Traditional Chinese medicine composition for treating cow endometritis and preparation method thereof

Abstract

The invention provides a traditional Chinese medicine composition for treating cow endometritis and a preparation method of the traditional Chinese medicine composition. The traditional Chinese medicine composition comprises the following traditional Chinese medicines: motherwort, ligusticum wallichii, golden cypress, codonopsis pilosula and semen plantaginis. According to in-vivo experiments, it is found that after the traditional Chinese medicine composition is used, the sick dairy cow secretes clear secretions in the vagina, mucosal hyperemia is relieved, and the proportion of neutrophils in uterine secretions is obviously reduced; the traditional Chinese medicine composition is prepared into a drug-containing serum mode for in-vitro experiments, the treatment effect of the traditional Chinese medicine composition on cow endometritis is proved, 15% of drug-containing serum of the traditional Chinese medicine composition acts on BEND cells induced by LPS for 24 h, the expression level of TNF-alpha and IL-6mRNA is obviously reduced compared with that of an LPS + blank serum group, and the treatment effect of the traditional Chinese medicine composition on cow endometritis is obviously improved. Therefore, the traditional Chinese medicine composition has the anti-inflammatory effect. The traditional Chinese medicine composition has a good treatment effect on cow endometritis, and the in-vivo and in-vitro experiment results can relieve the disease symptoms of the cow or relieve the inflammatory reaction, so that the traditional Chinese medicine composition can effectively treat the cow endometritis clinically.

Description

Technical Field

[0001] The invention relates to the field of traditional Chinese medicine compositions, and in particular to a traditional Chinese medicine composition for treating cow endometritis and a preparation method thereof. Background Art

[0002] Endometritis in dairy cows is a mucous or purulent inflammation of the uterus, which often leads to a decrease in the pregnancy rate and production performance of dairy cows. Compared with normal dairy cows, endometritis can increase the average interval from calving to the first insemination by nearly one week, delay the conception time by about four weeks, and nearly double the possibility of dairy cows being eliminated due to conception failure. Bacterial infection is the main factor causing endometritis in dairy cows, and pathogenic bacteria grow in large numbers in the uterine microbiome after delivery.

[0003] Cow endometritis is a common reproductive system disease in dairy cow farming, which has a serious impact on the health of dairy cows and the benefits of farming. At present, the existing technology for treating cow endometritis has the following shortcomings:

[0004] Endometritis in dairy cows can lead to lower pregnancy rates and reduced production performance. During a lactation period, the cost of raising diseased dairy cows will increase significantly, placing a heavy economic burden on farmers. For example, the economic losses caused by endometritis in dairy cows in the European Union amount to 1.4 billion euros each year, and in the United States to 650 million U.S. dollars.

[0005] Clinically, antibiotics are often used to treat endometritis in dairy cows. However, the use of antibiotics can lead to excessive antibiotic residues in milk, which not only affects the quality and safety of dairy products, but also poses a potential threat to consumers' health. As people's attention to food safety issues continues to increase, it is particularly important to find a residue-free treatment method.

[0006] As natural medicines, Chinese veterinary medicines have the advantages of low toxicity and side effects and a wide antibacterial spectrum. However, the current administration of Chinese medicine is relatively simple, and there is a relative shortage of Chinese medicine products with good efficacy and rapid effects. Traditional oral Chinese medicines have problems such as slow absorption and low bioavailability, which limit their clinical application.

[0007] Currently, most suppositories on the market use antibiotics as their main drug, and their large-scale use will cause the antibiotic content in milk to exceed the standard. In addition, excessive use of antibiotics may also cause pathogenic microorganisms to develop drug resistance, further increasing the difficulty of treatment.

[0008] There are many problems with existing methods for treating endometritis in dairy cows, such as large economic losses, food safety hazards, limitations of traditional Chinese medicine administration methods, and defects of existing suppositories. Therefore, it is urgent to develop a drug that can effectively treat endometritis in dairy cows and provide a more convenient administration method to reduce the economic losses and food safety issues caused by the disease, while improving the application effect of traditional Chinese medicine in clinical treatment. Therefore, we propose a traditional Chinese medicine composition for treating endometritis in dairy cows and a preparation method thereof. Summary of the invention

[0009] The purpose of the present invention is to solve the problems in the existing background technology. In order to achieve the above invention purpose, the present invention provides the following technical scheme: a Chinese medicine composition for treating cow endometritis, characterized in that it comprises the following ingredients: motherwort, chuanxiong, phellodendron, codonopsis, and plantain.

