Effect of harmine combined with rad51 inhibitor on the Echinococcus granulosus larvae and its DNA damage in vitro

doi:10.12140/j.issn.1000-7423.2024.06.006

Abstract

Abstract:

Objective To investigate the effect of harmine combined with rad51 inhibitor (RI-1) on Echinococcus granulosus in vitro and the level of DNA damage. Methods E. granulosus from the cysts of sheep liver were collected and randomly divided into groups. Each group contains a mixture of harmine (0, 20, 30, 40, 50, 100 μg/ml) and RI-1 (0, 5, 10, 20, 40, 80 μg/ml), cultured for 5 days, and stained with 0.1% methylene blue to calculate the survival rate. The group which is close to the half inhibitory concentration of harmine against E. granulosus was selected as the fixed concentration. The groups were treated against E. granulosus with a mixture of harmine and RI-1 in ratios of 40∶5, 40∶10 and 40∶20, respectively. The optimal synergistic ratio was screened. Set up a mixed drug group of harmine and RI-1, a harmine group and RI-1 group acting alone at the same concentration, and set up a positive control group (adding albendazole sulfoxide) and a blank control group. Observe the morphology of E. granulosus and vesicles after 3 days of intervention. DCFH-DA method was used to detect the reactive oxygen species content of E. granulosus after intervention in each group. Each group underwent comet assay (the positive control was doxorubicin), and the Olive tail moment was analyzed and measured. The reagent kit is used to detect the oxidative stress level and Caspase-3 content of E. granulosus after drug intervention, as well as the levels of adenosine triphosphate (ATP) and lactate dehydrogenase (LDH). qRT-PCR reaction is used to detect the mRNA expression levels of DNA damage repair related genes. Results The survival rates of E. granulosus after 5 days of intervention with 0, 20, 30, 40, 50 and 100 μg/ml harmine were (94.67 ± 1.25)%, (83.00 ± 3.27)%, (64.67 ± 3.86)%, (55.33 ± 3.40)%, (29.67 ± 2.49)%, and (9.33 ± 1.70)%, respectively. The survival rates of E. granulosus after 5 days of intervention with 0, 5, 10, 20, 40 and 80 μg/ml RI were (94.33 ± 0.94)%, (70.00 ± 2.94)%, (55.67 ± 3.86)%, and (30.00 ± 1.70)%, 5.09%, (2.67 ± 1.25)% and 0, respectively. After 3 days of intervention with a mixed drug ratio of 40 μg/ml∶10 μg/ml as the optimal synergistic ratio for E. granulosus, the survival rate of the mixed drug group was (48.67 ± 2.62)%, which was lower than that of the blank control group [(96.33 ± 1.25)%], positive control group [(91.67 ± 1.25)%], and harmine group [(72.67 ± 4.50)%] (F = 78.85, P < 0.01; F = 437.92, P < 0.05; F = 42.49, P < 0.01). The structure of the mixed drug group’s worm body collapsed, with severe morphological damage, significant shrinkage of the worm body, and insect like changes in the epidermal layer. The average fluorescence intensity of reactive oxygen species in the mixed drug group (45.71 ± 1.52) was enhanced compared with the blank control group (3.40 ± 0.55), the positive control group (12.98 ± 0.67), and the dehydroberberine group (39.79 ± 1.11) (F = 339.795, 773.96, 76.99, all P < 0.01). The Olive tail moment value of the mixed drug group (17.73 ± 1.11) was higher than that of the blank control group (0.03 ± 0.02), positive control group (6.46 ± 0.56), and dehydrocamel base group (8.80 ± 0.61) (F = 230.32, 165.59, 151.44, all P < 0.01). The SOD content in the mixed drug group was (0.71 ± 0.07) U/mg, which was lower than that in the blank control group [(2.32 ± 0.24) U/mg] and the dehydroberberine group [(1.26 ± 0.12) U/mg] (F = 39.57, 15.74, both P < 0.01). The MDA content in the mixed drug group was (0.62 ± 0.03) mmol/mg, which was lower than that in the blank control group [(0.82 ± 0.05) mmol/mg] and the dehydroberberine group [(0.70 ± 0.03) mmol/mg] (F = 22.78, 28.37, P < 0.01). The level of Caspase-3 in the mixed drug group was (1.82 ± 0.05) U/mg, which was higher than that in the blank control group [(0.70 ± 0.02) U/mg] and the dehydroberberine group [(1.36 ± 0.19) U/mg] (F = 26.24, P < 0.01; F = 7.52, P < 0.05). The mitochondrial ATP level of E. granulosus in the mixed drug group was (112.21 ± 2.72) µmol/ml, which was lower than that of the blank control group [(157.50 ± 5.23) µmol/ml] and the dehydrocoumarin group [(123.10 ± 4.73) µmol/ml] (F = 60.73, P < 0.01; F = 67.42, P < 0.05). The release of LDH from E. granulosus in the mixed drug group was (1.83 ± 0.09) mU, which was higher than that in the blank control group [(0.70 ± 0.05) mU] (F = 146.79, P < 0.01), but there was no statistically significant difference compared with the dehydrocoumarin group [(1.51 ± 0.05) mU] (F = 62.78, P > 0.05). The relative expression levels of h2ax, rad51, brca1 and mrel1 genes in the mixed drug group of E. granulosus were 2.34 ± 0.15, 1.19 ± 0.14, 1.35 ± 0.08, and 1.43 ± 0.10, respectively, which were upregulated compared with the blank control group (all 1.00 ± 0.00) (F = 23.79, P < 0.01; F = 8.96, P < 0.05; F = 30.75, P < 0.05; F = 39.81, P < 0.01). Conclusion The combination of harmine and rad51 inhibitor RI-1 can enhance the in vitro efficacy of harmine against E. granulosus, while also increasing DNA damage in E. granulosus.

Key words: Echinococcus granulosus, Harmine, rad51 inhibitor, Survival, DNA damage

CLC Number:

R383.3

GONG Yuehong, PAN Meichi, SUN Jiajia, MA Ruijia, WUSMANJIANG Aimaiti, ZHAO Yicong, ZHAO Jun, WANG Jianhua. Effect of harmine combined with rad51 inhibitor on the Echinococcus granulosus larvae and its DNA damage in vitro[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2024, 42(6): 726-736.

Figures/Tables 9

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名称 Name	序列（5'→3'） Sequences（5'→3'）
mre11	F：GAGTTCCAAGCAGTGACCAGTCTG
mre11	R：CCACTCGGCTGACGCAATCG
brca1	F：TGATTGCGACCTAAGAGAC
brca1	R：TTCCATACACAAGCCATCC
h2ax	F：TTTGGCTTCACGGCTGGCTATG
h2ax	R：TGAGTTTGCTGGAAGGGAAATGGG
rad51	F：GGCACTGATGGTGGCAATCTCC
rad51	R：CAGAAGATCCGCTCCACTTATCGC
β-actin	F：GCGATGTATGTAGCTATCCAGGCAGTGCTCTCGCT
β-actin	R：CAATCCAGACAGAGTATTTGCGTTCCGGAGGA

组别 Group	ATP/µmol·mg^-1	LDH/mU
空白对照组 Blank control group	157.50 ± 5.23	0.70 ± 0.05
去氢骆驼蓬碱组 Harmine group	123.10 ± 4.73	1.51 ± 0.05
RI-1组 RI-1 group	149.94 ± 1.94	1.16 ± 0.02
混合药物组 Mixed drug group	112.21 ± 2.72	1.83 ± 0.09