Application of baicalin in preparation of medicine for treating streptococcus agalactiae induced mastitis

Abstract

The invention provides application of baicalin in preparation of a medicine for treating streptococcus agalactiae induced mastitis, and relates to the technical field of medicine. A mouse is selected as an animal model to research the intervention effect and mechanism of the baicalin on the streptococcus agalactiae induced mouse mastitis inflammatory response, and it is clear that the baicalin can play a role in protecting the mouse mammary gland by regulating and controlling an NLRP3 inflammatory small body pathway; a reference basis is provided for treating the cow mastitis caused by streptococcus agalactiae infection by applying the baicalin.

Description

Technical Field

[0001] The invention provides application of baicalin in preparing a medicine for treating breast inflammation induced by Streptococcus agalactiae, belonging to the technical field of medicine. Background Art

[0002] Cow mastitis is a disease caused by multiple pathogens that causes infection in the mammary gland. It can cause a decrease in milk production and quality, abandonment of milk, premature culling of dairy cows, and increased costs for veterinary services and treatment, causing serious economic losses to the dairy industry and is one of the biggest problems threatening global dairy production. The most common pathogens that cause cow mastitis are Streptococcus, Escherichia coli, Klebsiella, Pseudomonas, Pasteurella, and Staphylococcus. Among them, Streptococcus agalactiae (GBS) is a highly contagious pathogen that can infect humans and cause neonatal meningitis, pneumonia, and septicemia. Infected cows cause mastitis in cows during the peak lactation period and postpartum cows. It can colonize in the mammary tissue of cows and produce biofilm structures, causing chronic infection and seriously affecting the production performance of dairy cows.

[0003] Traditional mastitis treatment is mainly antibiotic treatment, but there are problems such as bacterial resistance and antibiotic residues. Chinese medicine has the characteristics of low toxicity, low residue and not easy to produce drug resistance. At present, many studies have screened Chinese herbal medicines for the treatment of viral and bacterial infections, and it is expected to be one of the alternative antibiotics. Baicalin has anti-inflammatory, antiviral, anti-tumor, antioxidant and antibacterial effects, and is widely used in the treatment of hepatitis, pneumonia, enteritis, allergic reactions, dysentery, colds and respiratory infections. At present, the research on baicalin for mastitis induced by Streptococcus agalactiae infection is still incomplete. Summary of the invention

[0004] In view of this, the present invention provides the use of baicalin in the preparation of a drug for treating breast inflammation induced by Streptococcus agalactiae.

[0005] The present invention selects mice as an animal model to study the intervention effect and mechanism of baicalin on the inflammatory response of mouse mastitis induced by Streptococcus agalactiae, so as to clarify whether baicalin can protect the mouse mammary gland by regulating the NLRP3 inflammasome pathway, and provide a reference basis for the application of baicalin in the treatment of bovine mastitis caused by Streptococcus agalactiae infection.

[0006] Baicalin is a flavonoid isolated and extracted from the rhizome of Scutellaria baicalensis. It is one of the main active ingredients of Scutellaria baicalensis and has multiple pharmacological effects, such as anti-inflammatory, anti-tumor, antibacterial, and antiviral activities.

[0007] Studies have shown that in vivo administration of baicalin can reduce inflammatory damage to tissues and reduce the transcription and secretion levels of inflammatory factors IL-1β, IL-6 and TNF-α. The results of pathological observation of mammary tissue of mice in the baicalin groups at different concentrations in this study showed that baicalin could significantly improve the phenomenon of neutrophil infiltration in mammary tissue of mice caused by GBS infection. At the same time, the results of this experiment showed that compared with the infection group, the relative transcription levels of IL-6 in the L group and TNF-α in the M group were significantly increased, which was different from the theoretical prediction. The reason may be that IL-6 and TNF-α are both pro-inflammatory factors. While the expression of pro-inflammatory factors increases, the expression of IL-10 as an anti-inflammatory factor decreases. Although the drug was administered, the mice may still be in a period of vigorous secretion of pro-inflammatory factors. In addition, this study found that different concentrations of baicalin can reduce the relative transcription levels of IL-1β, IL-6, IL-10 and TNF-αmRNA to varying degrees. It can be seen that baicalin can achieve a protective effect on the mammary gland of mice by effectively reducing the transcription level of inflammatory-related factors.

