| Objective:To research the influence of autophagy in the infected cells of multi-drug resistant tuberculosis of yuehua pill(capsule).To explore and reveal the effect and mechanism of immunization of yuehua pill(capsule)on anti-tuberculosis,especially anti-multi-drug resistant refractory tuberculosis.Methods:(1)The preparation of yuehua pill(capsule)containing serum.According to the surface area of conversion between human and rat equivalent dose of 3 times the dose,the rat was given a stomach for 7 days,and the serum was prepared from the abdominal aorta.(2)Quality control.The qualitative and quantitative determination of the active components of main drugs Radix Rehmanniae,Radix Ophiopogonis,Radix Stemonae of yuehua pill(capsule)in drug-containing serum.(3)The determination of effective concentration of yuehua pill(capsule)containing serum.CCK-8method was used to detect the effect of yuehua pill(capsule)containing serum on the activity of monocyte-macrophage in mice(RAW264.7),the effective concentration of yuehua pill(capsule)containing serum was selected.(4)Cell infection.1μg 1×107·m L-1multi-drug resistant tuberculosis suspension was used to infect RAW264.7 cells.(5)Observation and detection of autophagy.RAW264.7 cells,which were infected with multi-drug resistant tuberculosis,was used to observe the effect of Yuehua pill(capsule)containing serum on autophagy after infection.Experimental grouping method: Except for the normal blank group were uninfected cells,RAW264.7 cells,which were infected with multi-drug resistant tuberculosis,were divided into 4 groups,a total of 5 groups:(1)negative control group(2)yuehua pill(capsule)containing serum group(3)yuehua pill(capsule)containing serum and autophagy inhibitor3-methyladenine(3-MA)group(4)positive control Rapamycin group(5)normal blank group.Index measurement:(1)Autophagosomes.The effect of the appearance and formation of autophagosomes of RAW264.7 cells infected with multi-drug resistant tuberculosis of yuehua pill(capsule)containing serum were observed by transmission electron microscope(TEM).(2)Key protein of autophagy.The expression of LC3,a key protein of autophagy,and the change of LC3Ⅱ/LC3Ⅰratio were detected by Western blot.Results:(1)The 12.05 g·kg·d gavage dose of yuehua pill(capsule)containing serum was prepared.The content of rehmannioside A,ophiopogonin D,protostemonine,detectedby HPLC,were 0.104,0.098,0.141mg·g-1in yuehua pill(capsule)containing serum.(2)The active effect of yuehua pill(capsule)containing serum on RAW264.7 cells showed that10% concentration cell proliferation effect is best,compared with the blank control group,the difference was statistically significant(P<0.05).(3)(1)According to TEM,there were no autophagosomes in negative control group,the blank control group and 3-MA group.The appearance and formation of autophagosomes were observed in 10% yuehua pill(capsule)containing serum group and rapamycin group.(2)Western blot showed that compared with other groups,the expression level of LC3Ⅱ protein in RAW264.7 macrophages was significantly increased and the ratio of LC3Ⅱ/LC3Ⅰwas significantly increased in 10% Yuehua pill(capsule)containing serum group,the difference was statistically significant(P<0.05).Conclusion:(1)Yuehua pill(capsule)containing serum contains the transfer of the active components of main drugs,could be carried on quality control.(2)Yuehua pill(capsule)containing serum can promote the proliferation and vitality of RAW264.7 cells.(3)Yuehua pill(capsule) containing serum can promote autophagy of RAW264.7 cells infected with multi-drug resistant tuberculosis.To induce autophagy in macrophages to kill mycobacterium tuberculosis may be the one of mechanisms of anti-tuberculosis and anti-multi-drug resistant tuberculosis on yuehua pill(capsule). |
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