阅读说明：本技术 曲格列酮在抗结核分枝杆菌药物中的应用 (Application of troglitazone in anti-mycobacterium tuberculosis drugs ) 是由 张国良 毕静 郭庆龙 李国保 王召钦 黄秀静 覃琳琇 于 2021-09-30 设计创作，主要内容包括：本发明公开了曲格列酮在抗结核分枝杆菌药物中的应用,包括以下步骤：THP-1巨噬细胞的分化：利用PMA对THP-1细胞进行诱导分化,然后加入曲格列酮进行培养；结核分枝杆菌的感染：结核分枝杆菌按MOI=10感染细胞,然后进行清洗,加入培养基进行继续培养；涂板计数:感染一段时间后,对细胞进行裂解,然后再进行梯度稀释涂板,再进行长时间培养,然后得到细菌存活数量。本申请测定了结核分枝杆菌强毒株H37Rv感染巨噬细胞后,曲格列酮在细胞内的抗结核分枝杆菌活性,结果显示曲格列酮显著增强巨噬细胞对结核分枝杆菌的清除作用,具有良好的开发价值。(The invention discloses an application of troglitazone in a mycobacterium tuberculosis resistant drug, which comprises the following steps: differentiation of THP-1 macrophages: inducing and differentiating THP-1 cells by using PMA, and then adding troglitazone for culturing; infection with mycobacterium tuberculosis: infecting cells with mycobacterium tuberculosis according to MOI =10, then cleaning, and adding a culture medium for continuous culture; and (4) plating counting, namely after a period of infection, cracking the cells, then performing gradient dilution plating, and then performing long-time culture to obtain the survival number of bacteria. The application determines the activity of troglitazone against mycobacterium tuberculosis in cells after macrophages are infected by a virulent strain H37Rv of mycobacterium tuberculosis, and the result shows that troglitazone obviously enhances the clearing effect of the macrophages on the mycobacterium tuberculosis and has good development value.)

1. The application of troglitazone in the anti-mycobacterium tuberculosis drugs is characterized by comprising the following steps:

differentiation of THP-1 macrophages: culturing and differentiating THP-1 cells by using PMA, and then adding troglitazone for culturing;

infection with mycobacterium tuberculosis: infecting the cells with Mycobacterium tuberculosis according to MOI 10, cleaning, adding culture medium, and continuing culturing;

and (4) plating counting, namely after a period of infection, cracking the cells, then performing gradient dilution plating, and then performing long-time culture to obtain the survival number of the cells.

2. The use of troglitazone in an anti-mycobacterium tuberculosis drug according to claim 1, wherein the formula of troglitazone is C₂₄H₂₇NO₅S, the relative molecular mass is 441.54, and the structural formula is as follows:

3. the use of troglitazone in anti-mycobacterium tuberculosis drugs according to claim 1, wherein in the differentiation step of THP-1 macrophages, a 1640 medium containing 10% fetal bovine serum is first used at 37 ℃ and 5% CO₂The cell culture chamber (2) of (2) was cultured in a 96-well plate of 5X 10 per well⁴The cells were plated and stimulated overnight with PMA at a final concentration of 100ng/ml to differentiate into macrophages, and 24h later replaced with complete medium.

4. The use of troglitazone in anti-mycobacterium tuberculosis drugs according to claim 3, wherein troglitazone at different concentrations is added after macrophage differentiation induction is continued to be cultured for 24h at 37 ℃ and 5% CO₂And (5) continuously culturing for 48h, and detecting the activity of the cells by using a cck-8 kit.

5. The use of troglitazone in an anti-mycobacterium tuberculosis drug according to claim 4, wherein the concentration of troglitazone is 5 μ M, 10 μ M, 20 μ M, 30 μ M, 50 μ M, 60 μ M, 70 μ M, 80 μ M and 100 μ M, respectively.

6. The use of troglitazone in anti-mycobacterium tuberculosis drugs according to claim 1, wherein, after THP-1 cells are induced into macrophages, troglitazone is added for pretreatment 1H before infection, mycobacterium tuberculosis H37Rv infects cells as MOI ═ 10, then PBS is used for washing, 1640 complete medium is added at 37 ℃ and 5% CO₂The culture was continued, during which time troglitazone addition was maintained.

7. The use of troglitazone in the preparation of anti-mycobacterium tuberculosis drugs as claimed in claim 1, wherein in the plating step, cells are lysed with 0.025% SDS at 4, 24, 48, 72 hours after infection, each by 10%^-2、10^-3The dilution was plated and the cells were incubated in a 37 ℃ incubator for about three weeks and counted.

Technical Field

The invention relates to the field of biomedical medicines, in particular to application of troglitazone in a mycobacterium tuberculosis resistant medicine.

