Application of sesquiterpenoids in preparation of medicines for treating thymus injury

Abstract

The invention discloses an application of sesquiterpenoids in preparation of drugs for treating thymus injury, and particularly relates to an application in preparation of drugs for treating acute thymus injury, an application in regulation of secretion of inflammatory factors in thymus cells and subgroups thereof and an application in increase of the number of immune cells. Wherein the extract of the sesquiterpenoids is a ligularia virgaurea seed, and is identified to be the Irimophenane type sesquiterpenoids. The medicinal value of the sesquiterpenoids WH-1 is further studied, and it is found that the sesquiterpenoids WH-1 can enhance the inflammatory iTECs cell viability, improve the inflammatory iTECs function and relieve acute thymus injury, so that the organism immunity is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of the application of traditional Chinese medicine extracts, and particularly relates to the application of sesquiterpenoid compounds in the preparation of drugs for treating thymic injury. Background Art

[0002] The thymus is an important central immune organ of the body and the main site for the differentiation, development, and maturation of T lymphocytes. Most thymocytes are cleared by the body during the maturation process, and only a very small number develop into naive T cells, which then migrate to peripheral immune organs to perform immune functions. Thymic epithelial cells (TECs) are the main components of the thymic microenvironment. They are derived from thymic epithelial progenitor cells and include cortical thymus epithelial cells (cTEC) and medullary thymus epithelial cells (mTEC). Among them, cTEC mainly mediates the positive selection of the thymus, enabling T lymphocytes to have the function of recognizing self-MHC molecule restriction. mTEC mediates negative selection, enabling T lymphocytes to have the characteristic of central immune tolerance. They jointly play an important role in promoting the differentiation, development, and selection of thymocytes. However, under physiological conditions, the thymus undergoes chronic atrophy with age. [5] Acute thymic atrophy mainly occurs under pathological conditions, mostly in infectious diseases such as viral, bacterial, and fungal infections. For example, sepsis caused by lipopolysaccharide in the outer membrane of Gram-negative bacteria can also lead to thymic function impairment, acute thymic atrophy, and a decrease in T lymphocytes during the process of causing systemic inflammation, thereby reducing the body's immunity.

[0003] Thymic atrophy is mainly manifested as a decrease in the volume of the thymus and the number of cells in the thymus, and the thymic microenvironment composed of TECs also changes. Eventually, the output of naive T cells decreases, leading to a decline in immune function. Therefore, alleviating thymic atrophy caused by acute inflammation and improving the body's immunity, thereby reducing secondary infections caused by infectious diseases, is crucial in the treatment of infectious diseases.

[0004] Guo et al. found that leucosceptroid N, the main sesterterpenoid component of Leucosceptrum canum, can significantly reduce the levels of cytokines IL-6 and TNF-α in the serum of septic mice at a dose of 25 mg / kg, effectively relieve the symptoms of multi-organ damage in diseased mice, and improve the survival rate of diseased mice (Kai Guo, Ting-Ting Zhou, Shi-Hong Luo, et al. Leucosceptrane sesterterpenoids as a new type of natural immunosuppressive agents in treating sepsis, J. Med. Chem. 2024, 67, 1, 513–528).

[0005] Zeng Xiangzhou's research found that artesunate has a significant protective effect on sepsis inflammation caused by lipopolysaccharide, heat-killed Escherichia coli, and Staphylococcus aureus attacking mice (Xiangzhou Zeng. Research on the effects and molecular mechanisms of artesunate on osteoclasts [D]. Southern Medical University, Guangzhou, 2015.).

[0006] The plants of the genus Ligularia are perennial herbs of the Compositae family, with diverse chemical compositions, including sesquiterpenoids, diterpenoids, triterpenoids, phenylpropanoids, flavonoids, steroids, etc., mainly concentrated in Yunnan, Sichuan, Hubei, Qinghai, and Gansu. The plants of the genus Ligularia generally have the effects of relieving cough and resolving phlegm, promoting blood circulation to remove stasis, and clearing heat and detoxifying. In recent years, domestic and foreign scholars have studied the plants of the genus Ligularia and found that the main components in their stems and leaves are sesquiterpenoid compounds, which have pharmacological activities such as anti-inflammatory, antioxidant, anti-cancer, and antibacterial. In recent years, it has been found that some natural active ingredients have a certain protective effect on organ damage caused by infectious diseases, can reduce the release of inflammatory factors, and improve the body's immunity to reduce mortality. However, the application of Ligularia veitchiana extract in thymic injury has not been discovered. Summary of the Invention

[0007] In view of the above technical problems, the present invention provides an application of a sesquiterpenoid compound in the preparation of a drug for treating thymic injury. The sesquiterpenoid compound WH-1 extracted from the seeds of Ligularia veitchiana can enhance the viability of inflammatory iTECs cells, improve the function of inflammatory iTECs, relieve acute thymic injury, and thus improve the body's immunity.

