CN113125763A - Application of combination marker in preparation of evaluation product for effect of cassia twig and poria cocos capsules on treatment of primary dysmenorrhea - Google Patents
Application of combination marker in preparation of evaluation product for effect of cassia twig and poria cocos capsules on treatment of primary dysmenorrhea Download PDFInfo
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Abstract
The cassia twig and tuckahoe capsule for treating primary dysmenorrheal lacks an objective curative effect evaluation index clinically. The inventor discovers that the cassia twig and poria cocos capsule regulates the internal flow of calcium ions in cells and intervenes COX2 to play an anti-inflammatory and analgesic effect to relieve primary dysmenorrhea through network pharmacological calculation prediction and experimental verification, and discovers that the cassia twig and poria cocos capsule intervenes endocrine hormone and microcirculation to regulate intrauterine circulation to play a sustained effect. The invention relates to application of a system (a combined biomarker consisting of TXB2, 6-Keto-PGF1 alpha, COX2, NO, ET-1 and intracellular calcium ions) in preparing a product for evaluating the effect of a cassia twig and poria cocos capsule on treating primary dysmenorrhea. In vitro experiments show that the cassia twig and poria cocos capsule can remarkably reduce the ratio of TXB2 to TXB2/6-Keto-PGF1 alpha in plasma, remarkably increase NO in uterine tissues, remarkably reduce ET-1 level, and inhibit COX2 enzyme activity and calcium ion inflow of uterine smooth muscle cells. Clinical tests show that the cassia twig and tuckahoe capsule has significantly better pain relieving effect than placebo and has obvious sustained effect after stopping taking the medicine.
Description
Technical Field
The invention relates to the field of medicines, and relates to application of a combined marker in preparation of a product for evaluating the effect of a cassia twig and poria cocos capsule on treating primary dysmenorrhea.
Background
Primary Dysmenorrhea (PD) refers to a functional Dysmenorrhea caused by organic lesions of non-reproductive organs, which seriously affects the normal work and quality of life of patients[1]. The pathogenesis of the disease involves changes of Prostaglandins (PGs), Oxytocin (OT), estrogen-progestogen, beta-endorphin (beta-EP) and the like[2]。
The cassia twig and tuckahoe capsule consists of five traditional Chinese medicines of cassia twig, tuckahoe, tree peony bark, white paeony root and peach kernel, and has the efficacies of promoting blood circulation by removing blood stasis, eliminating stagnation and eliminating mass[3]. The cassia twig and tuckahoe capsules have various pharmacological actions, and have been reported to have wide functions of immunoregulation, anti-tumor, anti-inflammatory, analgesia, smooth muscle regulation, endocrine regulation, hemorheology improvement and the like[4]The traditional Chinese medicine composition is mainly applied to the treatment of gynecological diseases such as hysteromyoma, pelvic inflammation, dysmenorrhea, endometriosis, ovarian cyst and hyperplasia of mammary glands in clinic. However, due to the complexity of the Chinese herbs system and the organism biological system, these studies cannot explain the mechanism of action of the Chinese herbs for treating the disease well[5]. How to identify objective curative effect indexes of the cassia twig and tuckahoe capsules for treating the primary dysmenorrhea is a problem to be solved urgently when the cassia twig and tuckahoe capsules are widely used clinically at present.
The network pharmacology 'network target-system regulation' research mode breaks through the limitation that the disease and the drug research are disjointed[6，7]And the method is more consistent with the essence of a biological system. Analyzing traditional Chinese medicine action machine through research mode of' network target-system regulationPreparation, a new method and a new idea are developed for the clinical pharmacological research of the traditional Chinese medicine[8，9]. The idea and method of network target-system regulation are well applied to understanding the action mechanism of traditional Chinese medicine prescription and natural medicine[10，11]Further provides a new idea and a new method for identifying objective curative effect indexes of the cassia twig and poria cocos capsules for treating the primary dysmenorrhea.
The inventor discovers an objective curative effect evaluation biomarker for treating the primary dysmenorrhea by using the cassia twig and poria cocos capsules through network pharmacological calculation prediction and experimental verification.