[0010] As a preferred technical solution of the present invention, the following ingredients are taken by weight: 12-15 parts of Leonurus japonicus, 8-12 parts of Phellodendron chinense, 6-8 parts of Chuanxiong, 6-8 parts of Plantago seed, and 3-6 parts of Codonopsis pilosula.

[0011] A method for preparing a traditional Chinese medicine composition for treating cow endometritis comprises the following steps: according to a formula, 12-15 parts of Leonurus japonicus, 8-12 parts of Phellodendron chinense, 6-8 parts of Chuanxiong, 6-8 parts of Plantago seeds and 3-6 parts of Codonopsis pilosula are respectively weighed, and the medicines are mixed and crushed.

[0012] As a preferred technical solution of the present invention, the specific steps for screening the optimal dose of the Chinese medicine composition are as follows:

[0013] Step 1: Select 20 dairy cows with endometritis from the farm, and screen them with the help of professional veterinarians according to the clinical diagnostic criteria for dairy cow endometritis by visually examining vaginal discharge, mucosal congestion, and rectal palpation of uterine horns);

[0014] Step 2: Randomly divided into 5 groups: control group, model group, Yigongqinggao, medium and low dose groups (concentrations of 220, 176 and 122 g / L);

[0015] Step 3: Each group was gavaged with 3L of drug, and the control group and the model group were gavaged with 3L of warm water at 7 am and 2 pm every day. On the 8th day, 10mL of blood was collected from the caudal vein of the cow and centrifuged to obtain plasma;

[0016] Step 4: Use a bovine IL-6 quantitative Elisa kit for detection; take uterine secretions of the cow, smear, and Swiss Giemsa staining. Microscopic examination shows that the neutrophil ratio is ≤14%, and the result is negative, thereby obtaining the optimal dose of the traditional Chinese medicine composition.

[0017] As a preferred technical solution of the present invention, the specific steps of preparing drug-containing serum are as follows:

[0018] Step 1: The high-dose group of Yigongqing is the most suitable dose for treatment, and this dose is used to prepare Yigongqing-containing serum;

[0019] Step 2: Six healthy dairy cows were selected and randomly divided into a blank group and a drug-containing serum group. The drug-containing serum group was gavaged with drugs at 7 am and 2 pm every day for 7 consecutive days. The blank group was gavaged with an equal amount of distilled water. The last gavage was performed at 7 am on the 8th day. Two hours later, 100 mL of blood was collected from the caudal vein of each cow using a negative pressure vacuum blood collection tube. The blood was left to rest for 2 hours and centrifuged at 3000 rpm for 15 minutes to obtain serum, which was then dispensed into 15 mL centrifuge tubes, placed in a 37°C water bath, inactivated for 15 minutes, and stored at -80°C.

[0020] As a preferred technical solution of the present invention, the cytotoxicity determination steps in the preparation method of the Chinese medicine composition are as follows:

[0021] Step 1: CCK-8 cell viability assay, cells were seeded on a 96-well plate at a density of 5×103 cells per ml. After the cell density reached 60%, 5%, 10%, 15%, 20% Yigongqing drug-containing serum and LPS (0, 5, 10, 15, 20, 25, 30 μg / mL) were used to act on BEND cells for 24 h, and CCK-8 reagent (10 μL) was added and incubated at 37°C for 1.5 h.

[0022] Step 2: Read the absorbance at 450 nm using a microplate reader.

[0023] As a preferred technical solution of the present invention, the cytokine detection steps in the preparation method of the Chinese medicine composition are as follows:

[0024] Step 1: After LPS (15 μg / mL) was applied to BEND cells (5×105 cells per well in a 6-well plate) for 24 h, different concentrations of Yigongqing drug-containing serum were used to treat the cells for 24 h and cultured at 37°C and 5% CO2;

[0025] Step 2: Total cell RNA was extracted using the TRIzol method, and qRT-PCR was performed using the SYBR Green dye method. The relative level of gene expression was calculated as △Ct=Ct(gene)-Ct(internal reference); the 2-△△Ct method was used to calculate the fold change of gene expression.