[0008] After confirming that baicalin has an anti-inflammatory effect on mouse mastitis, this study further explored the anti-inflammatory mechanism of baicalin. The activation of mouse NLRP3 inflammasome is closely related to inflammatory diseases, especially playing a key regulatory role in inflammatory damage caused by bacteria. Microorganisms release a large number of virulence factors that can activate NLRP3 inflammasomes. Studies have shown that clinical pathogenic isolates of bovine mastitis, such as Staphylococcus aureus, Escherichia coli, and Klebsiella, can activate NLRP3 inflammasomes in bovine or mouse mammary epithelial cells. Baicalin can inhibit the activation of NLRP3 inflammasomes in mouse colon tissue. During the inflammation process, the NF-κB pathway and NLRP3 inflammasomes affect the subsequent development of inflammation by regulating the expression of inflammatory factors TNF-α and IL-1, and the NF-κB pathway is an important pathway for activating NLRP3 inflammasomes. Unlike the NF-κB pathway, NLRP3 inflammasomes can promote the maturation and secretion of cytokines. Reactive oxygen species (ROS) can also activate NLRP3. Baicalin can reduce the activation of NLRP3 inflammasome and inhibit the release of IL-1β by reducing the content of ROS. Mycoplasma gallisepticum (MG) causes oxidative stress in chicken-like macrophages (HD11) and significantly increases the expression levels of ROS, NLRP3 inflammasome and autophagy-related proteins. After treatment with baicalin, the total ROS in HD11 cells decreased and the expression level of NLRP3 inflammasome was significantly downregulated. NLRP3 inflammasome is an important signaling pathway of the body's innate immune system and is essential for host defense against bacterial infection. Under the action of LPS, the expression of NLRP3, ASC and Caspase-1 is significantly increased. The inflammatory protein complex composed of NLRP3, adaptor protein ASC and effector protein Caspase-1 activates inactive pro-Caspase-1 to Caspase-1 and promotes IL-1β to activate inflammatory response. Subsequently, activated Caspase-1 can cleave GSDMD into GSDMD-N, and form pores in the plasma membrane through the aggregation of GSDMD-N. At the same time, IL-1β is released from the cell. Studies by Yan Liang et al. have shown that baicalin can also inhibit the activation of NLRP3 inflammasomes and the occurrence of cell pyroptosis. This study found that 20 mg / kg and 40 mg / kg baicalin can significantly or extremely significantly reduce the relative transcription levels of NLRP3, Caspase-1, ASC and IL-1β mRNA. However, the transcription level and secretion level of Caspase-1 mRNA in group H were inconsistent, which may be due to the fact that the transcription of its mRNA is not synchronized with the expression of protein. When detected, it was in the mRNA transcription stage and had not yet reached the protein expression stage.In addition, different concentrations of baicalin can significantly reduce the secretion of NLRP3, Caspase-1 and IL-1β proteins, indicating that baicalin can block the NLRP3 signaling pathway by reducing the activation of NLRP3 inflammasomes, thereby inhibiting the inflammatory response and protecting the mammary tissue from inflammatory damage. This experiment found that baicalin was not dose-dependent at the transcription level of NLRP3 pathway-related factors, indicating that the concentration range needs to be readjusted when exploring the effect of baicalin. The immunohistochemical experiment in this study further confirmed the above experimental results. It can be seen that baicalin is expected to become a potential drug for the treatment of bovine mastitis because of its strong anti-inflammatory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 A to E in the middle are pathological sections of mammary gland tissues of mice in the NC, Infection, L, M, and H groups, respectively;