[0008] To achieve the above object, the present invention provides an application of a sesquiterpenoid compound in the preparation of a drug for treating thymic injury, including at least one of the following: (a) Application in the preparation of a drug for treating acute thymic injury; (b) Application in regulating the secretion of inflammatory factors in thymic epithelial cells and their subsets; (c)Use in increasing the number of immune cells.

[0009] Preferably, the acute thymic injury described in (a) is acute thymic atrophy caused by infectious diseases.

[0010] More preferably, the infectious disease is sepsis caused by Gram-negative cell infection.

[0011] Preferably, the thymic epithelial cells and their subsets described in (b) are one or more of TECs, cTECs, and mTECs; the inflammatory factors include pro-inflammatory factors and anti-inflammatory factors.

[0012] More preferably, the pro-inflammatory factors are one or more of IL-1, IL-6, and TNF-α; the anti-inflammatory factor is IL-10.

[0013] Preferably, the immune cells described in (c) include one or more of thymocytes and their subsets, thymic epithelial cells and their subsets, and splenic T lymphocytes and their subsets.

[0014] More preferably, the thymocytes and their subsets include thymocyte DN, DP, CD4 + SP, CD8 + SP subsets; the thymic epithelial cells and their subsets include TECs, cTECs, mTECs, CD80 hi TECs, CD80 lo TECs, MHCII hi TECs, MHCII lo TECs, MHCII hi cTECs, MHCII lo cTECs, MHCII hi mTECs, MHCII lo mTECs subsets; the splenic T lymphocytes and their subsets are CD3 + T lymphocytes and their subsets CD4 + T, CD8 + T, CD4 + Naive T, CD8 + Naive T, CD4 + RTE and CD8 + RTE.

[0015] Preferably, the sesquiterpenoid compound is an eremophilane-type sesquiterpenoid compound with the molecular formula C 15 H 22 O.

[0016] Further preferably, the eremophilane sesquiterpenoid compound is extracted from the seeds of Ligularia veitchiana Even more preferably, the sesquiterpenoid compound is extracted from the petroleum ether extraction part of the seeds of Ligularia veitchiana.

[0017] Preferably, the effective concentration of the sesquiterpenoid compound in the drug is 5 - 20 μg / mL.

[0018] The beneficial effects of the present invention are as follows: 1. The sesquiterpenoid compound - WH - 1 was isolated and identified from the petroleum ether extraction part of the seeds of Ligularia veitchiana. Through cell experiments and animal experiments, it was found that WH - 1 has a certain therapeutic effect on acute thymic injury, can enhance the viability of inflammatory iTECs cells after treatment with Gram - negative bacteria LPS, improve the function of inflammatory iTECs, and inhibit the NF - κB inflammatory signaling pathway in iTECs cells; in addition, it was also found that WH - 1 can significantly increase the number of thymocytes and cells of each subset in neonatal rats with sepsis, the number of cells of each subset of thymic epithelial cells, and the number of spleen T lymphocytes and cells of each subset.

[0019] 2. The present invention screened and identified a natural drug suitable for acute thymic injury, which can be used to relieve thymic atrophy caused by the reduction of thymus volume and cell number, the degradation of the thymic microenvironment composed of TECs, etc., and helps to reduce the damage of acute infectious diseases to the thymus, thereby improving the body's immunity. Description of the Drawings

[0020] Figure 1 It is a schematic diagram of the chemical structure of WH - 1 in Example 1.

[0021] Figure 2 It is a high - performance liquid chromatography diagram of WH - 1 in Example 1.