[0026] An application of a traditional Chinese medicine composition for treating cow endometritis, and an application of the traditional Chinese medicine composition for anti-inflammatory effects on LPS-treated BEND cells.

[0027] As a preferred technical solution of the present invention, the Chinese medicine composition reduces the expression levels of TNF-α and IL-6 mRNA induced by LPS.

[0028] As a preferred technical solution of the present invention, the Chinese medicine composition is applied to the treatment of clinical cow endometritis.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows: In the scheme of the present invention: The present invention explores the clinical efficacy and the most suitable dosage of the Chinese medicine composition of the present invention through in vivo experiments, and then prepares the Chinese medicine composition of the present invention into a drug-containing serum model for in vitro experiments, further proving the therapeutic effect of the Chinese medicine composition of the present invention on cow endometritis, and providing a new treatment scheme for cow endometritis. The research results show that the high-dose group of the Chinese medicine composition of the present invention has obvious therapeutic effect and can be used for the preparation of drug-containing serum. LPS, as the main component of the outer membrane of Gram-negative bacteria, can promote the release of inflammatory factors, thereby inducing an inflammatory response. When the 15% drug-containing serum of the Chinese medicine composition of the present invention is applied to LPS-induced BEND cells for 24 hours, the expression levels of TNF-α and IL-6 mRNA are significantly reduced compared with the LPS+blank serum group, indicating that the Chinese medicine composition of the present invention has an anti-inflammatory effect.

[0030] In TCM pharmacology research, TCM ingredients are often directly added to in vitro cell or organ cultures. However, due to the complexity of TCM ingredients, the results of in vitro experimental studies with direct addition of TCM ingredients are usually different from those in vivo. TCM serum pharmacology is a modern development of TCM pharmacology, in which drugs or drug compounds are orally administered to animals, blood is collected after a certain period of time to separate serum, and then the drug serum is used for in vitro experimental studies. Many researchers believe that serum pharmacology is more scientific and more suitable for TCM research than traditional pharmacology. TCM serum pharmacology is now widely used in TCM research. For example, the study showed that the drug-containing serum of the FBR-2 prescription can inhibit H2O2 by regulating the balance of mitochondrial autophagy / cell apoptosis.

[0031] In summary, clinical experiments have shown that the Chinese medicine composition of the present invention has a good therapeutic effect on cow endometritis. The in vivo and in vitro experimental results can relieve the symptoms of the diseased cows or reduce the inflammatory response, indicating that the Chinese medicine composition of the present invention is effective in treating clinical cow endometritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 See schematic diagram for the sequence of primers provided by the present invention;

[0033] Figure 2 A schematic diagram of the uterine secretion smear microscopic examination results after treatment provided by the present invention;

[0034] Figure 3A schematic diagram of the protein expression level of IL-6 in the serum of dairy cows after treatment provided by the present invention;

[0035] Figure 4 A schematic diagram of the effect of LPS provided by the present invention on the viability of cow endometrial epithelial cells;

[0036] Figure 5 A schematic diagram of the effect of the drug-containing serum provided by the present invention on the viability of cow endometrial epithelial cells;

[0037] Figure 6 A, B, C, and D provided by the present invention are schematic diagrams of the IL-6 expression levels at 6, 12, 18, and 24 hours after drug action, respectively;

[0038] Figure 7 This is a schematic diagram of the effect of the drug-containing serum provided by the present invention on the expression level of TNF-α mRNA in BEND cells induced by LPS. DETAILED DESCRIPTION

[0039] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0040] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely represents some embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. It should be noted that the embodiments of the present invention and the features and technical solutions in the embodiments can be combined with each other without conflict. It should be noted that similar numbers and letters represent similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0041] Example 1: A Chinese medicine composition for treating cow endometritis, comprising the following ingredients: motherwort, chuanxiong, phellodendron, codonopsis, and plantain.

[0042] Take the following ingredients by weight: 12-15 parts of Leonurus japonicus, 8-12 parts of Phellodendron chinense, 6-8 parts of Chuanxiong, 6-8 parts of Plantago seed, and 3-6 parts of Codonopsis pilosula.

