This study investigates the effect of Bai Tou Weng Soup (BTWS) on the activation of TLR4/NF-κB inflammatory signaling pathway induced by dysbiosis of gut microbiota in UC rats. SD rats were randomly divided into a control group and a model group. A mouse model of acute ulcerative colitis was established by pushing 0.5ml of TNBS solution from the anus. HE staining was used to observe the pathological changes in colon tissue to determine the modeling effect. The results showed that BTWS could repair the physical barrier of colon tissue mucosa in mice with ulcerative colitis, improve local damage to colon tissue, and reduce TNF-α、IL-6 and IL-17A in serum (P < 0.05) decreased the mRNA and protein expression of the TLR4/NF-κB signaling pathway. This study indicates that BTWS has a therapeutic effect on ulcerative colitis rats by regulating the mRNA and protein expression of the TLR4/NF-κB signaling pathway.
Research Article
The effect of Bai Tou Weng Soup（BTWS）on the activation of TLR4/NF-κB inflammatory signaling pathway induced by dysbiosis of gut microbiota in UC rats
https://doi.org/10.21203/rs.3.rs-5229715/v1
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Bai Tou Weng Soup
ulcerative colitis
TLR4/NF-κB signaling pathway
Ulcerative colitis (UC) is an autoimmune disease targeting the colon, characterized by recurrent abdominal pain, diarrhea, mucus, pus, and bloody stools [1]. The etiology of ulcerative colitis is not yet clear, the pathogenesis is complex, and the disease is difficult to cure. Research has shown that ulcerative colitis is caused by immune imbalance mediated by pro-inflammatory cytokines and anti-inflammatory cytokines T cell activation [2, 3]. Abnormal presentation of epithelial cell antigens [4] and immune damage to colon mucosa [5, 6]. It plays an important role in the pathophysiology of ulcerative colitis. Research has shown that after external damage to the intestinal mucosa, macrophages and T lymphocytes in the submucosal lamina propria are activated to secrete a large amount of cytokines in the short term, including pro-inflammatory cytokines such as IL-1, IL-6, IL-17, IL-23, and tumor necrosis factor-α (TNF-α). After a large amount of expression, it stimulates further aggregation of immune cells, which in turn secretes more pro-inflammatory cytokines. Breaking the dynamic balance with anti-inflammatory cytokines such as IL-10 and IL-13, promoting the continuous formation of ulcers through inflammation[7].
At present, Western medicine treatment mainly focuses on drugs such as hormones, sulfonamides, and immunosuppressants [8]. However, hormones and sulfonamides have significant adverse reactions and are dependent and hormone resistant, making them difficult to use for a long time. Immunosuppressants are expensive. And its targeting ability needs to be improved. Traditional Chinese medicine provides an endless source of compounds with therapeutic and safety properties, which could potentially be developed as drugs for treating human diseases. Bai Tou Weng Soup(BTWS)has been used in Chinese medicine for thousands of years to treat diseases caused by bacteria. In recent decades, BTWS has become a famous traditional Chinese medicine formula for treating ulcerative colitis. According to indications, BTWS is currently used in clinical practice in China to treat gastrointestinal infections, including enteritis, bacterial dysentery, and intestinal amoebiasis [9, 10]. Specifically, it is a well-known traditional Chinese medicine formula for treating ulcerative colitis (UC), and its efficacy has been confirmed by many clinical observational studies [11].
However, the mechanism of action of BTWS in treating ulcerative colitis has not been systematically elucidated. Research has found that BTWS may reduce IL-6, IL-17, IL-23, and TNF-α by inhibiting the expression of TLR4/NF-κB signaling pathway- α Wait for release to promote the repair of intestinal mucosa and alleviate colonic inflammatory response [12]. TNF-α It is only a key pro-inflammatory cytokine that activates inflammatory cells by chemotactic neutrophils. It promotes and amplifies the inflammatory response, thereby promoting the occurrence of ulcerative colitis [13]. IL-6 is a key molecule that mediates inflammatory cell infiltration and apoptosis in colitis [14]. It can mediate the proliferation and differentiation of T and B cells to produce a large number of pro-inflammatory cytokines, leading to overexpression and immune damage to the mucosa of ulcerative colitis. IL-17A is one of the key pro-inflammatory cytokines in the inflammatory response of intestinal mucosa. It is produced by helper T cell 17 in response to IL-23 stimulation and can activate other pro-inflammatory cytokines, such as TNF-α. The expression of neutrophils is involved in the proliferation and chemotaxis of neutrophils, leading to inflammatory cell infiltration and abnormal mucosal immune response [15].