[0022] Figure 3 It is a detection result diagram of establishing an inflammatory iTECs cell model by LPS treatment in Example 2. In the figure, A1 is a representative diagram of cell subsets in inflammatory iTECs detected by flow cytometry; A2 is a statistical bar chart of the percentages of cTECs and mTECs in inflammatory iTECs detected by FCM; B1 is a representative diagram of the expression levels of Bcl - 2, IL - 10, Ki67, and Aire in inflammatory iTECs detected by FCM; B2 is a statistical diagram of the mean fluorescence intensity of the expression levels of Bcl - 2, IL - 10, Ki67, and Aire in inflammatory iTECs detected by FCM; C1 is a detection of the expression of related genes in iTECs cells changed by LPS by RT - qPCR; in the figure, Control vs LPS, * P< 0.05, ** P< 0.01, *** P<0.001。

[0023] Figure 4 This is the result graph of the effect of WH-1 on inflammatory iTECs cells in Example 3. In the figure, A is the result graph of the effect of different concentrations of WH-1 on the viability of iTECs cells detected by CCK8, and B is the result graph of the change in the expression level of inflammatory factors in the LPS-induced iTECs inflammation model detected by RT-qPCR at different times; in the figure, Control vs LPS, WH-1 vs LPS, * P< P < 0.05, ** P< P < 0.01, *** P< P < 0.001。

[0024] Figure 5 This is the result graph of the effect of WH-1 on inflammatory iTECs cells in Example 3. Figure A is a representative graph of the changes in each cell subset after WH-1 treatment of inflammatory iTECs detected by FCM; Figure B is a statistical graph of the changes in each cell subset after WH-1 treatment of inflammatory iTECs detected by FCM. Control vs LPS, WH-1 vs LPS, *P < 0.05, **P < 0.01, ***P < 0.001.

[0025] Figure 6 This is the result graph of the Western blot detection in Example 4. In the figure, A is a representative graph of the change in protein expression level in iTECs cells, and B is a statistical graph of the change in protein expression level in iTECs cells; in the figure, Control vs LPS, WH-1 vs LPS, * P< P < 0.05, ** P< P < 0.01, *** P< P < 0.001。

[0026] Figure 7 This is the graph of the thymus and spleen conditions of septic mice and the changes in the number of each subset of thymocytes in septic mice under different treatments in Example 5. In the figure, A1 and B1 are the general views of the morphology of the thymus and spleen of mice; A2 and B2 are the statistical graphs of the thymus index and spleen index; in the figure, C1 is a representative graph of the number of each subset of thymus cells after WH-1 treatment of septic mice detected by FCM; in the figure, C2 is a statistical graph of the number of each subset of thymus cells after WH-1 treatment of septic mice detected by FCM. Control vs LPS, WH-1 vs LPS, * P< P < 0.05, ** P< P < 0.01, *** P< P < 0.001。

[0027] Figure 8This is a graph showing the changes in the number of cells in each subpopulation of thymic epithelium and the expression of cytokines in thymic epithelial cells of septic mice under different treatments in Example 6. In the graph, A1 is a representative graph of the number of cells in each subpopulation of thymic epithelium detected by FCM after treating septic mice with WH-1; A2 is a statistical graph of the results of the number of cells in each subpopulation of thymic epithelium; B1 is a representative graph of the change in the expression level of inflammatory factors in thymic epithelial cells detected by FCM after treating septic mice with WH-1; B2 is a statistical graph of the results of the change in the expression level of inflammatory factors in thymic epithelial cells. In the graph, Control vs LPS, WH-1 vs LPS, * P< 0.05, ** P< 0.01, *** P< 0.001.

[0028] Figure 9 This is a graph showing the changes in the number of CD3 + T cells and their respective subpopulations in the spleens of septic mice under different treatments in Example 6. In the graph, A is a representative graph of the number of cells in each subpopulation of the spleen detected by FCM after treating septic mice with WH-1; B is a statistical graph of the flow cytometry results. Detailed implementation manners

[0029] The technical solutions of the present invention will be further explained and illustrated below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the following embodiments are only the preferred embodiments of the present invention and should not be construed as limiting the present invention. The protection scope of the present invention should be based on the content recorded in the claims. Modifications and substitutions made by those skilled in the art to the technical solutions of the present invention without creative efforts fall within the protection scope of the present invention.