[0043] A method for preparing a traditional Chinese medicine composition for treating cow endometritis comprises the following steps: according to a formula, 12-15 parts of Leonurus japonicus, 8-12 parts of Phellodendron chinense, 6-8 parts of Chuanxiong, 6-8 parts of Plantago seeds and 3-6 parts of Codonopsis pilosula are respectively weighed, and the medicines are mixed and crushed.

[0044] The specific steps for screening the optimal dose of the Chinese medicine composition are as follows:

[0045] Step 1: Select 20 dairy cows with endometritis from the farm, and screen them with the help of professional veterinarians according to the clinical diagnostic criteria for dairy cow endometritis by visually examining vaginal discharge, mucosal congestion, and rectal palpation of uterine horns);

[0046] Step 2: Randomly divided into 5 groups: control group, model group, Yigongqinggao, medium and low dose groups (concentrations of 220, 176 and 122 g / L);

[0047] Step 3: Each group was gavaged with 3L of drug, and the control group and the model group were gavaged with 3L of warm water at 7 am and 2 pm every day. On the 8th day, 10mL of blood was collected from the caudal vein of the cow and centrifuged to obtain plasma;

[0048] Step 4: Use a bovine IL-6 quantitative Elisa kit for detection; take uterine secretions of the cow, smear, and Swiss Giemsa staining. Microscopic examination shows that the neutrophil ratio is ≤14%, and the result is negative, thereby obtaining the optimal dose of the traditional Chinese medicine composition.

[0049] The specific steps for preparing drug-containing serum are as follows:

[0050] Step 1: The high-dose group of Yigongqing is the most suitable dose for treatment, and this dose is used to prepare Yigongqing-containing serum;

[0051] Step 2: Six healthy dairy cows were selected and randomly divided into a blank group and a drug-containing serum group. The drug-containing serum group was gavaged with drugs at 7 am and 2 pm every day for 7 consecutive days. The blank group was gavaged with an equal amount of distilled water. The last gavage was performed at 7 am on the 8th day. Two hours later, 100 mL of blood was collected from the caudal vein of each cow using a negative pressure vacuum blood collection tube. The blood was left to rest for 2 hours and centrifuged at 3000 rpm for 15 minutes to obtain serum, which was then dispensed into 15 mL centrifuge tubes, placed in a 37°C water bath, inactivated for 15 minutes, and stored at -80°C.

[0052] The steps of cytotoxicity determination in the preparation method of the Chinese medicine composition are as follows:

[0053] Step 1: CCK-8 cell viability assay, cells were seeded on a 96-well plate at a density of 5×103 cells per ml. After the cell density reached 60%, 5%, 10%, 15%, 20% Yigongqing drug-containing serum and LPS (0, 5, 10, 15, 20, 25, 30 μg / mL) were used to act on BEND cells for 24 h, and CCK-8 reagent (10 μL) was added and incubated at 37°C for 1.5 h.

[0054] Step 2: Read the absorbance at 450 nm using a microplate reader.

[0055] The cytokine detection steps in the preparation method of the Chinese medicine composition are as follows:

[0056] Step 1: After LPS (15 μg / mL) was applied to BEND cells (5×105 cells per well in a 6-well plate) for 24 h, different concentrations of Yigongqing drug-containing serum were used to treat the cells for 24 h and cultured at 37°C and 5% CO2;

[0057] Step 2: Total cell RNA was extracted using the TRIzol method, and qRT-PCR was performed using the SYBR Green dye method. The relative level of gene expression was calculated as △Ct=Ct(gene)-Ct(internal reference); the 2-△△Ct method was used to calculate the fold change of gene expression.

[0058] An application of a traditional Chinese medicine composition for treating cow endometritis, and an application of the traditional Chinese medicine composition for anti-inflammatory effects on LPS-treated BEND cells.

[0059] The Chinese medicine composition reduces the expression levels of TNF-α and IL-6 mRNA induced by LPS.

[0060] The Chinese medicine composition is applied to the clinical treatment of dairy cow endometritis.

[0061] Experimental example:

[0062] A traditional Chinese medicine composition for treating cow endometritis and a preparation method thereof.

