Application of pain and diarrhea essential prescription for regulating BLP + macrophage subpopulation to treat IBS-D

Abstract

The invention discloses application of a pain and diarrhea prescription to regulation and control of a BLP + macrophage subgroup to treat IBS-D. The pain and diarrhea prescription contains a liver soothing component (6-10 parts of pericarpium citri reticulatae and 6-10 parts of radix saposhnikoviae) and a spleen tonifying component (10-15 parts of rhizoma atractylodis macrocephalae and 10-15 parts of radix paeoniae alba) or a combination of the liver soothing component and the spleen tonifying component according to the ratio of 1: (1-2). By promoting the expression of anti-inflammatory cytokines IL-10 and TGF-beta, inhibiting the expression of pro-inflammatory cytokines IL-6 and TNF-alpha, activating an AMPK signal channel, inhibiting an NF-kappa B signal channel and promoting the expression of colonic mucosal barrier indexes ZO1 and OCLN, a colonic mucosal barrier is repaired, and the symptoms of IBS-D abdominal pain and diarrhea are relieved. The invention also provides a pharmaceutical composition containing the key active component, and the dosage forms comprise an aqueous solution, a tablet and the like. The traditional Chinese medicine composition solves the problems of large toxic and side effects, poor compliance and unknown mechanism in the existing IBS-D treatment, is definite and safe in curative effect, and has important clinical value and scientific significance.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of a traditional Chinese medicine formula for treating IBS-D by regulating BLP+ macrophage subsets. Background Technology

[0002] IBS-D is a chronic functional gastrointestinal disorder characterized by abdominal pain or discomfort accompanied by changes in bowel habits. It is characterized by long-term recurrent attacks and is difficult to cure. Its pathogenesis is not fully understood, the pathogenic factors are complex, there is a lack of specific biological markers, and the symptoms are overlapping and nonspecific, which brings many difficulties to the diagnosis and treatment of the disease.

[0003] Existing treatment methods have significant limitations: long-term use of antispasmodics, antidepressants, antidiarrheals, and other drugs has significant toxic side effects, poor patient tolerance, and is prone to relapse; lifestyle interventions such as low-FODMAP diet and stress management have long cycles and require a high degree of patient compliance and perseverance, which some patients find difficult to adhere to, resulting in poor treatment outcomes.

[0004] Traditional Chinese medicine (TCM) has rich clinical experience and efficacy advantages in the treatment of IBS-D. The method of soothing the liver and strengthening the spleen has been widely recognized by experts in both TCM and Western medicine. Tongxie Yaofang, as a classic formula for soothing the liver and strengthening the spleen, has a multi-target synergistic effect. It can regulate the brain-gut axis, protect the intestinal mucosal barrier, and improve visceral hypersensitivity. It can significantly relieve diarrhea and abdominal pain symptoms in IBS-D patients, with a total effective rate of over 80%.

[0005] Recent studies have found that a subset of colonic epithelial macrophages plays a crucial role in maintaining intestinal barrier function. A 2020 report in Cell stated that the BLP+Mφ subset in the distal colon can act as an epithelial cell "gatekeeper," maintaining the integrity of the colonic epithelial barrier by limiting the absorption of toxic substances; loss of function of this subset leads to epithelial cell apoptosis and impaired barrier function, directly participating in the pathogenesis of IBS-D.

[0006] Currently, existing technologies have not clearly defined the relationship between Tongxie Yaofang (a traditional Chinese medicine formula) and the BLP+Mφ subgroup, and there is a lack of technical solutions targeting "Tongxie Yaofang's regulation of the BLP+Mφ subgroup in the treatment of IBS-D." Furthermore, the complex composition of traditional Chinese medicine formulas makes it difficult to target and identify key active ingredients that regulate the BLP+Mφ subgroup using traditional screening methods, leading to a bottleneck in the study of Tongxie Yaofang's mechanism of action and formulation optimization. Therefore, developing an IBS-D treatment regimen based on Tongxie Yaofang and targeting the BLP+Mφ subgroup has significant clinical value and scientific importance. Summary of the Invention

[0007] The present invention provides an application of Tongxie Yaofang in regulating BLP+ macrophage subsets for the treatment of IBS-D, clarifies the association between Tongxie Yaofang and BLP+ Mφ subsets, elucidates its regulatory mechanism, optimizes the traditional Chinese medicine composition, and provides a new treatment plan with definite curative effect and high safety for IBS-D.

[0008] Technical solution: To solve the above technical problems, according to one aspect of the present invention, more specifically, it is an application of Tongxie Yaofang in regulating BLP+ macrophage subsets for the treatment of IBS-D, including the following: 1. Identification of new BLP+Mφ subsets related to IBS-D by single-cell sequencing Analyze the immune cells in the distal colon of IBS-D model rats by single-cell sequencing technology, and sort the new group of BLP+Mφ by flow cytometry.