[0030] Biomaterials: Immortalized thymic epithelial cell line (iTECs): Generously donated by Professor Gao Jianli of Zhejiang Chinese Medical University; SPF-grade C57BL / 6J mice: Purchased from the Animal Center of China Three Gorges University, and the mice were 7 days old.

[0031] Example 1 Extraction of WH-1 (1) Take Ligularia virgaurea seeds naturally dried at room temperature, divide them into several filter bags, and use a multi-functional extraction and concentration unit to perform hot reflux extraction 3 times with 95% ethanol at 75°C for 24 hours each time; (2) After concentrating the extraction solution in step (1) under reduced pressure to remove the solvent, obtain the crude ethanol extract of Ligularia virgaurea seeds; (3) Add 600 mL of pure water to disperse the crude ethanol extract of Ligularia virgaurea seeds, and then add petroleum ether (60 - 90°C), ethyl acetate, and n-butanol with the same volume as water for extraction in sequence; (4)The petroleum ether extract was concentrated under reduced pressure to remove the solvent, and an extract of the petroleum ether extraction part of Ligularia veitchiana seeds was obtained; (5)After mixing the extract of the petroleum ether extraction part of Ligularia veitchiana seeds with silica gel at a mass ratio of silica gel: extract of 1:1, normal-phase silica gel column chromatography (200 - 300 mesh) was used for separation, and gradient elution was carried out with petroleum ether∶ethyl acetate (V:V = 100:0 → 0:100) as the eluent to obtain 279 fractions; (6)Fraction 40 was separated and purified by preparative thin-layer chromatography with petroleum ether∶ethyl acetate (V:V = 10:1) as the developing agent to obtain Compound 3, named WH-1; (7)Identification: Compound WH-1: Colorless oily substance (CHCl3). EI-MS ( m / z ): 218 [M]+, and the molecular formula is C 15 H 22 O. 1 1H NMR (CDCl l3 , 400 MHz) δ: 0.95 (3H, d, J = 6.4 Hz, H-14), 0.97 (3H, s, H-15), 1.84 (3H, d, J = 1.6 Hz, H-13), 2.09 (3H, d, J = 2.0 Hz, H-12), 5.74 (1H, d, J = 1.6 Hz, H-9); 13 13C NMR (CDCl l3 , 100 MHz) δ: 15.6 (C-15), 16.2 (C-14), 22.2 (C-12), 22.7 (C-13), 26.7 (C-2), 30.7 (C-1), 32.7 (C-3), 41.2 (C-6), 42.1 (C-5), 42.7 (C-4), 126.3 (C-9), 128.4 (C-7), 142.4 (C-11), 168.9 (C-10), 192.5 (C-8). The above data are basically consistent with the NMR data of eremophila-7(11),9-dien-8-one, a type of eremophilane sesquiterpenoid compound (Xu Jianlong, Zou Kun, Yang Di, et al. Study on the eremophilane sesquiterpenoid chemical constituents in Ligularia veitchiana. Natural Product Research and Development, 2019, 31: 643 - 647.). Therefore, Compound WH-1 can be identified as eremophila-7(11),9-dien-8-one ( Figure 1 ); (8)The purity verification was carried out by high performance liquid chromatography (HPLC). Isocratic elution was performed with a mobile phase of acetonitrile: water = 60:60, and a single peak of WH-1 was obtained, with a purity above 95% ( Figure 2 ).