[0063] Experimental procedures

[0064] 1.1 Materials

[0065] 1.1.1 Experimental animals and cells Dairy cows at 15-30 days postpartum from a farm in the vicinity of Daqing City and bovine endometrial epithelial cells (BEND) were purchased from Beina Biotechnology (No.: 359233)

[0066] 1.1.2 Drugs and reagents: Preparation of Yigongqing drug: 12-15 parts of Leonurus japonicus, 8-12 parts of Phellodendron chinense, 6-8 parts of Chuanxiong, 6-8 parts of Plantago, and 3-6 parts of Codonopsis pilosula were mixed and crushed. All drugs were purchased from Daqing People's Hospital; Swiss Giemsa complex stain was purchased from Solebol, and Enhanced Cell Counting Kit-8 was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.; bovine interleukin 6 (IL-6) quantitative Elisa kit was purchased from Quanzhou Ruixin Biotechnology; Lipopolysaccharides (LPS) was purchased from SIGMA, DMEM / F12 (C11330500BT) culture medium and trypsin (T1320) were purchased from Gibco; TB Green Premix Ex Taq (RK21203) was purchased from TaKaRa; isopropanol and chloroform were purchased from Liaoning Quanrui Reagent Company; fetal bovine serum (FND500) was purchased from Excell.

[0067] 1.2 Experimental methods:

[0068] 1.2.1 Screening of the optimal dose of Yigongqing for the treatment of endometritis in dairy cows 20 dairy cows with endometritis from a farm around Daqing City were selected (with the help of professional veterinarians in the farm, according to the clinical diagnostic criteria for endometritis in dairy cows, visual examination of vaginal discharge, mucosal congestion, and rectal palpation of uterine horns were screened), and randomly divided into 5 groups: control group, model group, Yigongqing high, medium, and low dose (concentrations of 220, 176, and 122 g / L) groups, each group was gavaged with 3L of warm water, and the control group and model group were gavaged with 3L of warm water at 7 am and 2 pm every day. On the 8th day, 10mL of blood was collected from the caudal vein of the cow, and plasma was obtained by centrifugation, and tested using a bovine IL-6 quantitative Elisa kit. The uterine secretions of the dairy cow were taken, smeared, and processed with Swiss Giemsa staining, and the proportion of neutrophils was examined microscopically (≤14%, the result was negative).

[0069] The results of the preparation of Yigongqing drug-containing serum showed that the high-dose group of Yigongqing was the most suitable dose for treatment, and this dose was used to prepare Yigongqing drug-containing serum. Six healthy dairy cows were selected and randomly divided into a blank group and a drug-containing serum group. The drug-containing serum group was gavaged with an equal amount of distilled water every day for 7 consecutive days. The blank group was gavaged with an equal amount of distilled water, and the last gavage was performed at 7 am on the 8th day. After 2 hours, 100 mL of blood was collected from the caudal vein of each dairy cow using a negative pressure vacuum blood collection tube, and the blood was left to stand for 2 hours. The blood was centrifuged at 3000 rpm for 15 minutes to obtain serum, which was divided into 15 mL centrifuge tubes, placed in a 37°C water bath, inactivated for 15 minutes, and stored at -80°C.

[0070] Cytotoxicity Assay CCK-8 cell viability assay, cells were seeded on 96-well plates at a density of 5×103 cells per ml. After the cell density reached 60%, Yigongqing drug-containing serum 5%, 10%, 15%, 20% and LPS (0, 5, 10, 15, 20, 25, 30 μg / mL) were used to act on BEND cells for 24 h, and CCK-8 reagent (10 μL) was added and incubated at 37°C for 1.5 h. The absorbance at 450 nm was read using an enzyme reader.

[0071] Cytokine detection After LPS (15 μg / mL) was applied to BEND cells (5×105 cells per well in a 6-well plate) for 24 hours, different concentrations of Yigongqing drug-containing serum were replaced to treat the cells for 24 hours and cultured at 37°C and 5% CO2. Total RNA was extracted from cells using the TRIzol method, and qRT-PCR was performed using the SYBR Green dye method. The relative level of gene expression was calculated as △Ct=Ct(gene)-Ct(internal reference). The 2-△△Ct method was used to calculate the fold change of gene expression;

[0072] The sequences of the primers used are shown in Table 1:

[0073] Table 1 Primers of fluorescent quantitative

[0074]

[0075]

[0076] 1.2.7 Statistical analysis Graphpad prism 9.0 software was used for statistical analysis, and the results were expressed as mean ± standard deviation. One-way ANOVA was used for single-factor variance analysis, with P < 0.05 indicating a significant difference and P < 0.01 indicating an extremely significant difference.

