In vitro effects of Fasciola gigantica excretory-secretory products on human LO2 hepatocytes

Abstract

Abstract:

Objective To investigate the effect of Fasciola gigantica excretory-secretory products (FgESP) on the proliferation of LO2 hepatocytes and biochemical components related to hepatocyte injury in vitro. Methods LO2 cells were plated in a 96-well plate at 5 densities(1 000, 3 000, 5 000, 7 000 and 10 000 cells/well) while the blank control group was only added with culture medium. The growth curve was plotted to determine the optimal density for plating. FgESP was added to the wells at concentrations of 0.02, 0.10, 0.20, 0.40, and 1.00 mg/ml and incubated for 4, 8, 12, 24, 48 and 72 h followed by MTT assay. Each group had 4 replicates, and PBS was used as the negative control. Absorbance A₄₉₀ was measured in a microplate reader. In a 24-well plate, FgESP was added to the wells at concentrations of 0.02, 0.10, 0.20, 0.40, and 1.00 mg/ml and incubated for 4, 8, 12, 24, 48 and 72 h, the supernatant was then collected to assess the levels of phosphomonoesterase (ALP), alanine transaminase (ALT), glutamic oxalacetic transaminase (AST) and albumin (ALB). Each group had 3 replicates, and PBS was used as the negative control. Data were analyzed using GraphPad Prism 6.0 software. Results The optimal cell density was 5 000 cells/well based on the growth curve. After FgESP treatment for 72 h, the A₄₉₀ values for the five groups with FgESP concentrations of 0.02, 0.10, 0.20, 0.40 and 1.00 mg/ml were 1.29 ± 0.01, 1.28 ± 0.06, 1.13 ± 0.08, 0.97 ± 0.06 and 0.25 ± 0.01, respectively, which were all lower than the control group (1.45 ± 0.05) (P < 0.01). This suggested that FgESP significantly impaired the proliferation of LO2 cells in vitro, in a concentration- and time-dependent manner(P < 0.01). In addition, the contents of ALT in the 0.40 and 1.00 mg/ml FgESP group after 72 h were 2.00 ± 0.00, 3.67 ± 0.58, respectively, and the AST contents were 5.33 ± 0.58, 7.76 ± 0.58, respectively, which were higher than the control group (P < 0.01). The contents of ALT and AST in the 1.00 mg/ml FgESP group after 48 h were 2.00 ± 0.00 and 7.00 ± 0.00, significantly higher than that of control (0.33 ± 0.58, 3.67 ± 0.58 respectively) (P < 0.01). The ALP content significantly increased in comparison with the control within 12-72 hours of FgESP treatment at 0.10, 0.20, 0.40 and 1.00 mg/ml, while at 0.02 mg/ml, FgESP only increased at 72 h(P < 0.01). There was no significant difference in ALB content between the groups (P > 0.05). Conclusion FgESP could inhibit the proliferation of LO2 hepatocytes in vitro, and the extent of inhibition is positively correlated with the concentration of FgESP.

Key words: Fasciola gigantica, Excretory-secretory product, LO2 hepatocyte, MTT assay, Biochemical analysis, Cell injury

CLC Number:

R383.26

Wen-ping ZHAO, Xue-fang MEI, Wei SHI, Bin ZHU, Lin-jing HOU, Wei-yi HUANG. In vitro effects of Fasciola gigantica excretory-secretory products on human LO2 hepatocytes[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2018, 36(6): 619-624.

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组别Group	A₄₉₀值A₄₉₀ value
组别Group	4 h	8 h	12 h	24 h	48 h	72 h
对照组Control	0.09 ± 0.02	0.09 ± 0.02	0.09 ± 0.01	0.08 ± 0.02	0.09 ± 0.02	0.10 ± 0.03
1 000	0.14 ± 0.02^a	0.16 ± 0.02^a	0.19 ± 0.02^a	0.23 ± 0.02^a	0.38 ± 0.02^a	0.66 ± 0.04^a
3 000	0.24 ± 0.02^a	0.28 ± 0.03^a	0.36 ± 0.02^a	0.43 ± 0.02^a	0.73 ± 0.02^a	1.31 ± 0.07^a
5 000	0.34 ± 0.02^a	0.40 ± 0.02^a	0.49 ± 0.02^a	0.56 ± 0.02^a	1.01 ± 0.02^a	1.69 ± 0.05^a
7 000	0.44 ± 0.02^a	0.46 ± 0.10^a	0.60 ± 0.02^a	0.69 ± 0.02^a	1.27 ± 0.06^a	1.84 ± 0.10^a
10 000	0.58 ± 0.02^a	0.64 ± 0.07^a	0.78 ± 0.02^a	0.90 ± 0.05^a	1.56 ± 0.05^a	1.95 ± 0.03^a