[0010] Figure 2 The results of RT-qPCR detection of mRNA transcription levels of inflammatory factors in mammary gland tissues of mice in each group;

[0011] Figure 3 The results of RT-qPCR detection of mRNA transcription levels of NLRP3 inflammasome pathway-related factors in the mammary gland tissues of mice in each group;

[0012] Figure 4 The results of ELISA test on the secretion level of NLRP3 inflammasome pathway-related factors in the mammary gland tissue of each group of mice;

[0013] Figure 5 The results of immunohistochemistry test of mammary gland tissue of mice in each group (400×). DETAILED DESCRIPTION

[0014] In order to make the purpose, technical scheme and advantages of the present invention clearer, the preferred embodiments of the present invention are further described in detail in conjunction with the embodiments. 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.

[0015] 1 Materials and methods

[0016] 1.1 Drugs, strains and experimental animals

[0017] Baicalin (purity 95%, batch number B802696) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Streptococcus agalactiae CVCC1886 strain was cryopreserved by our laboratory. 7-week-old SPF BALB / c mice were purchased from Guangzhou Yancheng Biotechnology Co., Ltd.

[0018] 1.2 Main Reagents

[0019] RNA isolater Total RNA Extraction Reagent was purchased from Nanjing Novozyme Biotechnology Co., Ltd.;

[0020] BlasTaq TM 2×qPCR MasterMix and All-In-One 5×RT MasterMix were purchased from Zhenjiang Aibimeng Biotechnology Co., Ltd.;

[0021] Mouse Caspase-1, mouse IL-1β, and mouse NLRP3 ELISA assay kits were purchased from Shanghai ELISA Biotechnology Co., Ltd.;

[0022] Rabbit NLRP3 monoclonal antibody (MAb) was purchased from Abimat Pharmaceutical Technology (Shanghai) Co., Ltd.;

[0023] Immunohistochemical staining kit was purchased from Beijing Bioson Biotechnology Co., Ltd.

[0024] 1.3 Mouse grouping and treatment

[0025] Forty 8-10-week-old lactating mice 7-10 days after first mating were randomly divided into negative control (NC) group, infection (Infection) group, low (L, 20 mg / kg), medium (M, 40 mg / kg), and high (H, 80 mg / kg) baicalin groups. Each group of mice was anesthetized with 1% pentobarbital, and the mammary area of ​​mice was disinfected with 75% anhydrous ethanol. Then, the fourth nipple of mice in infection, L, M, and H groups was infused with 100 μL of Streptococcus agalactiae suspension (1×10 8 cfu / mL), and the NC group was perfused with 100 μL of sterile saline in the same way. At 24h, 30h, 36h and 42h after perfusion, the mice in the Infection group were perfused with 100 μL of sterile saline on each side of the fourth pair of mammary regions each time, and the mice in the L, M, and H groups were perfused with the corresponding dose of baicalin solution on each side of the fourth pair of mammary regions each time, and the mice in the NC group were perfused with 100 μL of sterile saline in the same way as above. 6h after the last perfusion, the mice in each group were killed and the mammary glands were removed, some of which were fixed in 4% paraformaldehyde, and the rest of the mammary glands were stored at -80℃.

[0026] 1.4 Observation of pathological changes in mouse mammary gland tissue

[0027] Part of the mammary gland tissue of each group of mice fixed in 4% paraformaldehyde for 48 h in 1.3 was obtained, and the tissue pathological sections were prepared by dehydration with different concentrations of ethanol, transparentization with xylene, wax immersion, embedding, 4 μm sectioning, and HE staining. The morphological structure of the mouse mammary gland was observed under a low power microscope, and the morphological structure of the mammary ducts and acinar cells was observed under a high power microscope.