[0032] Example 2 LPS inflammatory cell model (1)The immortalized thymic epithelial cell line (iTECs) was placed in DMEM complete medium containing 10% fetal bovine serum, 1% penicillin (1×10 5 U / L), and 1% streptomycin (100 mg / mL), and cultured in an incubator at 37 °C, 5% CO2, and saturated humidity. The medium was changed in a timely manner, and the cell viability and growth status were closely monitored until the cell density reached 70% - 80%; (2)Remove the DMEM medium, rinse the cells with PBS buffer solution, completely discard the PBS solution, and then add DMEM complete culture medium containing 0, 0.1, 0.5, 1.0 μg / mL LPS respectively, and incubate at 37 °C for 12 h; (3)Collect the cells of each treatment group, rinse them 3 times with PBS, and divide the TECs cells into two groups. For the first group of cells, first stain the cells with FVS-APC / cy7 for 10 min to identify cell viability, then incubate with purified CD16 / 32 antibody for 10 min to block Fc receptors. Incubate with fluorescently labeled mouse antibodies CD45-PE / cy7, EpCAM-Percp / Cy5.5, I-A / I-E-BV421, CD80-BV510 (CD80, namely B7 molecule, is a co-stimulatory factor for T cell activation), and Ly51-PE in the dark at 4 °C for 45 min. After fixing and permeabilizing the TECs cells with the kit from BD company (cat.no. 554714) according to the instructions, incubate with nuclear antibodies related to proliferation index Ki-67-647 and autoimmune regulator AIRE-488 in the dark for 30 min. Finally, resuspend with PBS for FCM detection of related indicators; For the second group of cells, first stain the cells with FVS-APC / cy7 to identify cell viability, then block Fc receptors with purified CD16 / 32 antibody, incubate in the dark at 4 °C for 45 min. After fixing and permeabilizing the TECs cells with the kit from BD company (cat.no. 554714) according to the instructions, incubate with nuclear antibodies Bcl-2-PE / cy7, inflammatory factors IL-10-PerCP / cy5.5, IL-1-FITC, TNFα-PE, and IL-6-APC in the dark at 4 °C for 30 min. Finally, resuspend with PBS for FCM detection of related indicators; (4)Collect the cells of each treatment group, rinse them 3 times with PBS buffer solution, extract total RNA with Trizol reagent, reverse transcribe to obtain cDNA, and perform RT-qPCR to detect the changes in the activity, inflammation and function-related gene expression levels of iTECs; using β-Actin as an internal reference, according to the formula: relative expression = 2 ﹣△△CT , calculate the relative expression levels of each gene.

[0033] Flow cytometry (FCM) results showed that the percentage of cTECs decreased and the percentage of mTECs increased ( Figure 3 A1, A2). The expression levels of anti-apoptosis indicators Bcl-2 and IL-10 decreased in cTECs; but the expression of Ki67 and Aire increased in mTECs ( Figure 3 B1, B2); and the optimal LPS modeling concentration screened was 0.1 μg / mL. RT-qPCR results showed that LPS decreased the target genes regulated by FOXN1 expressed by cTEC CCl25, CXCL12 , genes mediating T cell development CD40 , proliferation-related genes Fgfr2 , anti-apoptotic protein genes Bcl2 expression ( Figure 3 C1).

[0034] Example 3 WH-1 treatment of LPS inflammatory cell model (1) Take the inflammatory cell model obtained in Example 1 under 0.1 μg / mL LPS treatment, rinse the cells with PBS buffer solution, remove the DMEM medium, and then add complete DMEM solutions containing 0, 1.25, 2.5, 5, 10, 20, 40 μg / mL WH-1 (stock solution is dissolved 40 mg / mL WH-1) respectively (and ensure that the solution contains LPS with a final concentration of 0.1 μg / mL), and only add the solvent with an equal volume concentration to 0 μg / mL WH-1, and incubate at 37°C for 36 h; (2) Take the cells of each treatment group in step (1), add 10 μL of CCK8 solution to each well, gently shake the culture plate to mix evenly, and then place it in an incubator at 37°C for incubation. At 4 h, use an enzyme-labeled instrument to calculate the cell viability; (3)Collect the cells in each treatment group of 5, 10, and 20 μg / mL WH-1 in step (1), rinse them 3 times with PBS, divide the TECs cells into two groups. For the first group of cells, first stain the cells with FVS-APC / cy7 for 10 min to identify cell viability, then incubate with purified CD16 / 32 antibody for 10 min to block Fc receptors. Incubate with fluorescently labeled mouse antibodies CD45-PE / cy7, EpCAM-Percp / Cy5.5, I-A / I-E-BV421, CD80-BV510 (CD80, namely B7 molecule, is a co-stimulatory factor for T cell activation), and Ly51-PE in the dark at 4°C for 45 min. After fixing and permeabilizing the TECs cells with the kit from BD company (cat.no. 554714) according to the instructions, incubate with nuclear antibodies related to proliferation index Ki-67-647 and autoimmune regulator AIRE-488 in the dark for 30 min. Finally, resuspend with PBS for FCM detection of related indicators. For the second group of cells, first stain the cells with FVS-APC / cy7 to identify cell viability, then block Fc receptors with purified CD16 / 32 antibody, incubate in the dark at 4°C for 45 min. After fixing and permeabilizing the TECs cells with the kit from BD company (cat.no. 554714) according to the instructions, incubate with nuclear antibodies Bcl-2-PE / cy7, inflammatory factors IL-10-PerCP / cy5.5, IL-1-FITC, TNFα-PE, and IL-6-APC in the dark at 4°C for 30 min. Finally, resuspend with PBS for FCM detection of related indicators; (4)Collect the cells in each treatment group in step (1), rinse them 3 times with PBS buffer solution, extract total RNA with Trizol reagent, reverse transcribe to obtain cDNA, and perform RT-qPCR to detect the changes in the expression levels of genes related to iTECs activity, inflammation, and function; Use β-Actin as an internal reference, according to the formula: relative expression = 2 ﹣△△CT , calculate the relative expression levels of each gene.