[0077] 2 Results

[0078] 2.1 Screening of the optimal dose of Yigongqing

[0079] The number of neutrophils in the high- and medium-dose groups of Yigongqing was significantly less than that in the model group, and the number of neutrophils was ≤14%, which was negative. The therapeutic effect of the low-dose group of Yigongqing was not significant. Elisa-assisted diagnosis of IL-6 protein expression levels in cow plasma showed that the high- and medium-dose groups of Yigongqing were significantly lower than those in the model group (p < 0.05). After treatment, the cows in the high-dose group of Yigongqing excreted clearer secretions and had slight mucosal congestion. Taking all factors into consideration, this experiment used the high-dose group of Yigongqing to prepare drug-containing serum.

[0080] 2.2 Effects of Yigongqing medicated serum and LPS on cell viability

[0081] Cell viability was negatively correlated with LPS concentration. Compared with the 0μg / mL group, there was no statistical significance when the LPS concentration was 5 and 10μg / mL (P>0.05). When the concentration was greater than 15μg / mL, the cell viability decreased, which was statistically significant (p<0.05). When the concentration was greater than 20μg / mL, the cell viability decreased significantly. For the next experimental study, 15μg / mL was selected as the LPS concentration for subsequent experiments. 5%, 10%, 15%, and 20% concentrations of drug-containing serum can promote cell proliferation and have no effect on cell survival rate.

[0082] 2.3 Yigongqing-containing serum inhibits LPS-induced BEND cell inflammation

[0083] In order to study the anti-inflammatory effect of Yigongqing on LPS-treated BEND cells, the mRNA expression of IL-6 and TNF-α was measured by qRT-PCR. BEND cells were treated with blank serum 24 hours after LPS stimulation. The results showed that IL-6 and TNF-α were overproduced in BEND cells. Figure 4 As shown in the figure, when the drug-containing serum of each concentration acted on the BEND cells treated with LPS for 6 and 12 hours, the expression level of IL-6 was significantly higher than that of the blank serum group (p < 0.05). The four drug-containing serum concentrations were significantly lower than that of the LPS + blank serum group (p < 0.05), and the LPS + 10% and 15% drug-containing serum groups were significantly lower than the LPS + blank serum group (p < 0.05), and there was no significant difference with the blank serum group. Figure 5 In the experiment, when the drug-containing serum of each concentration acted on BEND cells treated with LPS for 6 and 12 hours, the expression level of TNF-α was significantly higher than that of the blank serum group. The four drug-containing serum groups were significantly lower than the LPS+blank serum group (p < 0.05), and there was no significant difference in the expression level of TNF-α between the drug-containing serum groups with concentrations of 10%, 15%, and 20% and the blank serum group. Figure 4 and Figure 5 As a result, the subsequent experiments will select blank serum group, LPS+blank serum group and LPS+15% drug-containing serum group for subsequent proteomic analysis.

[0084] Endometritis in dairy cows refers to superficial inflammation of the endometrium in the absence of systemic symptoms. It is a common postpartum uterine disease. In traditional Chinese veterinary theory, endometritis is a clinical manifestation of "blood stasis syndrome" and belongs to the category of "damp heat descending". Its cause is dampness and toxicity, filth and turbidity, and liver qi stagnation. The treatment is mostly composed of prescriptions of drugs that remove dead tissue, promote tissue regeneration, promote blood circulation and remove blood stasis, and resist inflammation and inhibit bacteria. Therefore, this study, based on the theory of traditional Chinese veterinary medicine, prescribes the right medicine for the right disease, and utilizes the effects of mutual assistance and mutual promotion to select five medicinal materials to prepare the traditional Chinese medicine formula Yigongqing. These five medicinal materials have different effects. Leonurus japonicus and Chuanxiong have the effects of promoting blood circulation and qi, dispersing blood stasis and relieving pain, and are mainly used to treat blood stasis and qi stagnation, and are the main medicines in this prescription; Phellodendron chinense clears away heat and dampness, Plantago asiatica has a diuretic and relieves stranguria, and is mainly used to treat damp-heat downward and dampness toxins downward, while Codonopsis pilosula has the effect of tonifying the middle and replenishing qi, can regulate liver qi stagnation, and can neutralize the effects of each traditional Chinese medicine to a certain extent. Therefore, this prescription has good effects of clearing heat and dampness, promoting blood circulation and removing blood stasis, tonifying the middle and replenishing qi, and has a good effect on endometritis. The effects of these five herbs have also been verified in modern medicine. Among them, leonurine, the main component of motherwort, has been proven to have anti-inflammatory effects in animal models of various inflammatory diseases. Phellodendron has anti-inflammatory, antibacterial, analgesic and other pharmacological effects. The various active ingredients in Chuanxiong have neuroprotective effects, anti-oxidative stress, anti-apoptosis, and anti-inflammatory effects. Codonopsis has the effects of enhancing hematopoietic function and regulating blood pressure. It can also be used clinically to prevent and treat functional uterine bleeding. The traditional Chinese medicine compound Yigongqing composed of these five herbs can treat cow endometritis to solve the problem of the non-persistence of antibiotic therapy and the problem of antibiotic residues affecting food safety.