Disclosure of Invention
The inventor discovers that the cassia twig and poria cocos capsule mainly influences five biological function network modules of hormone regulation, inflammatory reaction, microcirculation, calcium ion inflow and pain perception to treat primary dysmenorrhea by performing target prediction and biological function enrichment analysis on compounds contained in the cassia twig and poria cocos capsule. The inventor confirms that the cassia twig and poria cocos capsule regulates the internal flow of calcium ions in cells and intervenes COX2 to play an anti-inflammatory and analgesic role to relieve primary dysmenorrhea, and can intervene endocrine hormone and uterine microcirculation to play a continuous effect of treating primary dysmenorrhea.
In vitro experiments carried out by the inventor show that the cassia twig and poria cocos capsule can remarkably reduce the ratio of TXB2 and TXB2/6-Keto-PGF1 alpha in plasma, remarkably increase NO in uterine tissues, remarkably reduce ET-1 level, and inhibit COX2 enzyme activity and calcium ion influx of uterine smooth muscle cells. Clinical tests show that the cassia twig and tuckahoe capsule has significantly better pain relieving effect than placebo and has obvious sustained effect after stopping taking the medicine.
According to an aspect of the present invention, there is provided a use of an agent for detecting an expression level of a marker for the manufacture of a product for evaluating an effect of Guizhi Fuling Capsule for treating Primary dysmenorrhea, wherein the marker comprises at least one selected from the group consisting of:
the presence of the TXB2 in the blood,
6-Keto-PGF1 alpha in blood,
the COX2 in the blood is,
the presence of NO in the uterine tissue,
ET-1 in uterine tissue.
According to a further aspect of the invention, the above application comprises:
the objective curative effect of the Guizhi Fuling capsules on the primary dysmenorrhea is evaluated according to the content of TXB2 in blood and/or the change trend of the content, the content of 6-Keto-PGF1 alpha in blood and/or the change trend of the content, the content of COX2 in blood and/or the change trend of the content, the content of NO in uterine tissue and/or the change trend of the content, the content of ET-1 in uterine tissue and/or the change trend of the content and the concentration of calcium ions in uterine smooth muscle cells.
According to another aspect of the present invention, there is provided an immunohistochemical test kit for evaluating the efficacy of Guizhi Fuling Capsule for treating Primary dysmenorrhea, comprising a step of immunostaining at least one protein selected from the following protein molecules:
the presence of the TXB2 in the blood,
6-Keto-PGF1 alpha in blood,
the COX2 in the blood is,
the presence of NO in the uterine tissue,
ET-1 in uterine tissue.
According to still another aspect of the present invention, there is provided a system for evaluating the effect of Guizhi Fuling capsule for treating primary dysmenorrhea, comprising:
an immunohistochemical detection kit for evaluating the effect of the cassia twig and poria cocos capsules on treating primary dysmenorrhea is used for immunostaining at least one selected from the following protein molecules:
the presence of the TXB2 in the blood,
6-Keto-PGF1 alpha in blood,
the COX2 in the blood is,
the presence of NO in the uterine tissue,
the presence of ET-1 in the uterine tissue,
and the calcium ion signal measuring device is used for detecting the calcium ion concentration in the smooth muscle cells of the uterine tissue.
Drawings
Fig. 1 is a primary dysmenorrheal-related molecular network regulated by cassia twig and poria cocos capsules in multiple components.
FIG. 2 is a graph showing the results of experiments on the inhibition of the calcium ion influx in uterus smooth muscle cells by Guizhi Fuling Capsule.
FIG. 3 is the experimental verification chart of the key enzyme COX2 enzyme activity in the inhibition of inflammation and pain by Guizhi Fuling Jiaonang.
FIGS. 4A-4B are graphs of the results of clinical studies on Guizhi Fuling Capsule.