[0035] The CCK8 results showed that different concentrations of WH-1 could enhance the viability of iTECs cells, showing concentration dependence at 5, 10, and 20 μg / mL, and the effect was relatively significant. Therefore, in subsequent experiments, the concentrations of WH-1 were selected as 5, 10, and 20 μg / mL for treatment ( Figure 4 A).

[0036] When WH-1 and LPS were co-treated with iTECs cells, the RT-qPCR results showed that at 3, 9, and 12 h, WH-1 could reduce the expression of the pro-inflammatory factor IL-6 ( Figure 4 B).

[0037] After treating iTECs cells with WH-1 and LPS simultaneously for 36 h, the flow cytometry results showed that WH-1 could reduce the expression levels of pro-inflammatory factors IL-1, IL-6, and TNF-α in inflammatory iTECs, and could increase the expression levels of anti-inflammatory factor IL-10 and anti-apoptosis index Bcl-2 ( Figure 4 C). At the same time, WH-1 could upregulate the high expression of CD80 and MHCII in TECs, and upregulate the high expression of MHCII in cTECs and mTECs ( Figure 5 A and Figure 5 B).

[0038] Example 4 Effect of WH-1 on inflammatory signaling pathways (1) Take the iTECs cells treated with 5, 10, and 20 μg / mL WH-1 solutions for 36 h in Example 3, wash them twice with PBS, extract proteins with RAPI lysis buffer, and perform protein quantification by the BCA method; (2) Use polyacrylamide gel electrophoresis and load 25 μg of total protein into each lane; (3) After electrophoresis, transfer the proteins to a PVDF membrane, block it with 5% skim milk, incubate with the primary antibody overnight on a shaker at 4°C; wash three times with TBST, incubate with the secondary antibody; use ECL luminescent solution for development.

[0039] The Western blot results showed that WH-1 could reduce the expression of pattern recognition receptors TLR2 and TLR4 upstream of the NF-κB signaling pathway, and at the same time could reduce the expression of IKKα / IKKβ, p65, and p-p65 downstream of the pathway ( Figure 6 ), thereby inhibiting the inflammatory pathway.

[0040] Example 5 Effect of WH-1 on thymus and spleen in neonatal mouse sepsis model (1) Take 7-day-old SPF-grade C57BL / 6J neonatal mice and co-house them with their mothers under conditions of a temperature of 22 ± 2°C, a humidity of 60 ± 5%, and a simulated day-night change of light with low sensitivity; (2) Intraperitoneally inject 10 mg / kg LPS into 7-day-old neonatal mice to establish a model. Starting from the next day, intraperitoneally inject 5, 10, and 20 mg / kg of WH-1 (the mice treated with WH-1 are the drug administration group), and the control group only injects an equal volume of solvent (i.e., a DMSO / PBS solution with an equal volume concentration). Inject once a day for 5 consecutive days, and sacrifice the mice on the 6th day to obtain their thymus, spleen, peripheral blood, and kidneys and lungs for detection; It can be seen from Figure 7 that there was no significant difference in the thymus and spleen between the WH-1 drug administration group and the LPS model group ( Figure 7A1, B1), the thymus index increased but without statistical significance, and the spleen index showed no obvious change ( Figure 7 A2, B2).

[0041] Example 6 Effect of WH-1 on thymocytes in a neonatal mouse sepsis model Take the thymus, spleen, and peripheral blood of the mouse models in each group in Example 5, prepare single cell suspensions of mouse thymus, thymic epithelial cells, spleen, and peripheral blood, label the corresponding fluorescent antibodies, and use flow cytometry to detect the changes in the numbers and proportions of each subset of thymocytes, each subset of TECs, each subset of spleen T lymphocytes, and the changes in inflammatory factor markers in the peripheral blood of the model mice.