[0085] Mediated AEC oxidative stress damage; studies have shown that SX-1 prescription-containing serum may effectively improve CUMS-induced depressive-like behavior and liver damage by blocking liver IL-6 / JAK2 / STAT3 signaling; Yang Juanjuan et al.'s research showed that the Wu Lao Qi Shang prescription inhibits AS fibroblast proliferation and osteogenic differentiation by regulating the Wnt / β-catenin signaling pathway, thereby achieving the purpose of delaying AS pathological new bone formation.

[0086] This experiment explored the clinical efficacy and the most suitable dosage of Yigongqing through in vivo experiments, and then prepared Yigongqing into a drug-containing serum model for in vitro experiments, further proving the therapeutic effect of Yigongqing on dairy cow endometritis and providing a new treatment plan for dairy cow endometritis. The results showed that the high-dose group of Yigongqing had a significant therapeutic effect and could be used for the preparation of drug-containing serum. LPS, as the main component of the outer membrane of Gram-negative bacteria, can promote the release of inflammatory factors, thereby inducing an inflammatory response. When Yigongqing 15% drug-containing serum was applied to LPS-induced BEND cells for 24 hours, the expression levels of TNF-α and IL-6 mRNA were significantly reduced compared with the LPS+blank serum group, indicating that Yigongqing has an anti-inflammatory effect.

[0087] In TCM pharmacology research, TCM ingredients are often directly added to in vitro cell or organ cultures. However, due to the complexity of TCM ingredients, the results of in vitro experimental studies with direct addition of TCM ingredients are usually different from those in vivo. TCM serum pharmacology is a modern development of TCM pharmacology, in which drugs or drug compounds are orally administered to animals, blood is collected after a certain period of time to separate serum, and then the drug serum is used for in vitro experimental studies. Many researchers believe that serum pharmacology is more scientific and more suitable for TCM research than traditional pharmacology. TCM serum pharmacology is now widely used in TCM research. For example, the study showed that the drug-containing serum of the FBR-2 prescription can inhibit H2O2 by regulating the balance of mitochondrial autophagy / cell apoptosis.

[0088] In summary, clinical experiments have shown that Yigongqing has a good therapeutic effect on cow endometritis. The results of in vivo and in vitro experiments can relieve the symptoms of the disease in cows or reduce the inflammatory response, indicating that Yigongqing is effective in treating clinical cow endometritis.

[0089] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Although the present invention has been described in detail with reference to the above embodiments, the present invention is not limited to the above specific implementation methods. Therefore, any modification or equivalent replacement of the present invention; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the invention are included in the scope of the claims of the present invention.

Claims

1. A Chinese medicine composition for treating endometritis in dairy cows, characterized in that: Includes the following ingredients: Motherwort, Chuanxiong, Phellodendron, Codonopsis, and Plantago.

2. A Chinese medicine composition for treating cow endometritis according to claim 1, characterized in that: Take the following ingredients by weight: 12-15 parts of Leonurus japonicus, 8-12 parts of Phellodendron chinense, 6-8 parts of Chuanxiong, 6-8 parts of Plantago seed, and 3-6 parts of Codonopsis pilosula.

