日本血吸虫F类G蛋白偶联受体鉴定及平滑受体功能研究

摘要/Abstract

摘要： 目的　鉴定日本血吸虫F类G蛋白偶联受体（G protein⁃coupled receptor，GPCR），明确日本血吸虫平滑受体（Schistosoma japonicum Smoothened receptor，SjSMO）的生物学功能，为筛选新型抗血吸虫药物靶点提供依据。方法　利用隐马尔可夫模型（hidden Markov model，HMM）与基因组注释文件，从日本血吸虫GPCR库中筛选出6个F类GPCR，包括5个Frizzled（FZD）家族受体（SjFZD4、SjFZD5、SjFZD7、SjFZD8、SjFZD10）和1个SjSMO受体，均含保守的Frizzled结构域；在SMART网站进行蛋白质结构域分析，采用MEGA 11构建系统发育树并经iTOL网站美化。采用实时荧光定量PCR（reverse transcription quantitative real⁃time PCR，RT⁃qPCR）与原位杂交技术，分析F类GPCR的表达谱及组织定位。通过体内外RNA干扰（RNA interference，RNAi）探究SjSMO对虫体生长发育、生殖产卵的影响；体外实验选择日本血吸虫成虫开展双链RNA（double⁃stranded RNA，dsRNA）干扰，分别设置绿色荧光蛋白（green fluorescent protein，GFP）阴性对照组及SjFZD4、SjFZD5、SjFZD7、SjFZD8、SjFZD10、SjSMO dsRNA干扰组，观察各组虫体活力及状态，并统计雌雄合抱率；体内动物实验以6周龄SPF级雌性昆明小鼠作为实验动物，将小鼠随机分为GFP dsRNA处理的阴性对照组及SjFZD4、SjFZD5、SjFZD7、SjFZD8、SjFZD10、SjSMO dsRNA处理的干扰组，收集虫体与小鼠肝脏组织，测量虫体长度并统计虫荷数，使用明矾⁃卡红染色观察虫体生殖器官变化，统计小鼠肝脏虫卵数并观察肝脏病理损伤。结果　5个FZD家族受体和1个SjSMO受体均含保守的Frizzled结构域。系统发育分析显示SjSMO独立形成进化分支，与FZD家族进化距离较远。RT⁃qPCR检测结果表明SjSMO在雄虫中表达水平稳定，在雌虫中随虫体发育而逐渐下降；原位杂交技术检测结果显示SjSMO呈全身性高表达。体外RNAi结果显示不同组间雌雄合抱率存在显著差异（[χ2] = 130.530，P < 0.000 1）；两两比较结果显示，仅SjSMO干扰组合抱率（20.00%）低于对照组（94.74%）（P < 0.000 1），且虫体活力降低；FZD家族基因干扰组合抱率与对照组差异均无统计学意义（P均> 0.05）。体内RNAi结果显示，在感染后1 ～< 26 d干扰SjSMO可导致虫体长度显著缩短，干扰组雄虫、雌虫体长分别为（6.51 ± 1.77）、（5.49 ± 1.36） mm，与对照组［（9.62 ± 1.44）、（8.68 ± 2.60） mm］相比分别缩短了32.27%、36.77%（t = 7.196、5.953，P均< 0.000 1），雄虫睾丸及雌虫卵巢发育受阻；在感染后26 ～< 42 d干扰SjSMO可显著降低小鼠肝脏虫卵负荷，SjSMO干扰组小鼠每克肝脏虫卵数为（2 824 ± 844）个，与对照组［（5 548 ± 797）个］相比减少49.10%（t = 4.064，P < 0.05），且SjSMO干扰组肉芽肿形成及肝纤维化程度减轻。结论　SjSMO是调控日本血吸虫生长发育、生殖的关键受体，可作为抗血吸虫药物开发的潜在新靶标。