[0009] Detect the intracellular cytokine levels of this new group by cytokine protein chip, identify the new group of BLP+Mφ related to IBS-D regulated by Tongxie Yaofang, and determine its cytokine expression profile.

[0010] 2. Elucidation of the pharmacodynamic mechanism of Tongxie Yaofang in regulating new BLP+Mφ subsets Construction and intervention of IBS-D animal models: Establish IBS-D animal models by the three-factor method, set up a model group, a control group, and a traditional Chinese medicine intervention group (aqueous solutions of different components of Tongxie Yaofang), and the intervention period is 6 weeks.

[0011] Histological detection of colon epithelium: After the pharmacodynamic detection is completed, isolate the distal colon epithelium, and clarify the improvement of the colon epithelial mucosal permeability by different components of Tongxie Yaofang through HE staining and electron microscopy observation.

[0012] Observation of myeloid cell mobilization: Extract myeloid cells from the bone marrow and blood of each group of model rats, perform flow cytometry staining and labeling, and observe the mobilization of hematopoietic stem cells - myeloid progenitor cells - mononuclear macrophages.

[0013] Sorting and determination of the regulation spectrum of new BLP+Mφ subsets: Isolate and digest the distal colon epithelium, sort BLP+Mφ by flow cytometry after CD11c labeling, sort the new subsets according to the determined markers, and clarify the regulation spectrum of different components of Tongxie Yaofang on this new subset.

[0014] Detection of the influence on colon mucosal permeability: Collect primary neonatal rat distal colon epithelial cells, co-culture them with the new BLP+Mφ subsets regulated by different components of Tongxie Yaofang for 48 hours, and detect colon mucosal permeability indexes such as ZO1 and OCLN by qPCR to determine the influence of the new subsets on colon epithelial mucosa.

[0015] Molecular mechanism identified: Transcriptome sequencing (RNA-Seq) was used to detect the transcriptome profile of the selected new BLP+Mφ subpopulation. After enriching the core signal, Western blot was used to verify the molecular mechanism by which different components of Tongxie Yaofang regulate this new subpopulation.

[0016] 3. Validation of the efficacy of different components of Tongxie Yaofang in treating IBS-D model Animal model preparation and grouping intervention: IBS-D animal model was prepared, and the animals were administered physiological saline, predicted component group and positive control drug by gavage, while a normal control group was set up.

[0017] Serum and mucosal permeability index detection: Colonic epithelial mucosa was isolated, and the expression of permeability-related indexes was detected to determine the effect of optimized components on colonic mucosal permeability in the IBS-D model.

[0018] BLP+Mφ Subgroup Analysis: By binding antibodies to novel BLP+Mφ subgroup markers, flow cytometry was used to analyze the regulatory effects of different components of Tongxie Yaofang on the BLP+Mφ subgroup.

[0019] Molecular biological assays: Western blot was used to detect key transcription factors of the BLP+Mφ subgroup, and Chip-PCR was used to detect transcription factor binding regions to clarify the effects of different components of Tongxie Yaofang on key targets of the BLP+Mφ subgroup in the IBS-D model.

[0020] Specific deletion / inhibition verification: Based on markers or key transcription factors of new BLP+Mφ subpopulations, custom marker DTR mice or transcription factor KO mice were used to delete / inhibit the relevant BLP+Mφ subpopulations to establish the model. Normal SD rats were selected as the control group, and the above-mentioned related indicators were detected after 6 weeks of gavage.

[0021] Bone marrow transplantation reverse verification: Through bone marrow transplantation experiments, the effects of different components of Tongxie Yaofang on the BLP+Mφ subgroup were verified in reverse.

[0022] The beneficial effects of this invention's application of a traditional Chinese medicine formula for treating IBS-D by regulating BLP+ macrophage subsets are as follows: (1) This invention clarifies the association between Tongxie Yaofang and the BLP+Mφ subgroup, and reveals a new mechanism by which Tongxie Yaofang repairs the colonic mucosal barrier and treats IBS-D by regulating this subgroup, filling the gap in the existing technology.

[0023] (2) This invention utilizes advanced technologies such as single-cell sequencing and transcriptome sequencing to target and discover key active ingredients that regulate the BLP+Mφ subgroup in the Tongxie Yaofang formula, solving the problem of complex components and difficult screening of traditional Chinese medicine compound formulas, and providing a scientific basis for formulation optimization.

[0024] Tongxie Yaofang, a classic Chinese medicine compound, is highly safe and has few toxic side effects. The IBS-D treatment plan developed based on it can overcome the shortcomings of poor long-term tolerance of existing drugs, resulting in higher patient compliance.