3. A method for preparing a Chinese medicine composition for treating endometritis in dairy cows, characterized in that: The following steps are involved: According to the formula, 12-15 parts of Leonurus japonicus, 8-12 parts of Phellodendron chinense, 6-8 parts of Chuanxiong, 6-8 parts of Plantago seed and 3-6 parts of Codonopsis pilosula were weighed respectively, and the drugs were mixed and crushed.

4. The method for preparing a Chinese medicine composition for treating endometritis in dairy cows according to claim 3, characterized in that: The specific steps for screening the optimal dose of the Chinese medicine composition are as follows: Step 1: Select 20 dairy cows with endometritis from the farm, and screen them with the help of professional veterinarians according to the clinical diagnostic criteria for dairy cow endometritis by visually examining vaginal discharge, mucosal congestion, and rectal palpation of uterine horns); Step 2: Randomly divided into 5 groups: control group, model group, Yigongqinggao, medium and low dose groups (concentrations of 220, 176 and 122 g / L); Step 3: Each group was gavaged with 3L of drug, and the control group and the model group were gavaged with 3L of warm water at 7 am and 2 pm every day. On the 8th day, 10mL of blood was collected from the caudal vein of the cow and centrifuged to obtain plasma; Step 4: Use a bovine IL-6 quantitative Elisa kit for detection; take uterine secretions of the cow, smear, and Swiss Giemsa staining. Microscopic examination shows that the neutrophil ratio is ≤14%, and the result is negative, thereby obtaining the optimal dose of the traditional Chinese medicine composition.

5. The method for preparing a Chinese medicine composition for treating endometritis in dairy cows according to claim 4, characterized in that: The specific steps of preparing drug-containing serum are as follows: Step 1: The high-dose group of Yigongqing is the most suitable dose for treatment, and this dose is used to prepare Yigongqing-containing serum; Step 2: Six healthy dairy cows were selected and randomly divided into a blank group and a drug-containing serum group. The drug-containing serum group was gavaged with drugs at 7 am and 2 pm every day for 7 consecutive days. The blank group was gavaged with an equal amount of distilled water. The last gavage was performed at 7 am on the 8th day. Two hours later, 100 mL of blood was collected from the caudal vein of each cow using a negative pressure vacuum blood collection tube. The blood was left to rest for 2 hours and centrifuged at 3000 rpm for 15 minutes to obtain serum, which was then dispensed into 15 mL centrifuge tubes, placed in a 37°C water bath, inactivated for 15 minutes, and stored at -80°C.

6. The method for preparing a Chinese medicine composition for treating cow endometritis according to claim 5, characterized in that: The steps of cytotoxicity determination in the preparation method of the Chinese medicine composition are as follows: Step 1: CCK-8 cell viability assay, cells were seeded on a 96-well plate at a density of 5×103 cells per ml. After the cell density reached 60%, 5%, 10%, 15%, 20% Yigongqing drug-containing serum and LPS (0, 5, 10, 15, 20, 25, 30 μg / mL) were used to act on BEND cells for 24 h, and CCK-8 reagent (10 μL) was added and incubated at 37°C for 1.5 h. Step 2: Read the absorbance at 450 nm using a microplate reader.

7. The method for preparing a Chinese medicine composition for treating endometritis in dairy cows according to claim 6, characterized in that: The cytokine detection steps in the preparation method of the Chinese medicine composition are as follows: Step 1: After LPS (15 μg / mL) was applied to BEND cells (5×105 cells per well in a 6-well plate) for 24 h, different concentrations of Yigongqing drug-containing serum were used to treat the cells for 24 h and cultured at 37°C and 5% CO2; Step 2: Total cell RNA was extracted using the TRIzol method, and qRT-PCR was performed using the SYBR Green dye method. The relative level of gene expression was calculated as △Ct=Ct(gene)-Ct(internal reference); the 2-△△Ct method was used to calculate the fold change of gene expression.

8. An application of a Chinese medicine composition for treating endometritis in dairy cows, characterized in that: Application of Chinese herbal medicine composition in anti-inflammatory effect on LPS-treated BEND cells.

9. The use of a Chinese medicine composition for treating cow endometritis according to claim 8, characterized in that: The Chinese medicine composition reduces the expression levels of TNF-α and IL-6 mRNA induced by LPS.

10. The use of a traditional Chinese medicine composition for treating cow endometritis according to claim 9, characterized in that: The Chinese medicine composition is applied to the clinical treatment of dairy cow endometritis.

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