Detailed Description
The inventor discovers a network regulation mechanism of the cassia twig and tuckahoe capsule for treating the primary dysmenorrhea by performing target prediction and biological function enrichment analysis on compounds contained in the cassia twig and tuckahoe capsule. Furthermore, the experiment proves that the cassia twig and poria cocos capsule is a biomarker for treating primary dysmenorrhea. The work performed by the present inventors includes:
(1) cassia twig and poria cocos capsule component target prediction and biological function enrichment analysis
(2) Experiments prove that the cassia twig and tuckahoe capsule reduces calcium ion signals in uterus smooth muscle cells
(3) Experiments prove that the cassia twig and tuckahoe capsule inhibits the activity of COX2
(4) Experiments prove that the cassia twig and tuckahoe capsule improves the uterine microcirculation disturbance
(5) Clinical experiments prove that the pain relieving effect of the cassia twig and tuckahoe capsule component combination is obviously superior to that of a placebo, and the cassia twig and tuckahoe capsule component combination has an obvious sustained effect after the medicine is stopped.
Example 1: network regulation mechanism of cassia twig and poria cocos capsules for treating primary dysmenorrhea
The inventor collects the ingredients contained in the cassia twig and tuckahoe capsules from the literature and predicts the target spectrum of the ingredients of the cassia twig and tuckahoe capsules by using a high-precision medicine target prediction method. And (4) calculating the biological functions influenced by the cassia twig and poria cocos capsule compound by utilizing enrichment analysis.
1. Collection of Compound contained in Guizhi Fuling Capsule
The cassia twig and tuckahoe capsule comprises 5 traditional Chinese medicines of cassia twig, tuckahoe, tree peony bark, white paeony root and peach kernel together, which are obtained from the literature[13]The compounds contained in the Chinese medicaments are collected. 122 compounds are collected, wherein 30 compounds contained in cassia twig, 33 compounds contained in tuckahoe, 66 compounds contained in moutan bark and compound contained in white paeony root57 kinds of compounds and 28 kinds of compounds contained in peach kernel.
2. Compound target prediction and biofunctional enrichment analysis
In the invention, the inventor uses a high-precision compound target prediction algorithm to calculate a target spectrum for predicting the direct action or indirect action of the traditional Chinese medicine monomeric compound. The inventor utilizes Fisher accurate test to perform enrichment analysis to calculate the biological functions influenced by the cassia twig and poria cocos capsule compound. Specifically, a Gene set under a predicted target and a Gene ontology Biological process (GO BP) entry of a cassia twig tuckahoe capsule compound is enriched, 4-6 grades of GO BP entries are reserved for reflecting a more specific Biological process and a remarkable Biological process (Pvalue < 0.05) is screened out. Then, the inventor finds that the cassia twig and poria cocos capsule mainly affects five biological function network modules of hormone regulation, inflammatory reaction, microcirculation, calcium ion influx and pain perception according to keyword mapping and functional description of terms, as shown in table 1. Thereby identifying five biological functions related to the regulation of the primary dysmenorrhea by the cassia twig and poria cocos capsules, as shown in figure 2.
TABLE 1 Cassia twig and Poria cocos capsule compound prediction target spectrum biological function enrichment analysis
Example 2: cassia twig and poria cocos capsule for reducing calcium ion signals in uterus smooth muscle cells
1. Experimental methods
The experimental design was as follows: ICR female mice were injected with estradiol benzoate injection for two days (0.1 mg per injection). ICR female mice were sacrificed by cervical dislocation, sterilized with 75% alcohol, and the uteri removed and placed in PBS freshly added to double antibody. Stripping fat and connective tissues, longitudinally cutting open the uterus, gently scraping the endometrium with a scalpel, and washing with PBS for 1-2 times. Buffer was discarded, the ophthalmic scissors cut the muscle strips into 3 minced pieces of 1-2mm, 2ml of 0.025 g.L-1 collagenase II was added and transferred into Erlenmeyer flasks, and 5 ml of collagenase II was used to wash the scissors. Moving the conical flask to digest in a water bath at 37 deg.C for 1h, and blowing with a pasteur pipette for 5min every half an hour. Equal volumeStopping digestion of complete culture medium containing 15% serum, filtering with 200 mesh cell sieve, centrifuging filtrate 1000r × 10min, discarding supernatant, suspending cells from complete culture medium, and inoculating into 25cm2 cell bottle for culture. Every two days, PBS is washed for 2 times, dead cells and impurities are removed, complete culture medium is added, and the culture is continued in a 5% CO2 cell culture box at 37 ℃. Cells were cultured to day 5, the supernatant was discarded, 2ml of 0.025 g.L-1 trypsin +0.002 g.L-1 EDTA was added for digestion for 5min, digestion was stopped with an equal volume of complete medium, centrifugation was carried out at 1000 r.times.3 min, the supernatant was discarded, complete medium was added, cells were suspended, 1) plated at 1X 105/ml density in a black 96-well plate, and cultured overnight in a 5% CO2 cell incubator at 37 ℃. The supernatant was discarded, 100. mu.L of Calcium 6-QF solution was added, and the mixture was incubated at 37 ℃ for 2 hours. Grouping according to need, adding the drugs for action for 15min, use
3, detecting dynamic calcium ion change by a calcium flow workstation: the instrument was set to run for 20s and 50. mu.L PBS was added to the blank and 50. mu.L PGF 2. alpha. 50 was added to the remaining groups to a final concentration of 420 nM, and the change in calcium ion was measured over 100 s.
