| Tuberculosis(TB) is a major infectious disease, serious harm to human health, in this century is still a public health and social issues of global concern. Since the anti-tuberculosis drugs were used to treat TB,there have been reports of drug-resistant tuberculosis, but this problem did not caused enough attention. In recent years, due to the mortality rate of multidrug-resistant TB(MDR-TB) rise, the development of new effective anti-TB drugs is imminent.Verapamil is a kind of phenylalkylamine calcium channel blocker, a specific inhibitor of L- type calcium channel(LTCC), combines with alpha1 subunit receptor of the LTCC to reduce opening rate of calcium channel.Clinically, it is mainly used for the treatment of cardiovascular diseases.Recent studies found that the verapamil showed potential therapeutic value on pulmonary tuberculosis in combination with chemotherapy. It could enhance the effect of chemotherapy, shorten the course of treatment, and reduce drug resistance. The sufficient research data showed that the LTCC is a very promising therapeutic target of anti-TB drug, has become a new direction on new drug research.In this study, the expression changes of calcium channel on macrophages infected with Mycobacterium tuberculosis(M.tb) were detected by quantitative reverse transcription-polymerase chain reaction(RT-PCR) method, and the effect of verapamil on macrophage’s immune function was studied. We understood the expression of proinflammatory genes and the secretion of inflammation-related cytokines in theM.tb-infected macrophages under the effects of different concentrations of verapamil.1. The expression of calcium channel of macrophages infected with Mycobacterium tuberculosisFifty-six of mice were randomly divided into seven groups, infected with M.tb virulent strain H37 Rv, attenuate strain H37 Ra, M. bovis BCG(BCG), staphylococcus aureus(S.aureus), respectively. The macrophages were harvested by peritoneal lavage at 24 hours after infection. The expression of macrophage LTCC mRNA was determined by RT-PCR method. The results showed LTCC gene expression levels of macrophages infected by M.tb obviously increased in comparation with normal control group and S.aureus group(P < 0.05). The LTCC gene expression levels of macrophages infected by S.aureus had no significant change(P > 0.05).2. The effect of verapamil on macrophage’s immune functionSixty of mice were randomly divided into two groups, one group was normal control group, and another group was infected with M.tb H37Rv(infection group). The macrophages were harvested by peritoneal lavage at24 hours after infection. The macrophages harvested in normal control group and infection group were randomly divided into 9 subgroups,respectively. 0 μg/ml, 1 μg/ml, 5 μg/ml, 10 μg/ml, 25 μg/ml, 50 μg/ml, 100μg/ml, 200 μg/ml, and 300 μg/ml of verapamil were added to cultured macrophages each subgroup, respectively. At 24 hours after culture, the macrophages were collected, then the IL-12 and nuclear factor-kappa B(NF-κB) mRNA expression levels of macrophage were determined by semi-quantitative RT-PCR method. The supernatants of cultured macrophages and cell lysates were collected, the expression levels of IL-12(p40), tumor necrosis factor-α(TNF-α), IL-1β, and NF-κBp65(pS536), NF-κBp65(Total) in the supernatants or cell lysates were detected by ELISA method. The results showed as follow: Compared with normal control group without verapamil, the expression levels of IL-12 and NF-κBgenes in infection group significantly increased(P < 0.05), the IL-12(p40)level in macrophage culture supernatant and the levels of NF-κBp65( p S536) and NF-κBp65(Total) in cell lysates also significantly increased(P < 0.05). When a certain concentration range of verapamil were added into macrophage cultures, the expression of IL-12 mRNA and NF-kappa B mRNA, the IL-12(p40) level in macrophage culture supernatant, and the levels of NF-κBp65( pS536) and NF-κBp65(Total) in macrophage lysates in infection groups were significantly higher than those in normol control group without verapamil. When 50 μg/ml or more verapamil was added,these gene expressions or cytokine levels had no significant difference with normol control group without verapamil(P > 0.05). Compared with the normal group without verapamil, the IL-1β level and TNF-α level in infection groups were significantly higher(P < 0.01). The IL-1β level and TNF-α level in infection groups decreased dose-dependently with increase of verapamil concentrations. When 25 μg/ml or more verapamil was added,the IL-1β level and TNF-α level in infection groups had no significant difference with the normal group without verapamil(P < 0.05). Verapamil had less impact on the secretion of cytokines in the normal macrophages.The results mentioned above showed that the expression of LTCC, the gene expression or the secretion of IL-12 and NF-kappa B, and the secretion of IL-1β and TNF-α was all significantly increased in the macrophages infected with M.tb. Calcium channel blocker verapamil in a certain concentration range, IL-12 and NF-κB both in gene level or at cell level elevated with the increase of verapamil concentration. However, the secretion of IL-1β and TNF-α decreased with the increase of verapamil concentration in a dose-dependent manner. Calcium channel blocker verapamil could enhance the immunity of macrophages infected with M.tb. |
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