[0028] 1.5 RT-qPCR detection of mRNA transcription levels of inflammatory factors and NLRP3-related factors in mouse mammary tissue

[0029] The ultra-low temperature frozen mouse mammary tissue was added to grinding beads and RNA isolater for thorough grinding, and then centrifuged at 12000g for 5 min at 4℃. The supernatant was aspirated to extract the total RNA of mouse mammary tissue, which was reverse transcribed into cDNA as a template. The corresponding primers in Table 1 were used to detect the transcription levels of inflammatory-related factors TNF-α, IL-6, IL-10, and NLRP3 pathway-related factors IL-1β, Caspase-1, NLRP3, and ASC gene mRNA in the mammary tissue of each group of mice by RT-qPCR.

[0030] RT-qPCR primers were designed based on the relevant gene sequences in GenBank and synthesized by Sangon Biotech (Shanghai) Co., Ltd. β-actin was used as the internal reference gene. -ΔΔCT The relative transcription level of each gene was calculated.

[0031] Table 1 Primer sequences

[0032]

[0033] 1.6 ELISA determination of the secretion level of NLRP3 pathway-related factors in mouse mammary gland tissue

[0034] The remaining mammary gland was weighed and placed in pre-cooled PBS (tissue: PBS = 1:9) for tissue homogenization. The supernatant was taken after centrifugation at 6000g and 4°C for 5 min, and the expression levels of NLRP3, Caspase-1, and IL-1β in mouse mammary gland tissue were detected using corresponding ELISA kits.

[0035] 1.7 Immunohistochemical detection of NLRP3 protein distribution in mouse mammary gland tissue

[0036] The paraffin sections prepared in 1.4 without HE staining were dewaxed with xylene, dehydrated with different concentrations of ethanol, antigen repaired with 0.01 mol / L citrate, acted with 3% H2O2 endogenous enzyme blocker for 10 min to 15 min, and blocked with 5% BSA for 1 h. The rabbit anti-NLRP3 MAb (1:500) in the immunohistochemical staining kit was used as the primary antibody (PBS was used instead of the primary antibody for the negative control), and biotin-labeled goat anti-rabbit IgG (1:200) was used as the secondary antibody to detect the distribution of NLRP3 protein in breast tissue by immunohistochemical assay.

[0037] 1.8 Statistical analysis of data

[0038] SPSS23 software was used to analyze the experimental data of each group, and one-way ANOVA was used for statistical analysis. P<0.05 was considered significant, and P<0.01 was considered extremely significant. The experimental data were expressed as "mean ± standard deviation" and graphs were drawn using Graphpadprism9.

[0039] 2 Results

[0040] 2.1 Observation results of pathological changes in mammary gland tissue of mice in each group

[0041] 6 hours after the last milk duct perfusion, mice in each group were killed and part of the mammary tissue was taken. HE staining was used to observe the pathological changes of the mammary tissue of mice in each group. The results showed that the mammary tissue interstitial edema, thickening of the alveolar wall, inflammatory cell infiltration in the alveolar wall, and the alveoli were filled with inflammatory secretions ( Figure 1 B). Compared with the Infection group, the inflammatory cell infiltration in the mammary gland tissue of mice in the baicalin L, M, and H groups was significantly reduced, the interstitial edema was relieved, the alveolar wall became narrower, and the alveolar structure became gradually obvious. In addition, the degree of inflammatory lesions in the mammary gland tissue of the H group was lower than that of the L and M groups ( Figure 1 C~ Figure 1 E). Compared with the NC group, the walls of the mammary gland alveoli of mice in the L, M, and H groups were thickened, and inflammatory secretions were distributed in the alveoli ( Figure 1 A). This indicates that GBS infection in the mammary gland of mice can lead to mammary gland inflammation, and intervention with baicalin can alleviate mammary gland inflammation.

