] (9) Centrifuge at 7500 × g for 5 min at 4 °C, discard the supernatant and keep the precipitate, let it stand to dry at room temperature, and then dissolve the precipitate in an appropriate amount of DNase / RNase-free water;

[0039] (10) 1% agarose gel electrophoresis was used to analyze the degree of RNA degradation and whether there was any contamination. The purity and concentration of RNA were detected by Nanodrop 2000 / 2000C spectrophotometer. An OD260 / 280 ratio between 1.8 and 2.0 indicated that the sample was qualified and the RNA was not contaminated by impurities such as protein and phenol. The RNA sample concentration was also obtained. The integrity of RNA was accurately detected using Agilent 2100.

[0040] 4. Library Construction and Library Testing

[0041] After the sample is tested and qualified, press Ultra TMThe RNA library preparation kit instructions were used to construct the sequencing library, and the index code was added to the attribute sequence of each sample. Magnetic beads with Oligo (dT) were used to enrich the mRNA of eukaryotic organisms by binding to the ployA tail of mRNA through AT complementary pairing. Then, the fragmentation buffer was added to break the mRNA into short fragments. Using mRNA as a template, random hexamers and M-MuLV reverse transcriptase (RNase H-) were used to synthesize the first-strand cDNA. Then, buffer, dNTPs and DNA polymeraseⅠ were added to synthesize the second-strand cDNA. Then, the double-stranded cDNA was purified using AMPure XP beads. The purified double-stranded cDNA was then end-repaired, A-tailed and connected to the sequencing adapter. Then, the fragment size was selected using AMPure XP beads, and finally, PCR enrichment was performed to obtain the final cDNA library.

[0042] After the library construction is completed, Qubit2.0 is used for preliminary quantification to dilute the library to 1 ng / uL, and then the insert size of the library is detected using Agilent 2100. After the insert size meets expectations, the effective concentration of the library is accurately quantified using the Q-PCR method (library effective concentration>2nM) to ensure the quality of the library.

[0043] 5. Sequencing

[0044] All samples were sequenced on the Illumina HiSeq 4000 platform. The sequencing process was performed at Beijing Novogene Co., Ltd., and the original sequencing sequences were finally obtained.

[0045] 6. Sequencing data filtering

[0046] The original sequencing sequence obtained by sequencing contains low-quality reads with adapters. In order to ensure the quality of information analysis, it is necessary to filter the raw reads to obtain clean reads. Subsequent analysis is based on clean reads. The data filtering steps are as follows:

[0047] (1) Remove reads with adapters;

[0048] (2) remove reads with a ratio of N (N means the base information cannot be determined) greater than 10%;

[0049] (3) Remove low-quality reads (reads in which the number of bases with Qphred < 20 accounts for more than 50% of the entire read length).

[0050] 7. Quantitative Analysis

[0051] HISAT 2.0.4 software was used to align the filtered clean reads to the human reference genome to predict new transcripts. After the clean reads were aligned to the reference genome, the parameters and processes recommended by Cufflinks 2.1.1 software were used to assemble transcripts. HTSeq 0.9.1 software was used to calculate the number of reads mapped to each gene, and then the FPKM of each gene was calculated based on the length of the gene, and the reads were mapped to the gene. FPKM is the number of fragments per kilobase length from a gene per million fragments. It takes into account the effects of sequencing depth and gene length on fragment counts and is currently the most commonly used method for estimating gene expression levels.

[0052] 8. Screening of differentially expressed genes

[0053] First, the original read count is normalized, mainly to correct the sequencing depth. Then the hypothesis test probability (P-value) is calculated through the statistical model, and finally the multiple hypothesis test correction (BH) is performed to obtain the FDR value (false discovery rate, padj is its common form). The DESeq2 software is used to determine the differential expression in digital gene expression data using a model based on the negative binomial distribution. Generally speaking, if the expression difference of a gene in two groups of samples is more than twice, we believe that such a gene has an expression difference. In this experiment, the differential gene screening criteria used |log2(FoldChange)|>1&padj<0.05.