Therefore, this study intends to use UC rats to investigate the regulatory effect of BTWS on the activation of TLR4/NF-κB inflammatory signaling pathway induced by dysbiosis of the microbiota, in order to briefly elucidate the mechanism of BTWS in treating ulcerative colitis and provide scientific basis for its clinical rational use.
2.1 Experimental instruments and reagents
Electronic balance (DY15K), desktop low-speed centrifuge (SF-TDL-5C). Zoletil 50 was purchased from Vic, France, with item number FBS060, Cyperazine hydrochloride injection (20220301) was purchased from Shengda Animal Medicine Co., Ltd, TNBS (MB5547) and sulfasalazine (SASP) (MB5634) were purchased from Dalian Boglin Biotechnology Co, Ltd.
2.2 Reagent preparation
Mixed anesthetic: Mix 250mg of Shutai 50 with 2.5ml of Serazine Hydrochloride (100mg/ml), dilute the above mixed solution with 22.5ml of sterile physiological saline to form a mixed solution of Shutai 50 (10mg/ml) and Serazine Hydrochloride (10mg/ml), and set aside.
10% neutral formaldehyde: Dilute the formaldehyde solution concentration to 10% using 1 x PBS, and store at 4 ℃ for later use.
TNBS: Weigh 275mg of TNBS, dissolve it in 50% ethanol and make up to 12ml (2.3%). Prepare it as needed.
SASP: Weigh 13g of sulfasalazine, dissolve in physiological saline and make up to 130ml (10%). Store at 4 ℃ for later use.
BTWS: 15g of Baitouweng, 12g of Huangbai, 6g of Huanglian, 12g of Qinpi, 15g of Codonopsis pilosula, 10g of Poria cocos, 15g of stir fried Atractylodes macrocephala, and 10g of roasted licorice, made into 200ml liquid.
2.3 UC model establishment and evaluation
38 healthy male SD rats aged 6–8 weeks were randomly divided into the following two groups:Normal control group (11 rats): Fasted for 24 hours before modeling, and rats were anesthetized by intramuscular injection of a mixed anesthetic (1ml/kg); Gently insert a hose with a diameter of about 2mm into the anus, push in 0.5ml of 50% ethanol solution, pinch the anus tightly, lift the tail upside down for 3 minutes, and then put it back into the cage to naturally wake up.
UC model group (27 rats): Fasted for 24 hours before modeling, and rats were anesthetized by intramuscular injection of a mixed anesthetic (1ml/kg); Gently insert a hose with a diameter of about 2mm into the anus, push in 0.5ml of TNBS solution, pinch the anus tightly, lift the tail upside down for 3 minutes, and then put it back into the cage to naturally wake up. Three days after modeling, use a mixed anesthetic (1ml/kg) for intramuscular injection anesthesia; After the animals enter stable anesthesia, collect colon tissues from 3 rats in the control group and 3 rats in the model group, and observe the pathological changes of the tissues after fixation, paraffin embedding, sectioning, and HE staining.
2.4 Animal experiments
32 rats with successfully established models were divided into four groups (8 in each group, n = 8): control group, UC model group, UC model + SASP intervention group, UC model + BTWS intervention group. All groups of rats underwent gavage once a day for 14 consecutive days. The control group and UC model group were given 10ml/kg of double distilled water by gavage, the UC model + SASP intervention group was given 10ml/kg of SASP by gavage, and the UC model + BTWS intervention group was given 10ml/kg of BTWS by gavage.
After the administration of each group of rats, a mixed anesthetic (1ml/kg) was used for intramuscular injection anesthesia; After the animals enter stable anesthesia, collect the following samples for subsequent experimental testing:
(1) Blood sample collection: After collecting blood from the abdominal aorta, the animals were euthanized. The blood was left to stand overnight at 4 ℃ and centrifuged at 3000 rpm for 5 minutes to obtain serum. The serum samples were extracted and stored in a -80 ℃ freezer. Subsequently, TNF-α 、IL-6 and IL-17 levels in the serum of each group of rats was detected using an ELISA kit.