[0042] 2.2 Detection results of mRNA transcription levels of inflammatory factors in mammary gland tissue of mice in each group

[0043] RT-qPCR was used to detect the transcription levels of TNF-α, IL-10 and IL-6 mRNA in the mammary gland tissues of mice in each group. The results showed that compared with the NC group, the transcription levels of IL-6, IL-10 and TNF-α mRNA in the mammary gland tissues of mice in the Infection group were significantly increased (P<0.01) ( Figure 2 A~ Figure 2 C). Compared with the Infection group, the IL-6 mRNA transcription level in the mammary gland tissue of mice in the L group was significantly increased (P<0.01), and the IL-6 mRNA transcription levels in the mammary gland tissue of mice in the M and H groups were significantly decreased (P<0.01), and the IL-6 mRNA transcription levels in the mammary gland tissue of mice in the L, M, and H groups showed a gradual downward trend; compared with the NC group, the IL-6 mRNA transcription level in the mammary gland tissue of mice in the L group was significantly increased (P<0.01) ( Figure 2A). Compared with the Infection group, the IL-10 mRNA transcription levels in the mammary gland tissues of mice in the L, M, and H groups were extremely significantly decreased (P<0.01); compared with the NC group, the IL-10 mRNA transcription levels in the mammary gland tissues of mice in the L and H groups were extremely significantly decreased (P<0.01), and the IL-10 mRNA transcription level in the mammary gland tissues of mice in the M group was significantly decreased (P<0.05) ( Figure 2 B). Compared with the Infection group, the TNF-α mRNA transcription levels in the mammary gland tissues of mice in the L and H groups were extremely significantly decreased (P<0.01), and the TNF-α mRNA transcription level in the mammary gland tissues of mice in the M group was extremely significantly increased (P<0.01); compared with the NC group, the TNF-α mRNA transcription levels in the mammary gland tissues of mice in the L and H groups were significantly increased (P<0.05), and the TNF-α mRNA transcription level in the mammary gland tissues of mice in the M group was extremely significantly increased (P<0.01) ( Figure 2 C). The results showed that GBS infection could upregulate the mRNA transcription levels of inflammatory factors IL-6, IL-10, and TNF-α in the mammary tissue of mice. All doses of baicalin treatment groups could downregulate the mRNA transcription levels of inflammatory factors after intervention, among which the downregulation effect of baicalin H group (80 mg / kg) was the most significant.

[0044] 2.3 Results of mRNA transcription level detection of NLRP3 pathway-related factors in mammary gland tissue of each group of mice

[0045] The RT-qPCR method was used to detect the mRNA transcription levels of IL-1β, Caspase-1, NLRP3, and ASC in the mammary gland tissues of mice in each group. The results showed that compared with the NC group, the mRNA transcription levels of IL-1β and NLRP3 in the mammary gland tissues of mice in the Infection group were significantly increased (P<0.01), and the mRNA transcription levels of Caspase-1 and ASC were significantly increased (P<0.05) ( Figure 3 A~ Figure 3 D). Compared with the Infection group, the IL-1β mRNA transcription level in the mammary gland tissue of mice in the L and H groups was extremely significantly decreased (P<0.01), and the IL-1β mRNA transcription level in the mammary gland tissue of mice in the M group was significantly decreased (P<0.05); compared with the NC group, there was no significant difference in the IL-1β mRNA transcription level in the mammary gland tissue of mice in the L, M, and H groups (P>0.05) ( Figure 3A). Compared with the Infection group, the transcription level of Caspase-1 mRNA in the mammary gland tissue of mice in the L and M groups was significantly decreased (P<0.05), while the transcription level of Caspase-1 mRNA in the H group was significantly increased (P<0.05); compared with the NC group, the transcription level of Caspase-1 mRNA in the mammary gland tissue of mice in the H group was significantly increased (P<0.05), while there was no significant difference in the transcription level of Caspase-1 mRNA in the mammary gland tissue of mice between the L and M groups (P>0.05) ( Figure 3 B).