[0042] The FCM detection results showed that WH-1 increased the numbers of DN, DP, CD4 + SP, CD8 + SP subsets in the thymocytes of septic mice, and the 5 mg / kg group was more significant ( Figure 7 C1, C2).

[0043] The FCM detection results showed that WH-1 increased the numbers of TECs, cTECs, mTECs, CD80 hi TECs, CD80 lo TECs, MHCII hi TECs, MHCII lo TECs, MHCII hi cTECs, MHCII lo cTECs, MHCII hi mTECs, MHCII lo in the subsets of mTECs in the thymic epithelial cells of septic mice ( Figure 8 A1, A2), and also increased the expression of the inhibitory inflammatory factor IL-10 in the thymic epithelial cells of septic mice ( Figure 8 B1, B2).

[0044] The FCM detection results showed that WH-1 could increase the number of CD3 + T lymphocytes in the spleen of septic mice, and at the same time increased the numbers of CD4 + Naive T, CD8 + Naive T and CD4 + RTE, CD8 + in the RTE subsets in the spleen of septic mice ( Figure 9 A, B).

Claims

1. Use of a sesquiterpenoid compound in the preparation of a drug for treating thymus damage, characterized in that: Include at least one of the following: (a) Use in the preparation of drugs for treating acute thymus injury; (b) Application in regulating the secretion of inflammatory factors in thymocytes and their subsets; (c) Application in increasing the number of immune cells.

2. Use of a sesquiterpenoid compound according to claim 1 in the preparation of a drug for treating thymus damage, characterized in that: The acute thymic injury described in (a) is acute thymic atrophy caused by infectious diseases.

3. Use of a sesquiterpenoid compound according to claim 1 in the preparation of a drug for treating thymus damage, characterized in that: The thymic epithelial cells and their subpopulations described in (b) are one or more of TECs, cTECs and mTECs; the inflammatory factors include pro-inflammatory factors and anti-inflammatory factors.

4. Use of a sesquiterpenoid compound according to claim 3 in the preparation of a drug for treating thymus damage, characterized in that: The pro-inflammatory factor is one or more of IL-1, IL-6 and TNF-α; the anti-inflammatory factor is IL-10.

5. Use of a sesquiterpenoid compound according to claim 1 in the preparation of a drug for treating thymus damage, characterized in that: The immune cells described in (c) include one or more of thymocytes and their subsets, and splenic T lymphocytes and their subsets.

6. Use of a sesquiterpenoid compound according to claim 5 in the preparation of a drug for treating thymus damage, characterized in that: The thymocytes and their subpopulations include thymocytes DN, DP, CD4 + SP、CD8 + SP subgroups; the thymic epithelial cells and their subgroups include TECs, cTECs, mTECs, CD80 hi TECs, CD80 lo TECs, MHCII hi TECs, MHCII lo TECs, MHCII hi cTECs、MHCII lo cTECs、MHCII hi mTECs、MHCII lo mTECs subsets; the spleen T lymphocytes and their subsets are CD3 + T lymphocytes and their subsets CD4 + T, CD8 + T, CD4 + Naive T、CD8 + Naive T, CD4 + RTE and CD8 + RTE.

7. Use of a sesquiterpenoid compound according to claim 1 in the preparation of a drug for treating thymus damage, characterized in that: The sesquiterpenoid compound is an erimophenan-type sesquiterpenoid compound, and the molecular formula is C 15 H 22 O.

8. Use of a sesquiterpenoid compound according to claim 7 in the preparation of a drug for treating thymus damage, characterized in that: The erimopane-type sesquiterpenoid compounds are extracted from the seeds of Ligularia officinalis.

9. Use of a sesquiterpenoid compound according to claim 8 in the preparation of a drug for treating thymus damage, characterized in that: The sesquiterpenoid compounds are extracted from the petroleum ether extraction part of Ligularia japonica seeds.

10. Use of a sesquiterpenoid compound according to claim 1 in the preparation of a drug for treating thymus damage, characterized in that: The effective concentration of the sesquiterpenoid compound in the drug is 5-20 μg / mL.

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