Background

Tuberculosis, particularly drug-resistant tuberculosis, is the key and difficult point of current tuberculosis treatment, host-oriented treatment is a hotspot in the research field of the drug-resistant tuberculosis at present, and the core idea is to treat the drug-resistant tuberculosis by interfering the host gene function to enhance the protective immune response or inhibit excessive inflammatory reaction. On the basis of deep knowledge of host anti-tuberculosis immune characteristics and regulation mechanisms, the development of immunotherapy or more generalized new strategies for treating tuberculosis based on host gene targeting provides a new vision for treating tuberculosis, especially for overcoming multi-drug resistant tuberculosis.

Disclosure of Invention

Aiming at the defects in the technology, the invention provides a method for inhibiting mycobacterium tuberculosis by using troglitazone, which is characterized in that after macrophage is infected by a virulent strain H37Rv of mycobacterium tuberculosis, the activity of the troglitazone in the mycobacterium tuberculosis in cells is determined, and the result shows that the troglitazone has obvious activity of resisting the mycobacterium tuberculosis, and the elimination of the mycobacterium tuberculosis by the macrophage is obviously enhanced; the novel function of troglitazone for resisting mycobacterium tuberculosis is found for the first time, and the troglitazone is a potential anti-tuberculosis drug targeting a host.

In order to achieve the purpose, the invention provides application of troglitazone in anti-mycobacterium tuberculosis drugs, which is characterized by comprising the following steps:

differentiation of THP-1 macrophages: culturing and differentiating THP-1 cells by using PMA, and then adding troglitazone for culturing;

infection with mycobacterium tuberculosis: infecting cells with Mycobacterium tuberculosis according to MOI 10, cleaning, adding culture medium containing troglitazone, and continuously culturing;

and (4) counting the panels, namely cracking the cells after a period of infection, then coating the panels by dilution times, and then culturing for a long time to obtain the survival number of the cells.

Preferably, troglitazone has the formula C₂₄H₂₇NO₅S, the relative molecular mass is 441.54, and the structural formula is as follows:

preferably, in the differentiation step of THP-1 macrophage, a 1640 medium containing 10% fetal bovine serum is first used at 37 ℃ and 5% CO₂The cell culture chamber (2) of (2) was cultured in a 96-well plate of 5X 10 per well⁴The cells were plated and stimulated overnight with PMA at a final concentration of 100ng/ml to differentiate into macrophages, and 24h later replaced with complete medium.

Preferably, induced differentiation macrophages are cultured for 24h and then troglitazone of different concentrations is added at 37 ℃ and 5% CO₂And (5) continuously culturing for 48h, and detecting the activity of the cells by using a cck-8 kit.

Preferably, the concentration of troglitazone is 5. mu.M, 10. mu.M, 20. mu.M, 30. mu.M, 50. mu.M, 60. mu.M, 70. mu.M, 80. mu.M and 100. mu.M, respectively.

Preferably, after inducing THP-1 cells into macrophages, 1H before infection, troglitazone is added for pretreatment, Mycobacterium tuberculosis H37Rv infects cells at MOI 10, then PBS is used for washing, 1640 complete medium is added at 37 ℃ and 5% CO₂The culture was continued, during which time troglitazone addition was maintained.

Preferably, in the plating step, cells are lysed with 0.025% SDS 24, 48, and 72 hours after infection, each 10 th time^-2、10^-3The dilution was plated and the cells were incubated in a 37 ℃ incubator for about three weeks and counted.

The invention has the beneficial effects that: the technical scheme mainly includes that troglitazone is applied to anti-mycobacterium tuberculosis medicines, after macrophage is infected by a mycobacterium tuberculosis virulent strain H37Rv, the anti-mycobacterium tuberculosis activity of troglitazone in cells is determined, and the result shows that troglitazone has obvious anti-mycobacterium tuberculosis activity, and the elimination of mycobacterium tuberculosis by macrophages is obviously enhanced; and has the following advantages: the invention discovers the new function of troglitazone anti-tuberculosis mycobacteria for the first time, and is a potential anti-tuberculosis drug targeting a host; the invention provides the application of troglitazone in antituberculosis drugs for the first time; the invention discovers that troglitazone can obviously enhance the clearing function of macrophages on mycobacterium tuberculosis.

Drawings

FIG. 1 is a dose response curve for troglitazone of the present invention;

FIG. 2 shows the inhibition of Mycobacterium tuberculosis H37Rv by troglitazone;

FIG. 3 is a plate coating count test of the effect of troglitazone in combination with isoniazid on the clearance of M.tuberculosis by macrophages.

Detailed Description

In order to more clearly describe the present invention, the present invention is further described below with reference to the accompanying drawings and detailed description.

Troglitazone is an agonist of PPAR γ, and can increase chromatin condensation, enhance caspase-3 activity and down-regulate Bcl-2 expression in MIA Paca2 and PANC-1 cells. Troglitazone promotes TRAIL-mediated apoptosis in lung adenocarcinoma cells, and autophagy inhibitors can block troglitazone-enhanced TRAIL-induced apoptosis. Troglitazone is a thiazolidinedione drug, and can improve insulin resistance, increase the sensitivity of human tissues to insulin and enhance the action of insulin. Animal experiments show that the action target site reduces the insulin resistance by combining with a nuclear peroxisome proliferator activated receptor (involved in the transcription of insulin-responsive genes and regulating the differentiation and lipid metabolism of lipid cells); the molecular formula of troglitazone is C₂₄H₂₇NO₅S, the relative molecular mass is 441.54, and the structural formula is as follows:

however, the prior art mainly applies troglitazone to insulin, but no research or report indicates that the troglitazone can obviously enhance the scavenging function of macrophages to mycobacterium tuberculosis in the process of carrying out an anti-mycobacterium-binding test.