[0025] (3) This invention uses the BLP+Mφ subgroup as a specific target, and the treatment plan is highly targeted. It can effectively improve the colonic mucosal barrier function, relieve symptoms such as diarrhea and abdominal pain in IBS-D patients, and improve the treatment effect. It has broad clinical application prospects. Attached Figure Description

[0026] The present invention will now be described in further detail with reference to the accompanying drawings and specific implementation methods.

[0027] Figure 1 This is a schematic diagram of the structure of the present invention. Detailed Implementation

[0028] The present invention will be described in detail below with reference to the accompanying drawings and embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in the present application can be combined with each other.

[0029] To make the technical solution of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0030] Example 1 Identification of the new subpopulation of BLP+Mφ IBS-D model rats were constructed, and their distal colon tissue was collected to isolate immune cells.

[0031] Immune cells were analyzed using single-cell sequencing technology, and BLP+Mφ cell populations were obtained by flow cytometry sorting.

[0032] The intracellular cytokine levels of this cell population were detected using a cytokine protein chip, and a new subpopulation of BLP+Mφ associated with IBS-D and regulated by the Tongxie Yaofang formula was identified, and its cytokine expression profile was determined.

[0033] Example 2 A Study on the Pharmacodynamic Mechanism of Tongxie Yaofang in Regulating New Subgroups of BLP+Mφ An IBS-D animal model was constructed using the three-factor method. The rats were randomly divided into a model group, a control group, a group containing the complete formula of Tongxie Yaofang, a group containing the liver-soothing formula, and a group containing the spleen-strengthening formula. Ten rats were in each group. The control group consisted of normal rats. Each group was given the corresponding component aqueous solution by gavage for 6 consecutive weeks.

[0034] After the intervention, the distal colonic epithelium of rats in each group was isolated, and the tissue morphology was observed by HE staining. The ultrastructure of the mucosa was observed by electron microscopy to assess the improvement of colonic epithelial mucosal permeability.

[0035] Myeloid cells were extracted from the bone marrow and blood of rats in each group, and flow cytometry staining was performed to observe the differences in mobilization of hematopoietic stem cells, myeloid progenitor cells, and monocytes / macrophages.

[0036] Distal colonic epithelium was isolated and digested, and a new subgroup of BLP+Mφ was sorted by flow cytometry after CD11c labeling. The regulatory effects of different components of Tongxie Yaofang on this subgroup were analyzed.

[0037] Primary rat distal colon epithelial cells were collected and co-cultured with a new subpopulation of BLP+Mφ regulated by different components of Tongxie Yaofang for 48 hours. The expression levels of ZO1 and OCLN were detected by qPCR.

[0038] Transcriptome sequencing (RNA-Seq) was performed on the new BLP+Mφ subpopulation to enrich the core signaling pathways, and the expression of key proteins was verified by Western blot to clarify the molecular mechanism.

[0039] Example 3 Efficacy verification of different components of Tongxie Yaofang in treating IBS-D An IBS-D animal model was established, and animals were randomly divided into a model control group (administered via gavage with physiological saline), a predicted component group, a positive drug control group, and a normal control group, with 10 animals in each group.

[0040] Six weeks after gavage intervention, the colonic epithelial mucosa was separated, and the expression of permeability-related indicators was detected.

[0041] The effects of different components of Tongxie Yaofang on the BLP+Mφ subset were analyzed by flow cytometry using antibody-conjugated markers of a new BLP+Mφ subset.

[0042] Key transcription factors of the BLP+Mφ subset were detected by Western blot, and transcription factor binding regions were detected by Chip-PCR to clarify the role of key targets.

[0043] Customized marker DTR mice or transcription factor KO mice were used to establish a model by deleting / inhibiting the relevant BLP+Mφ subset. These mice, along with a normal SD rat control group, were administered different components of the Tongxie Yaofang formula by gavage for 6 weeks, and the aforementioned related indicators were measured.

[0044] A bone marrow transplantation experiment was conducted to reverse-engineer the effects of different components of the Tongxie Yaofang formula on the BLP+Mφ subgroup.