[0046] Compared with the infection group, the NL-RP3 mRNA transcription levels in the mammary gland tissues of mice in the L, M, and H groups were significantly decreased (P<0.01); compared with the NC group, the NLRP3 mRNA transcription levels in the mammary gland tissues of mice in the L and M groups were significantly decreased (P<0.01), while there was no significant difference in the H group (P>0.05) ( Figure 3 C). Compared with the Infection group, there was no significant difference in the ASC mRNA transcription level in the mammary gland tissue of mice in the L, M, and H groups (P>0.05); compared with the NC group, the ASC mRNA transcription level in the H group was extremely significantly increased (P<0.01), while there was no significant difference between the L and M groups (P>0.05) ( Figure 3 D). The results showed that GBS infection could cause an increase in the mRNA transcription levels of IL-1β, Caspase-1, NLRP3 and ASC, factors related to the NLRP3 inflammasome pathway, in the mammary tissue of mice. All doses of baicalin intervention could promote the secretion of anti-inflammatory factors, and 20 mg / kg and 40 mg / kg doses of baicalin had better effects.

[0047] 2.4 Detection results of secretion levels of NLRP3 pathway-related factors in mammary gland tissue of each group of mice

[0048] After partial mammary tissue homogenate was taken and centrifuged to obtain the supernatant, the secretion of NLRP3-related factors was detected by ELISA kit. The results showed that the secretion levels of Cas-pase-1, IL-1β and NLRP3 in the mammary gland tissue of mice in the Infection group were significantly higher than those in the NC group (P<0.01)( Figure 4 A~ Figure 4 C); The secretion level of Caspase-1 in the mammary gland tissue of mice in the L, M, and H groups was significantly lower than that in the Infection group (P<0.01), and had no significant difference with the NC group (P>0.05) ( Figure 4A). Compared with the Infection group, the secretion level of IL-1β in the mammary gland tissue of mice in the M and H groups was significantly decreased (P<0.01), while there was no significant difference in the L group (P>0.05); compared with the NC group, the secretion level of IL-1β in the mammary gland tissue of mice in the L group was significantly increased (P<0.01), and there was no significant difference between the M and H groups (P>0.05) ( Figure 4 B). The NLRP3 secretion levels in the mammary gland tissues of mice in the L, M, and H groups were significantly lower than those in the Infection group (P<0.01). There was no significant difference in the NLRP3 secretion levels in the mammary gland tissues of mice in the M, H, and NC groups (P>0.05). The NLRP3 secretion level in the mammary gland tissues of mice in the L group was significantly lower than that in the NC group (P<0.01). Figure 4 C). This indicates that GBS infection can cause an increase in the secretion level of factors related to the NLRP3 inflammasome pathway in the mammary tissue of mice. After intervention with various doses of baicalin, the secretion level of factors related to the NLRP3 pathway decreased, and the secretion level could be restored to the state before infection. Among them, 40 mg / kg and 80 mg / kg doses of baicalin had better therapeutic effects.

[0049] 2.5 Immunohistochemical detection results of NLRP3 protein distribution in mammary gland tissue of each group of mice

[0050] Immunohistochemical detection was performed on the fixed mammary tissues of mice in each group. The results showed that the positive signals of NLRP3 protein in the mammary tissues of mice were mainly present in the infection group, L group, M group and H group. Diffuse brown particles were distributed in the area of ​​inflammatory cell infiltration, and there were also a small amount of NLRP3 positive signals in the alveolar wall. Among them, the distribution of NLRP3 positive signals in the mammary tissues of mice in the M group was lower than that in the infection group and other dose drug groups, which was consistent with the results of the relative transcription level of NLRP3 mRNA in 2.4. No or only a few NLRP3 positive signals were found in the mammary tissues of mice in this group ( Figure 5 The results further showed that all doses of baicalin could downregulate the expression of NLRP3 protein and effectively alleviate the mammary tissue damage of mice caused by GBS infection, among which 40 mg / kg baicalin had the best therapeutic effect.

[0051] The embodiments described above are part of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected 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.

Claims

1. Application of baicalin in the preparation of drugs for treating breast inflammation induced by Streptococcus agalactiae.

2. The use according to claim 1, characterized in that: The drug dosage form is an injection.

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