关键词: 日本血吸虫, G蛋白偶联受体, SjGPR89, 双链RNA干扰, 生长发育

Abstract:

Objective To preliminarily explore the role of G protein coupled receptors （SjGPR89） in the growth and development of Schistosoma japonicum. Methods The protein structure and transmembrane structure of SjGPR89 were predicted using the SMART website and TMHMM website. Forty femal Kunming mice at 6-8 week old of specific pathogen free (SPF) type were infected with S. japonicum cercariae 200 ± 10 per mouse through abdominal skin, and the schistosomes were collected on the 14, 16, 18, 20, 22, 24, 26, 28, and 30 days post-infection (dpi), respectively. The worm RNA was extracted and reverse transcribed into cDNA, and the relative expression level of SjGPR89 mRNA was determined by qRT-PCR. Twelve SPF Kunming mice were infected with (60 ± 2) cercariae each, and then randomly assigned into SjGPR89 double-stranded RNA (dsRNA) group (interference group) and a green fluorescent protein (GFP) control group at even. To each of the interference group mice, 10 μg SjGPR89 dsRNA was injected through the tail vein on 1, 6, 10, 14, 18, 22, and 26 dpi. On day 30, the worm RNA was extracted from 5 male and 5 female worms and reverse transcribed into cDNA, and the relative expression level of SjGPR89 mRNA was determined by qRT-PCR. The males and females separately manually were fixed using acetate formalin alcohol (AFA) to measure the worm body length using Image J software and to count the worm burden; the male and femal were stained with carmine to observe the developmental changes of gonads with fluorescence microscope. Additional 12 femal SPF Kunming mice of 6-8 week old were infected with 40 ± 2 cercariae each mouse, and then randomly assigned evenly into the interference group and control group; to each of the interference group mice, 10 μg dsRNA was injected through the tail vein, on the 26 30, 34 and 38 dpi, and on day 42 for collection of liver tissue to determine the relative transcription levels of collagen Ⅰ, collagen Ⅲ, and α-smooth muscle actin (α-SMA) mRNA by qRT-PCR. The liver tissues were digested with 5% NaOH to count the eggs and calculate the egg number/g tissue; liver sections were prepared and stained with Masson to observe the status of fibrosis. Results The TMHMM website predicted that SjGPR89 is a conserved nine-times transmembrane G protein-coupled receptor, and the SMART website predicted that SjGPR89 protein has four transmembrane regions, one GPHR_structure domain, one low complexity region, and one abscisic acid GPCR domain. SjGPR89 transcription in female and male S. japonicum at different developmental stages (14-30 days) in mice were found at a relatively stable level detected by qRT-PCR; continuous interference with tail vein injection of SjGPR89-dsRNA showed that the relative mRNA expression of SjGPR89 in interferece females (307.70 ± 58.21) and males and (97.88 ± 11.38) were significantly lower than the GFP control group (767.10 ± 142.79) and (182.02 ± 7.42) (t = 5.96, 12.39; P < 0.01), with knockdown levels of 59.89% and 46.23%, respectively. Image J measurement showed that the lengths of the female (12.13 ± 2.67) and male (10.00 ± 1.72) in the interference group were significantly shorter than that of the control group (13.67 ± 1.74), (11.48 ± 1.94) (t = 4.10, 5.09; P < 0.01), the female and male parasites were 11.22% and 12.93% shorter, respectively. The number of parasite loads of the female and male parasites in the interference group (23.00 ± 1.83), (23.75 ± 2.99) was less than that of the control group (26.75 ± 0.96), (31.00 ± 3.56) (t = 3.64, 3.12; P < 0.05) significantly, and the degree of reduction of male and female parasite loads were 14.02% and 23.39%. The results of the carmine staining revealed that the gonads of the females in the interference group were delayed in development, the coloring of the ovaries and vitelline glands was lighter, and the area of the ovaries was significantly less than that of the control group; the male gonads were immature and smaller than those of the control group. The number of eggs per gram liver tissue in the interference group (2 777.33 ± 197.94) was significantly less than that in the control group (5 871.32 ± 875.25) (t = 5.97, P < 0.01), with a reduction of 52.70%, and the degree of liver fibrosis in the interference group was further observed to be significantly less than that in control group using Masson staining. qRT-PCR detected the relative expressions of Collagen Ⅰ, Collagen Ⅲ, and α-SMA in the liver tissues of the interference group (530.20 ± 246.81), (825.26 ± 139.82), (551.59 ± 189.94) were significantly lower than those of the control group (t = 3.81, 2.50, 4.72; P < 0.05). Conclusion S. japonicum development, viability, and egg production were impacted by interference with the SjGPR89 protein. Additionally, the host’s liver is far less likely to sustain pathological damages.

Key words: Schistosoma japonicum, G protein coupled receptors, SjGPR89, Double-stranded RNA interference, Growth and development