2. Results of the experiment
The experimental results prove that the cassia twig and poria cocos capsule can reduce calcium ion signals in uterine smooth muscle cells, and the results are shown in figure 3. The above results suggest that the Guizhi Fuling Capsule can inhibit the intracellular calcium ion influx caused by PGF2 alpha or Oxytocin (OT) to relax the uterine smooth muscle and reduce the uterine smooth muscle spasm contraction, thereby alleviating the symptoms of dysmenorrhea.
Example 3: substance for inhibiting COX2 enzyme activity by cassia twig and poria cocos capsule
1. Experimental methods
The experimental design was as follows: by adopting a biological evaluation method based on the activity of human recombinant COX2 enzyme detected by fluorescence, the cassia twig and poria cocos capsules are detected to have the activity of inhibiting COX 2. The technology detects the influence of the cassia twig and poria cocos capsules on the effect of COX2 according to the specification of a cyclooxygenase 2(COX2) inhibitor screening kit.
2. Results of the experiment
The experimental result verifies the inhibition effect of the cassia twig and tuckahoe capsule on COX2 which is a key enzyme in inflammation and pain, and is shown in figure 4. The results indicate that the cassia twig and poria cocos capsules can exert the anti-inflammatory and analgesic effects and improve the primary dysmenorrhea by influencing the expression and activity of a key enzyme COX2 in inflammation and pain and further influencing the synthesis of Prostacyclin (PGs) in uterus.
Example 4: cassia twig and poria cocos capsule for improving uterine microcirculation disturbance
1. Experimental methods
The experimental design was as follows: 3-month-old SD male rats in estrus, model group and administration group were injected with estradiol benzoate continuously for 5d s subcutaneously, each 0.2mg daily. Administration was started on the day of estrogen injection, and rats were given 1 time ig per day for 5 consecutive days. 1h after the last day of administration of each group of animals, rats were anesthetized by injecting 10% chloral hydrate, whole blood was taken by using an anticoagulation tube, plasma was taken by centrifuging at 3500rpm for 15min, uterine tissue was taken rapidly, washed well in pre-cooled physiological saline, and frozen in a refrigerator at-80 ℃, wherein the plasma was assayed for the levels of thromboxane B2(TXB2) and 6-Keto-prostaglandin F1 alpha (6-Keto-PGF1 alpha) by using a kit, and the level of Nitric Oxide (NO) and endothelin (ET-1) was assayed by taking the supernatant from the homogenate of the uterine tissue using a kit.
2. Results of the experiment
The experimental result shows that the cassia twig and poria cocos capsule can remarkably reduce the ratios of TXB2 and TXB2/6-Keto-PGF1 alpha in plasma, remarkably increase NO in uterine tissues and remarkably reduce ET-1 levels. As shown in tables 1 and 2. Therefore, the cassia twig and tuckahoe capsule improves the uterine microcirculation disturbance.
