| Objective To observe and explore the effect of Yuehua Pills(capsules)on key proteins of autophagy,such as LC3 and Beclin-1,in mononuclear macrophages infected with multidrug-resistant tuberculosis,and to reveal multidrug-resistant tuberculosis from the perspective of serum pharmacology and molecular biology.The role and mechanism of autophagy in the infection of macrophages provides an experimental basis for clarifying the effect of traditional Yuehua Pill against tuberculosis.Methods Preparation of drug-containing serum of Yuehua pills(capsules).The equivalent dose was converted from human to rat according to body surface area.The dose of 3.02 mg kg-1 was given to rats once a day for 7 days.The drug-containing serum was prepared by abdominal aorta blood sampling.(2)Determination of the effective concentration of drug-containing serum.CCK-8 method was used to detect the effect of Yuehua pill(capsule)containing serum on the activity of mononuclear macrophages in mice.The effective concentration of Yuehua pill(capsule)containing serum was selected.(3)Cell model was constructed according to cell: bacteria = 1:10 infection.(4)Autophagy key proteins LC3 B,Beclin-1 and their genes were determined.Yuehua pill(capsule)containing serum was used to interfere with the monocyte macrophages of mice infected with Mycobacterium tuberculosis.The experiment was divided into five groups:(1)negative control group;(2)10% Yuehua pill(capsule)containing serum group;(3)autophagy inhibitor group: autophagy inhibitor 3-methyladenine(3-MA)+Yuehua pill(capsule)containing serum group;Autophagy inducer group:rapamycin(Rep)group;_normal group: uninfected cell group.After 8 hours of culture,the expression of LC3 B protein,Beclin-1 protein and their genes were detected by indirect immunofluorescence,Western blot and real-time fluorescence quantitative RT-q PCR.Result:(1)CCK-8 assay of Yuehua pill(capsule)drug-containing serum on the activity of mouse mononuclear macrophages(RAW264.7)showed that 10% drug-containing(2)serum concentration had a better effect on cell proliferation,compared with the blank control group,the difference was statistically significant(P < 0.05).(2)Indirect immunofluorescence test showed that no LC3 B protein granules were found in the cytoplasm of the negative control group.Compared with negative control group,the number of LC3 B protein granules in cytoplasm of 10% Yuehua pill(capsule)containing serum group was significantly increased;the number of LC3 B protein granules in cytoplasm of autophagy inducer group was the largest;and the number of LC3 B protein granules in cytoplasm of autophagy inhibitor group was not significantly increased.(2)Western blot showed that compared with negative control group,normal group and autophagy inhibitor group,the ratio of Beclin-1 and Beclin-1/beta-actin of 10% Yuehua capsule containing serum group and autophagy inducer group increased significantly,and the expression of Beclin-1 protein increased significantly(P < 0.05);the ratio difference among negative control group,normal group and autophagy inhibitor group was significant(P < 0.05).There was no significant difference(P > 0.05).(3)RT-q PCR showed that compared with normal group,negative control group and autophagy inhibitor group,the expression of LC3 and Beclin-1 in 10% Yuehua pill-containing serum group and autophagy inducer group was significantly higher than that of internal reference gene,while the expression of LC3 and Beclin-1 was significantly higher than that of internal reference gene(p < 0.05);the difference was statistically significant in normal group,negative control group and autophagy inhibition group(p < 0.05).There was no significant difference in each value between the preparation groups(p > 0.05).Conclusion(1)Yuehua Pill(capsule)drug-containing serum can promote the proliferation of mouse monocyte macrophage(RAW264.7)and promote its vitality.(2)10% Yuehua Pill(capsule)containing drug serum regulation Beclin-1,LC3 m RNA and protein expression increased.It is proved that Yuehua Pill(capsule)can induce autophagy of macrophages infected by multidrug-resistant tuberculosis.Detection of changes in the expression of these proteins in multidrug-resistant tuberculosis infections can help provide new treatments for MDR-TB. |
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