Example one

S1 differentiation of THP-1 macrophages: THP-1 cells were purchased from cell banks of Chinese academy of sciences using 1640 medium containing 10% fetal bovine serum at 37 ℃ with 5% CO₂The cell culture chamber (2) of (2) was cultured in a 96-well plate of 5X 10 per well⁴The cells were plated and stimulated overnight with PMA (propylene glycol methyl ether acetate) at a final concentration of 100ng/ml to differentiate into macrophages, and 24h later the medium was replaced with complete medium.

S2 determination of cell viability: continuously culturing the above induced and differentiated macrophage for 24 hr, adding troglitazone at concentrations of 5 μ M, 10 μ M, 20 μ M, 30 μ M, 50 μ M, 60 μ M, 70 μ M, 80 μ M and 100 μ M respectively; and (3) repeating three times for each concentration, taking troglitazone solvent DMSO (dimethyl sulfoxide) as a control group, taking a cell-free culture medium as a blank group, continuously culturing for 48 hours at 37 ℃ in 5% CO2, detecting the activity of the cells by using a cck-8 kit, adding 10 mu l of cck-8 into each hole, culturing for 2 hours in an incubator, and measuring the absorbance of OD450nm by using a microplate reader.

Then calculating the cell survival rate of troglitazone with different concentrations according to the following formula, drawing a dose response curve by utilizing GraphPad software and calculating IC₅₀。：

The cell survival rate is [ (As-Ab)/(Ac-Ab) ]. times.100%

Wherein As: experimental wells (medium containing cells, CCK-8, troglitazone at various concentrations); ac: control (cell-containing medium, CCK-8, DMSO); ab: blank wells (medium without cells, CCK-8).

FIG. 1 is a dose response curve of troglitazone, showing that troglitazone is IC for THP-1 cells₅₀The concentration was 41.14. mu.M.

Example two

S3 branch rod for tuberculosisInfection with bacteria: THP-1 cells were plated in 24-well plates at 2.5X 10 per well⁵Inducing the number of cells into macrophage by the above method, adding troglitazone for pretreatment 1 hr before infection, infecting the cells with Mycobacterium tuberculosis H37Rv at MOI of 10, washing with PBS for 3 times 4 hr, adding 1640 complete culture medium, culturing at 37 deg.C and 5% CO₂The culture was continued in the incubator, during which time troglitazone addition was maintained.

S4, coating plate counting CFU: after infection for 4, 24, 48, 72 hours, cells were lysed with 0.025% SDS, 10 each^-2、10^-3Dilution was plated and CFU was counted at 37 ℃ in a bacterial incubator for about three weeks.

FIG. 2 shows the inhibition of the troglitazone on Mycobacterium tuberculosis H37Rv, and the results show that the survival rate of the Mycobacterium tuberculosis is obviously reduced after the troglitazone is added compared with that of a DMSO control group, and the inhibition effect on the Mycobacterium tuberculosis is also obviously increased along with the increase of the concentration of the troglitazone. Therefore, the conclusion can be drawn that troglitazone can effectively inhibit the growth of mycobacterium tuberculosis in macrophages, thereby providing a new research direction for the anti-tuberculosis treatment.

Example three:

infection with mycobacterium tuberculosis: THP-1 cells were plated in 24-well plates at 2.5X 10 per well⁵Inducing the number of cells into macrophage by the above method, adding troglitazone for pretreatment 1 hr before infection, infecting the cells with Mycobacterium tuberculosis H37Rv at MOI of 10, washing with PBS for 3 times 4 hr, adding 1640 complete culture medium, culturing at 37 deg.C and 5% CO₂The culture was continued in the incubator of (1), during which the addition of troglitazone/isoniazid was maintained.

Plate coating counting CFU: 24, 48, and 72 hours after infection, cells were lysed with 0.025% SDS, 10 each^-2、10^-3Dilution was plated and CFU was counted at 37 ℃ in a bacterial incubator for about three weeks.

FIG. 3 shows the inhibition of Mycobacterium tuberculosis H37Rv by combination of troglitazone and isoniazid, and the results show that the single use of isoniazid or troglitazone can effectively inhibit or kill Mycobacterium tuberculosis in macrophages, and that the combination of isoniazid and troglitazone can significantly enhance the killing effect of isoniazid.

The above disclosure is only for a few specific embodiments of the present invention, but the present invention is not limited thereto, and any variations that can be made by those skilled in the art are intended to fall within the scope of the present invention.