关键词: 日本血吸虫, F类G蛋白偶联受体, 平滑受体, 生长发育, 药物靶点

Abstract: Objective　To characterize Schistosoma japonicum Class F G protein⁃coupled receptors (GPCRs), and to decipher the biological function of S. japonicum Smoothened receptor (SjSMO), so as to provide insights into screening of novel anti⁃schistosomal drug targets. Methods　Six Class F GPCRs were identified from the S. japonicum GPCR library using a hidden Markov model (HMM) and genome annotation filese, including five frizzled (FZD) receptors (SjFZD4, SjFZD5, SjFZD7, SjFZD8, SjFZD10) and one Smoothened receptor (SjSMO). Protein domain analysis was performed at the SMART website, and a phylogenetic tree was built with the software MEGA 11 and refined with the Interactive Tree Of Life (iTOL) tool. The expression profile and tissue localization of S. japonicum class F GPCRs were analyzed using the real⁃time fluorescence⁃based quantitative PCR (RT⁃qPCR) assay and in situ hybridization, and the effects of SjSMO on parasite growth, development, reproduction, and oviposition were examined using in vivo and in vitro RNA interference (RNAi). S. japonicum adult worms were selected for double⁃stranded RNAi (dsRNA) in in⁃vitro experiments, and a green fluorescent protein (GFP) negative control group and SjFZD4, SjFZD5, SjFZD7, SjFZD8, SjFZD10, and SjSMO dsRNAi groups were assigned to observe the parasite vitality and state and calculate the female and male rating rate. For in⁃vivo animal experiments, 6⁃week⁃old SPF female mice of the Kunming strain were randomly divided into a GFP dsRNA negative control group and SjFZD4, SjFZD5, SjFZD7, SjFZD8, SjFZD10, and SjSMO dsRNAi groups. Worms and liver tissues were collected for measurement of the worm length and estimation of the worm burdens, and changes in the parasite reproductive organs were observed using alum carmine staining. In addition, the number of S. japonicum eggs in mouse livers was counted and the pathological damages of mouse livers were recorded. Results　The five FZD receptors and one Smoothened receptor all contained a conserved Frizzled domain. Phylogenetic analysis revealed that SjSMO formed an independent evolutionary clade, which exhibited a far genetic distance from the FZD family. RT⁃qPCR assay quantified stable SjSMO expression in male worms but gradually decreased in female worms with worm development, and in situ hybridization revealed high systemic expression of SjSMO. In vitro RNAi results revealed a significant difference in pairing rates among all groups ([χ2] = 130.530, P < 0.000 1). Only the SjSMO interference group exhibited a markedly reduced worm pairing rate of 20.00%, which was significantly lower than that of the control group (94.74%) (P < 0.000 1), accompanied by decreased worm vitality. In contrast, no statistically significant differences in pairing rates were observed between each FZD family gene interference group and the control group (all P > 0.05). In vivo RNAi assay revealed that SjSMO dsRNAi 1 to 26 days post⁃infection resulted in a significant reduction in worm length, with reductions in the male worm length by 32.27% and female worm length by 36.77% as compared to controls [male worms: (6.51 ± 1.77) mm vs. (9.62 ± 1.44) mm, t = 7.196, P < 0.000 1; female worms: (5.49 ± 1.36) mm vs. (8.68 ± 2.60) mm, t = 5.953, P < 0.000 1], as well as arrest of testicular development in male worms and ovarian development in females. In addition, SjSMO dsRNAi 26 to 42 days post⁃infection was found to significantly reduce hepatic egg burdens in mice, with a 49.10% reduction in the count of liver eggs in the SjSMO dsRNAi group relative to the GFP dsRNA negative control group [(2 824 ± 844) eggs per gram vs. (5 548 ± 797) eggs per gram; t = 4.064, P < 0.05], and to alleviate granuloma formation and severity of hepatic fibrosis. Conclusion　SjSMO is a key receptor that regulates the growth, development, reproduction, and oviposition of S. japonicum, which provides a new potential target for development of anti⁃schistosomal drugs.

Key words: Schistosoma japonicum, Class F G protein?coupled receptor, Smoothened receptor, Growth and development, Drug target

中图分类号:

R383.24

王馨然, 鲁振潇, 蔡明岐, 王晓玲, 胡薇. 日本血吸虫F类G蛋白偶联受体鉴定及平滑受体功能研究[J]. 中国血吸虫病防治杂志（中英文）, 2026, 38(4): 407-416, 454.

WANG Xinran, LU Zhenxiao, CAI Mingqi, WANG Xiaoling, HU Wei. Characterization of Class F G protein⁃coupled receptors and functional analysis of Smoothened receptor in Schistosoma japonicum[J]. Chinese Journal of Schistosomiasis Control, 2026, 38(4): 407-416, 454.

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日本血吸虫F类G蛋白偶联受体鉴定及平滑受体功能研究

Characterization of Class F G protein⁃coupled receptors and functional analysis of Smoothened receptor in Schistosoma japonicum

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