[0045] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. An application of a traditional Chinese medicine formula for treating IBS-D by regulating BLP+ macrophage subsets, characterized in that: (1) The formula for treating diarrhea includes components for soothing the liver and strengthening the spleen; The composition and weight ratio of the liver-soothing components are as follows: 6-10 parts of dried tangerine peel and 6-10 parts of saposhnikovia root; The composition and weight ratio of the spleen-strengthening components are as follows: Atractylodes macrocephala 10-15 parts, Paeonia lactiflora 10-15 parts; When it is a combination of liver-soothing and spleen-strengthening components, the weight ratio of the two is 1:(1-2); (2) The drug targets the colonic BLP+ macrophage (BLP+Mφ) subset as a specific target, and repairs the colonic mucosal barrier by regulating the functional activity of the BLP+Mφ subset, thereby exerting the therapeutic effect of IBS-D. The regulation includes: Regulation of cytokine expression profile: promotes the expression of anti-inflammatory cytokines IL-10 and TGF-β, and inhibits the expression of pro-inflammatory cytokines IL-6 and TNF-α; Regulation of transcriptional pathways: Activation of the AMPK signaling pathway, inhibition of the NF-κB signaling pathway, and regulation of the expression of transcription factors FOXO3 and NRF2; It promotes the mRNA and protein expression of colonic mucosal barrier-related markers ZO1 and OCLN, and reduces colonic mucosal permeability.

2. The application of the Tongxie Yaofang formula for regulating BLP+ macrophage subsets in the treatment of IBS-D according to claim 1, characterized in that: The key active ingredients of the Tongxie Yaofang formula are: hesperidin (≥5% in tangerine peel) and cimicifugain (≥2% in saposhnikovia root) in the liver-soothing component; atractylodes lactone III (≥1% in atractylodes macrocephala) and paeoniflorin (≥8% in peony root) in the spleen-strengthening component. The key active ingredients have been verified by single-cell sequencing, transcriptome sequencing and Western blot to specifically regulate the BLP+Mφ subset.

3. The application of the Tongxie Yaofang formula according to claim 1 in regulating BLP+ macrophage subsets for the treatment of IBS-D, characterized in that: The prescribed formula for treating diarrhea regulates the BLP+Mφ subgroup in the following ways: (1) Mobilize bone marrow hematopoietic stem cells to differentiate into myeloid progenitor cells, promote the maturation of myeloid progenitor cells into monocytes and macrophages, and increase the proportion of BLP+Mφ subset in total macrophages in colon tissue by ≥20%; (2) Inhibit colonic epithelial cell apoptosis through the AMPK / NF-κB signaling pathway (apoptosis rate decreased by ≥35%) and reduce colonic mucosal permeability (transepithelial resistance TER value increased by ≥40%). (3) After 4 weeks of medication, according to the IBS-SSS scoring criteria, the patient’s abdominal pain score decreased by ≥30% and the number of daily diarrhea episodes decreased by ≥40%.

4. The application of the Tongxie Yaofang formula according to claim 1 in regulating BLP+ macrophage subsets for the treatment of IBS-D, characterized in that, It also includes a pharmaceutical composition for treating IBS-D, said pharmaceutical composition: (1) The active ingredient is the key active ingredient in Tongxie Yaofang that can target and regulate the BLP+Mφ subgroup. The key active ingredient is derived from the liver-soothing component, the spleen-strengthening component or a combination thereof. Liver-soothing components: 6-10 parts of dried tangerine peel and 6-10 parts of saposhnikovia root, with key active ingredients being hesperidin (≥5%) and cimicifugoside (≥2%). Spleen-strengthening components: 10-15 parts of Atractylodes macrocephala and 10-15 parts of Paeonia lactiflora. The key active ingredients are atractylodes lactone III (≥1%) and paeoniflorin (≥8%). The combined weight ratio of the liver-soothing component to the spleen-strengthening component is 1:(1-2); (2) The excipients are pharmaceutically acceptable and are selected from one or more of the following: fillers (lactose, starch), binders (hydroxypropyl methylcellulose), disintegrants (crospovidone) or lubricants (magnesium stearate); (3) The dosage form is aqueous solution, tablet, capsule or granule, wherein each tablet contains a total amount of key active ingredients of 50-200mg and each capsule contains a total amount of key active ingredients of 50-200mg.

5. The application of the Tongxie Yaofang formula according to claim 4 in regulating BLP+ macrophage subsets for the treatment of IBS-D, characterized in that, The pharmaceutical composition is prepared by the following method: the liver-soothing component and spleen-strengthening component of the Tongxie Yaofang are extracted and purified respectively to obtain the key active ingredients, and then mixed with excipients and prepared into the target dosage form according to conventional pharmaceutical processes; When the pharmaceutical composition is used for patients with IBS-D, it is administered once or twice daily, with each dose equivalent to 50-200 mg of the total amount of the key active ingredient. The extraction method of the liver-soothing component is as follows: after pulverizing tangerine peel and saposhnikovia root, extract twice with 70% ethanol for 1.5 hours each time, combine the extracts, concentrate under reduced pressure to a relative density of 1.10-1.15 (60℃), purify with macroporous resin, and freeze-dry to obtain the key active ingredient powder.

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