(2) Colon tissue samples: After euthanizing rats, take the anus to the cecal colon, cut along the longitudinal axis of the mesentery, rinse thoroughly with cold physiological saline, fix in a 4% paraformaldehyde solution, embed a part of the intestinal tissue in paraffin, and evaluate inflammation and ulcers under HE staining microscopy; According to the instructions of the reagent, the total RNA of cells was extracted using the Trizol chloroform method and reverse transcribed to obtain cDNA. Real time fluorescence quantitative PCR (qPCR) reaction was performed on a PCR instrument using cDNA as a template, with the internal reference group being the housekeeping gene Gapdh−ΔΔ Analyze the expression levels of mRNA TLR4 and NF-κB p65 using the Ct method; Add an appropriate amount of RIPA lysis solution to fully lyse cells for 5 minutes. Subsequently, place on ice for 30 minutes, centrifuge at 4 ℃ and 12000 rpm for 10 minutes, and collect the total protein supernatant. Mix well with 5 x reducing protein loading buffer, denature in a boiling water bath for 5 minutes, and freeze the obtained sample at -20 ℃ for later use. Prepare polyacrylamide concentrate gel and denatured gel, and set the electrophoresis program to: concentrate gel at 80 V and separate gel at 100 V. Incubate skim milk at room temperature for 60 minutes, primary antibody at 4 ℃ overnight, and secondary antibody at room temperature for 60 minutes at 100V constant pressure transfer membrane. Add the mixed luminescent solution and fully react for 2 minutes before imaging in a chemiluminescence instrument. Scan and archive the film, and analyze the obtained image data. The remaining intestinal tissue was frozen at -80 ℃.
(3) Fecal collection: Before euthanizing rats, collect 3 fecal samples from each group of rats, transfer them to centrifuge tubes, freeze them in liquid nitrogen, and store them at -80 ℃.
3.1 UC model mouse established successfully
The commonly used evaluation index to determine the success of establishing a UC animal model is to conduct histopathological examination and analysis, observe the colon structure under an optical microscope, and evaluate the modeling effect. Under the microscope, the morphology of the intestinal mucosa in the blank control group was intact and the structure was clear. No clear abnormalities were found in the submucosal layer, muscular layer, and serosal layer. Compared with the blank control group, the structure and boundaries of each layer of the intestinal wall in the model group were unclear, and some intestinal walls were completely necrotic with purulent inflammation and fibrous granulation tissue proliferation (Fig. 1). Based on the above results, it can be concluded that the UC model was successfully established.
3.2 HE and blood results after intervention in UC model mice
After 14 days of gastric lavage in each group of rats, the colon tissue of each group was observed under a microscope. In the Fig. 2, the blank group had intact intestinal mucosa morphology, clear structure, and no clear abnormalities were found in the submucosal, muscular, and serosal layers; The model group had severe necrosis of the entire intestinal wall, with unclear structures and boundaries of each layer. Most of the mucosal glands and lamina propria were damaged or missing, and a large number of neutrophils infiltrated the submucosal, muscular, and serosal layers. Abscesses formed in some areas, and some interstitial edema accompanied by granulation tissue proliferation; Compared with the model group, the degree of intestinal mucosal ulcer was significantly reduced in both the BTWS group and the SASP group, and the number of neutrophils in the intestinal wall was significantly reduced. The glandular structure and morphology of the intestinal mucosa were better in both groups; There was no significant difference in intestinal mucosal lesions between the BTWS group and the SASP group, indicating that both BTWS and SASP have therapeutic effects on ulcerative colitis.
|
Group |
TNF-α(pg/ml) |
IL-6(pg/ml) |
IL-17A(pg/ml) |
|---|---|---|---|
|
Blank group |
6.653 ± 2.282 |
22.625 ± 3.753 |
3.253 ± 1.090 |
|
UC group |
23.984 ± 6.340△ |
107.902 ± 20.827△ |
16.100 ± 3.629△ |
|
SASP group |
10.846 ± 1.644△▲ |
37.205 ± 20.860▲ |
8.170 ± 2.003△▲ |
|
BTWS group |
12.257 ± 2.414△▲ |
49.254 ± 17.699▲ |
9.786 ± 2.272△▲ |
| Note: △, P < 0.05;▲, P < 0.05. | |||
)Related studies have shown that the immune regulatory effect of BTWS on cytokines in ulcerative colitis mainly involves pro-inflammatory cytokine regulation [16]. In order to investigate the immunomodulatory effect of BTWS on cytokines in ulcerative colitis, ELISA was used to detect some cytokines in the serum of SD rats on the 14th day of gastric lavage TNF-α、IL-6、IL-17A levels. As shown in the figure, the serum TNF-α of rats in each intervention group (Table 1). The average levels of IL-6 and IL-17A in water were significantly lower than those in the model group, and the levels of BTWS were also lower than those in the intervention group with SASP. These data indicate that both BTWS and SASP can reduce TNF-α、The expression of IL-6 and IL-17A can alleviate the immune inflammatory response in ulcerative colitis (Fig. 3).