TABLE 2 Effect on rat model dysmenorrhea plasma TXB2, 6-Keto-PGF1 alpha ((R))
n＝10)
Comparison with the control group:#P＜0.05##p is less than 0.01; comparison with model groups:*P＜0.05**P＜0.01
TABLE 3 Effect on rat dysmenorrhea model uterus NO, ET-1: (
n＝10)
Comparison with the control group:#P＜0.05##p is less than 0.01; comparison with model groups:*P＜0.05**P＜0.01
example 5: clinical experiments prove that the composition of the cassia twig and tuckahoe capsule components has significantly better analgesic effect than placebo, and has obvious sustained effect after drug withdrawal
1. Experimental methods
The clinical test aims at evaluating the effectiveness of the cassia twig and tuckahoe capsule in treating the primary dysmenorrhea. The primary efficacy index is the change in the maximum VAS (simulated visual score) value compared to the baseline period at the end of the treatment period and at the end of the follow-up period. The secondary efficacy index is the change in response rate (percentage of patients with greater than or equal to 50% total pain reduction) during each phase of treatment and follow-up compared to baseline. The clinical test of the period comprises a screening period, a baseline period, a treatment period and a follow-up period, wherein the period is four, namely, about 9 months, a subject starts to visit 1 in the screening period, an electronic diary is recorded every day, and after the evaluation is qualified through two complete baseline periods, the subject is randomly divided into a high dose group, a low dose group and a placebo group, wherein the low dose group is the domestic clinical dose, the high dose group is 1.5 times of the low dose group, the three groups enter the follow-up period through three treatment periods, and the test is finished after the follow-up period is 3 months. Visit 3a and visit 7a blood draw time points for hormone determination, visit 3b dispensed test drugs, and after the third menstrual cycle was completed, the subjects began to take the drugs. 612 patients with primary dysmenorrhea were screened, and the patients were enrolled into 280 patients, completed 200 patients in the treatment period, and completed 178 patients in the follow-up period.
2. Results of the experiment
The test results show that: at the end of the follow-up period, the maximum VAS values were statistically different (P <0.001) in the high dose group compared to the placebo group, and at the end of the follow-up period, the high and low dose combination group compared to the placebo group. Clinical experiments prove that the composition of the cassia twig and tuckahoe capsule has a significantly better pain relieving effect than a placebo and has an obvious sustained effect after the medicine is stopped.
Reference
[1]Burnett M，Lemyre M.No.345-Primary Dysmenorrhea Consensus Guideline[J].Journal of Obstetrics and Gynaecology Canada，2017，39(7)：585-595.
[2] Modern medical understanding and treatment research progress of primary dysmenorrhea [ J ] world latest medical information abstracts, 2019, 19 (07): 29-30.
[3] The Meta analysis of the effect of chenchen crystal, songshiang, zhuang, guo, weiluo, irish, wangxiao, cinnamomi poria capsules on primary dysmenorrhea [ J ]. journal of traditional chinese medicine in hunan, 2019, 35 (08): 126-131.
[4] Suzhenzhen, Lina, Cao Lian et al, Guizhi Fuling Capsule, the main pharmacological action and clinical application research progress [ J ] Chinese traditional medicine journal, 2015 (06): 9-12.
[5]S Li.Network Systems Underlying Traditional Chinese Medicine Syndrome and Herb Formula[J].Curr Bioinfo，2009，4(3)：188-196.
[6] Prune, network target: one entry point in the network pharmacological study of traditional Chinese medicine formulae [ J ]. journal of Chinese traditional medicine, 2011, 36 (15): 2017-2020.
[7]S Li，Zhang B.Traditional Chinese medicine network pharmacology：theory，methodology and application[J].Chin J Nat Med，2013，11(2)：110-120.
[8]Shao Li.Mapping ancient remedies：Applying a network approach to traditional Chinese medicine[J].Science，2015，350(6262Suppl)：S72-S74.
[9]S Zhao，Li S.Network-based relating pharmacological and genomic spaces for drug target identification[J].PLoS One，2010，5(7)：e11764.
[10]Y Guo，Bao C，Ma D，et al.Network-Based Combinatorial CRISPR-Cas9 Screens Identify Synergistic Modules in Human Cells[J]ACS Synth Biol，2019，8(3)：482-490.
