Infectious Diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, Kolkata 700032, India.
J Antimicrob Chemother. 2021 Jan 1;76(1):135-145. doi: 10.1093/jac/dkaa406.
To evaluate the antileishmanial efficacy of genipin, which specifically inhibits uncoupling protein 2 (UCP2) that is induced in leishmaniasis to neutralize reactive oxygen species (ROS).
The effect of genipin was assessed against intracellular parasites in cultured macrophages and in suppressing spleen and liver parasite burdens in a BALB/c mouse model of visceral leishmaniasis by microscopic evaluation of intracellular amastigotes stained with Giemsa. ROS and mitochondrial membrane potential were measured by H2DCFDA- and JC-1-based fluorometric analysis. ELISA was performed for various Th1 and Th2 cytokines in both in vitro and in vivo infected conditions to evaluate the type of immunological responses. The role of UCP2 was assessed by lipofectamine-mediated transfection and overexpression in macrophages and short hairpin RNA-mediated knockdown of UCP2 in infected animals.
Genipin reduced the infection-induced UCP2 levels in macrophages, with optimum effect at 100 μM. Genipin reversed parasite-induced ROS suppression and mitochondrial membrane potential disruption. It has no inhibitory effect on promastigote or axenic amastigote forms, but markedly suppressed amastigote multiplication within macrophages, which was reversed by the ROS scavenger N-acetyl cysteine. Genipin administration (30 mg/kg/day) in infected mice showed significant suppression of liver and spleen parasite burdens with an enhanced host-favourable cytokine balance in a ROS-p38 mitogen-activated protein kinase-dependent manner. Co-treatment with genipin plus a sublethal dose of sodium antimony gluconate (SAG50) showed almost a curative reduction in spleen and liver parasite burden.
These results suggest the effectiveness of genipin as a synergistic agent for the front-line antileishmanial drug SAG in circumventing the resistance and toxicity problems associated with its high curative dose.
评估京尼平抑制解偶联蛋白 2（UCP2）的抗利什曼原虫疗效，UCP2 在利什曼病中诱导产生以中和活性氧（ROS）。
通过吉姆萨染色评估细胞内无鞭毛体，在 BALB/c 内脏利什曼病小鼠模型中通过显微镜评估细胞内利什曼原虫，评估京尼平对培养的巨噬细胞内寄生虫和抑制脾、肝寄生虫负荷的作用。通过基于 H2DCFDA 和 JC-1 的荧光分析测量 ROS 和线粒体膜电位。在体外和体内感染条件下进行各种 Th1 和 Th2 细胞因子的 ELISA 检测，以评估免疫反应类型。通过脂转染介导的转染和过表达以及感染动物的短发夹 RNA 介导的 UCP2 敲低评估 UCP2 的作用。
京尼平降低了巨噬细胞中感染诱导的 UCP2 水平，在 100μM 时效果最佳。京尼平逆转了寄生虫诱导的 ROS 抑制和线粒体膜电位破坏。它对前鞭毛体或无鞭毛体形式没有抑制作用，但明显抑制了巨噬细胞内无鞭毛体的增殖，ROS 清除剂 N-乙酰半胱氨酸可逆转这种抑制作用。感染小鼠给予京尼平（30mg/kg/天）治疗后，显示出显著抑制肝和脾寄生虫负荷，同时以 ROS-p38 丝裂原激活蛋白激酶依赖性方式增强有利于宿主的细胞因子平衡。京尼平联合亚致死剂量的葡萄糖酸锑钠（SAG50）联合治疗显示出对脾和肝寄生虫负荷几乎治愈性的降低。
这些结果表明京尼平作为前线抗利什曼原虫药物 SAG 的增效剂的有效性，可规避其高治疗剂量相关的耐药性和毒性问题。
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