[0054] IX. KEGG enrichment analysis

[0055] KEGG (Kyoto Encyclopedia of Genes and Genomes) is a database that integrates genomic, chemical and system functional information. The KEGG pathway database (https: / / www.kegg.jp / kegg / pathway.html) that we use most often stores a large amount of functional information. The KEGG pathway database contains seven pathways: metabolism, genetic information processing, environmental information processing, cellular processes, biological systems, human diseases and drug development. The differential gene-pathway-disease network was constructed using cytoscape software. The KEGG pathway enrichment uses padj less than 0.05 as the threshold for significant enrichment.

[0056] 10. Experimental Results

[0057] Through differential gene analysis and KEGG enrichment analysis of up-regulated genes, it was found that compared with the blank control group, the gene expression of the gonadotropin-releasing hormone receptor pathway in the Eucommia leaf extract treatment group changed significantly, as shown in the following table. Figure 1 and Table 1.

[0058] in, Figure 1 This is the result of gene expression sequencing of the gonadotropin-releasing hormone receptor pathway in the group treated with Eucommia ulmoides leaf extract. The arrow indicates the gonadotropin-releasing hormone receptor pathway. Figure 1 It can be seen that the gene expression of multiple signaling pathways in the group treated with Eucommia ulmoides leaf extract changed significantly.

[0059] Table 1 lists the 15 genes with significant changes in expression in the gonadotropin-releasing hormone receptor pathway in the group treated with eucommia leaf extract.

[0060] Table 1

[0061] Gene Average expression log2(FoldChange) P-value RAC1 40.54 -0.69 0.004 ID3 26.35 -0.69 0.009 PTK2 6.94 -0.65 0.017 MAP3K4 44.07 -0.62 0.006 EGR1 30.55 -0.49 0.047 JUNB 90.13 0.34 0.050 DUSP1 0.96 0.34 0.027 TCF7 221.87 0.39 0.003 SLC2A1 1.08 0.43 0.019 PLA2G6 2.27 0.44 0.048 MAP2K3 22.65 0.54 0.028 BMP4 60.45 0.56 0.002 MIF 7.87 0.71 0.011 MAPK3 23.51 0.75 0.002 FST 52.39 0.80 0.000

[0062] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. Use of Eucommia ulmoides leaves in preparing products for regulating the gonadotropin-releasing hormone receptor pathway.

2. The use according to claim 1, characterized in that The method of regulating the gonadotropin-releasing hormone receptor pathway comprises: Regulates the expression of genes involved in the gonadotropin-releasing hormone receptor pathway.

3. The use according to claim 2, characterized in that: The gonadotropin-releasing hormone receptor pathway-related genes include: RAC1 gene, ID3 gene, PTK2 gene, MAP3K4 gene, EGR1 gene, JUNB gene, DUSP1 gene, TCF7 gene, SLC2A1 gene, PLA2G6 gene, MAP2K3 gene, BMP4 gene, MIF gene, MAPK3 gene, FST gene.

4. The use according to claim 3, characterized in that The regulation of the expression of genes related to the gonadotropin-releasing hormone receptor pathway comprises: Down-regulate the expression of RAC1, ID3, PTK2, MAP3K4 and EGR1 genes; and / or, up-regulating the expression of JUNB gene, DUSP1 gene, TCF7 gene, SLC2A1 gene, PLA2G6 gene, MAP2K3 gene, BMP4 gene, MIF gene, MAPK3 gene and FST gene.

5. The use according to any one of claims 1 to 4, characterized in that The product is prepared from raw materials including eucommia leaves and / or eucommia leaf extracts.

6. The use according to claim 5, characterized in that The product uses eucommia leaves and / or eucommia leaf extract as the only active ingredient.

7. The use according to claim 5, characterized in that The product includes at least one of medicine, food or health care product.

Images