[11]X Liang，Li H，Li S.Anovel network pharmacology approach to analyse traditional herbal formulae：the Liu-Wei-Di-Huang pill as a case study[J].Mol Biosyst，2014，10(5)：1014-1022.
[12]ZHAO S，LI S.Network-based relating pharmacological and genomic spaces for drug target identification[J].PLoS One，2010，5(7)：e11764.
[13] The former phytochemistry of traditional Chinese medicine is divided into [ M ] and [ 2009 ] scientific press.
Claims (5)
1. Use of an agent for detecting the expression level of a marker for the manufacture of a product for evaluating the effect of Guizhi Fuling Capsule for treating Primary dysmenorrhea, wherein the marker comprises at least one selected from the group consisting of:
the presence of the TXB2 in the blood,
6-Keto-PGF1 alpha in blood,
the COX2 in the blood is,
the presence of NO in the uterine tissue,
ET-1 in uterine tissue.
2. Use according to claim 1, characterized in that it comprises:
determining the objective curative effect of the Guizhi Fuling capsules on treating the primary dysmenorrhea according to the content and/or the change of the content of TXB2 in blood, the content and/or the change of the content of 6-Keto-PGF1 alpha in blood, the content and/or the change of the content of COX2 in blood, the content and/or the change of the content of NO in uterine tissue, the content and/or the change of the content of ET-1 in uterine tissue and the calcium ion concentration in uterine smooth muscle cells.
3. An immunohistochemical detection kit for evaluating the effect of the cassia twig and poria cocos capsules on treating primary dysmenorrhea is used for immunostaining at least one of the following protein molecules:
the presence of the TXB2 in the blood,
6-Keto-PGF1 alpha in blood,
the COX2 in the blood is,
the presence of NO in the uterine tissue,
ET-1 in uterine tissue.
4. An evaluation system for the effect of cassia twig and poria cocos capsules on treating primary dysmenorrhea is characterized by comprising the following components:
an immunohistochemical detection kit for evaluating the effect of the cassia twig and poria cocos capsules on treating primary dysmenorrhea is used for immunostaining at least one selected from the following protein molecules:
the presence of the TXB2 in the blood,
6-Keto-PGF1 alpha in blood,
the COX2 in the blood is,
the presence of NO in the uterine tissue,
the presence of ET-1 in the uterine tissue,
and the calcium ion signal measuring device is used for detecting the calcium ion concentration in the smooth muscle cells of the uterine tissue.
5. The evaluation system of the effect of Guizhi Fuling Jiaonang on treating primary dysmenorrhea according to claim 4, further comprising:
an evaluation device for determining the objective therapeutic effect of the Guizhi Fuling Capsule for treating Primary dysmenorrhea based on at least one selected from the following values:
the amount of TXB2 in the blood and/or changes in this amount,
the amount of 6-Keto-PGF1 alpha in blood and/or the change in the amount,
the level of COX2 in the blood and/or changes in this level,
the amount of NO in the uterine tissue and/or the change in this amount,
the amount of ET-1 in the uterine tissue and/or the change in this amount,
uterine smooth muscle intracellular calcium ion concentration.
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| CN1689614A (en) * | 2004-04-21 | 2005-11-02 | 广州中医药大学第二附属医院 | Chinese medicinal composition for treating endometriosis and its preparation method |
| CN108159148A (en) * | 2018-01-22 | 2018-06-15 | 吉林大学 | A kind of guizhi fuling pill and preparation method and application |
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| CN1689614A (en) * | 2004-04-21 | 2005-11-02 | 广州中医药大学第二附属医院 | Chinese medicinal composition for treating endometriosis and its preparation method |
| CN108159148A (en) * | 2018-01-22 | 2018-06-15 | 吉林大学 | A kind of guizhi fuling pill and preparation method and application |
| CN110531076A (en) * | 2019-07-19 | 2019-12-03 | 江苏康缘药业股份有限公司 | A method of COX-2 enzymatic activity in detection medicine analgesic evaluation procedure |
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