|
Group |
TLR4 |
NF-κB p65 |
|---|---|---|
|
Blank group |
1.012 ± 0.164 |
1.005 ± 0.114 |
|
UC group |
1.718 ± 0.189△ |
1.600 ± 0.237△ |
|
SASP group |
1.170 ± 0.204▲ |
1.141 ± 0.177▲ |
|
BTWS group |
1.282 ± 0.128△▲ |
1.253 ± 0.158△▲ |
| Note: △, P < 0.05;▲, P < 0.05. | ||
)Recent studies have shown that BTWS may by inhibit TLR4/NF-κB Expression of signaling pathway, thereby reducing TNF-α Wait for release to promote the repair of intestinal mucosa and alleviate colonic inflammatory response. Therefore, in this study, the gene expression level was detected by RT-qPCR, and the effects of BTWS on TLR4 and NF-κB p65 in colon tissue cells of UC rats were studied (Table 2).
As shown in Fig. 4, RT-qPCR is used to detect TLR4 and NF-κB p65 after treatment with SASP or BTWS The expression of. Compared with the model group, the mRNA TLR4 and NF-κB p65 in the treatment group treated with BTWS or SASP were significantly higher.
Western blot analysis showed that TLR4 and p-NF-κB p65 in the experimental group The protein level of p-NF-κB p65 was downregulated compared to the model group (Fig. 5), but NF-κB p65 The level of protein did not change significantly, indicating that BTWS or SASP did not participate in the regulation of NF-κB p65. These results indicate that treatment with BTWS or SASP can effectively inhibit the expression of TLR4 and p-NF-κB p65 in colon tissue cells of UC rats with intestinal microbiota.
In recent years, UC has attracted the attention of medical researchers due to its recurrent onset. Although SASP tablets are indicated for use in ulcerative colitis, there are many adverse reactions [9]. As a traditional Chinese medicine ingredient, BTWS has been widely used in UC patients for a long time with minimal side effects. Scholars at home and abroad have conducted extensive research on the regulatory effect of BTWS on cytokines in the treatment of ulcerative colitis. Studies have shown that BTWS has a regulatory effect on pro-inflammatory cytokines, and studies suggest that it inhibits the expression of pro-inflammatory cytokines. Restoring immune dynamic balance may be one of the mechanisms by which BTWS treats ulcerative colitis.
TLR4/NF-κB The inflammatory response involved in the signaling pathway affects various stages of UC occurrence and development. TLR4 is the main receptor associated with immunity and inflammation, which can be activated through multiple pathways. This study selected TNF-α、 IL-6 and IL-17A were used as inflammatory cytokine detection indicators and were found to be associated with TLR4 and NF-κB The decrease in of TLR4 and p-NF-κB p65 protein expression leads to a decrease in the inflammatory factor in the treatment group of rats TNF-α、 IL-6 and IL-17A levels, as well as TLR4 and NF-κB The expression of showed statistically significant differences compared to the model group rats (P < 0.05).
In summary, this study preliminarily confirms that BTWS can restore the morphology of intestinal villi, glandular structure of intestinal mucosa, reduce the number of neutrophils, regulate the level of inflammatory factors, and reduce TLR4/NF-κB The mRNA and protein levels of the B signaling pathway have therapeutic effects on rats with ulcerative colitis. The strategy of using BTWS for ulcerative colitis is feasible, effective, and safe.
Funding Declaration
The research work was supported by The natural science foundation of the Autonomous Region plans special training projects for ethnic minorities(2021D3016).
Competing interests
The authors claim no competing interests. All the authors have read and approved this manuscript. The work has not been submitted to or is not considered for publication elsewhere.
Ethical Approval
This animal experiment scheme has been audited by the experimental animal welfare ethics Committee and conforms to the national animal protection, animal welfare and ethical principles. The experimental scheme was approved by Zhejiang Animal Experimental Ethics Committee (approval number: ZJCLA-IAUC-20040102).
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No competing interests reported.