组别Group	A₄₉₀值A₄₉₀ value
组别Group	4 h	8 h	12 h	24 h	48 h	72 h
对照组Control	0.25 ± 0.02	0.26 ± 0.02	0.30 ± 0.03	0.47 ± 0.01	0.89 ± 0.05	1.45 ± 0.05
0.02 mg/ml	0.22 ± 0.02	0.25 ± 0.01	0.37 ± 0.01	0.59 ± 0.01	1.00 ± 0.04	1.29 ± 0.01^a
0.10 mg/ml	0.21 ± 0.03	0.24 ± 0.01	0.42 ± 0.01	0.66 ± 0.01	1.01 ± 0.04	1.28 ± 0.06^a
0.20 mg/ml	0.21 ± 0.03	0.24 ± 0.01	0.44 ± 0.02	0.77 ± 0.01	0.97 ± 0.02	1.13 ± 0.08^a
0.40 mg/ml	0.23 ± 0.01	0.24 ± 0.01	0.41 ± 0.03	0.79 ± 0.01	0.90 ± 0.01	0.97 ± 0.06^a
1.00 mg/ml	0.22 ± 0.01	0.22 ± 0.03	0.35 ± 0.01	0.76 ± 0.03	0.50 ± 0.02^a	0.25 ± 0.01^a

组别Group	A₄₉₀值A₄₉₀ value
组别Group	4 h	8 h	12 h	24 h	48 h	72 h
对照组Control	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.33 ± 0.58	0.33 ± 0.58
0.02 mg/ml	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
0.10 mg/ml	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.33 ± 0.58	0.00 ± 0.00
0.20 mg/ml	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.33 ± 0.58	1.00 ± 0.00	1.00 ± 0.00
0.40 mg/ml	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.33 ± 0.58	0.33 ± 0.58	2.00 ± 0.00^a
1.00 mg/ml	0.33 ± 0.58	0.33 ± 0.58	0.67 ± 0.58	1.00 ± 0.00	2.00 ± 0.00^a	3.67 ± 0.58^a

组别Group	A₄₉₀值A₄₉₀ value
组别Group	4 h	8 h	12 h	24 h	48 h	72 h
对照组Control	4.33 ± 1.15	3.33 ± 0.58	3.33 ± 1.53	3.67 ± 1.15	3.67 ± 0.58	3.67 ± 0.58
0.02 mg/ml	4.67 ± 0.58	4.33 ± 1.15	3.33 ± 0.58	4.33 ± 1.15	4.33 ± 0.58	4.00 ± 0.00
0.10 mg/ml	4.67 ± 1.15	4.00 ± 0.00	4.00 ± 0.00	3.67 ± 1.15	4.00 ± 0.00	4.33 ± 0.58
0.20 mg/ml	4.00 ± 0.00	4.33 ± 1.53	4.33 ± 0.58	4.33 ± 0.58	5.00 ± 0.00	5.00 ± 1.00
0.40 mg/ml	4.00 ± 1.00	5.00 ± 1.00	4.67 ± 2.08	4.33 ± 0.58	5.33 ± 0.58^a	5.33 ± 0.58^a
1.00 mg/ml	4.67 ± 0.58	5.00 ± 0.00	5.67 ± 1.73	6.67 ± 0.58	7.00 ± 0.00^a	7.67 ± 0.58^a

组别Group	A₄₉₀值A₄₉₀ value
组别Group	4 h	8 h	12 h	24 h	48 h	72 h
对照组Control	6.00 ± 1.00	6.00 ± 1.00	6.00 ± 1.00	6.33 ± 0.58	7.00 ± 1.00	7.67 ± 1.15
0.02 mg/ml	6.33 ± 0.58	6.33 ± 0.58	6.00 ± 0.00	8.00 ± 2.00	11.00 ± 3.00	12.00 ± 1.00^a
0.10 mg/ml	6.00 ± 1.00	6.33 ± 0.58	9.50 ± 0.50^a	11.33 ± 0.58^a	12.00 ± 1.00^a	11.00 ± 0.00^a
0.20 mg/ml	6.33 ± 0.58	5.67 ± 0.58	9.67 ± 0.58^a	11.67 ± 0.58^a	16.00 ± 0.00^a	17.00 ± 2.00^a
0.40 mg/ml	6.00 ± 1.00	6.33 ± 0.58	11.00 ± 1.00^a	12.00 ± 0.00^a	14.67 ± 0.58^a	21.33 ± 1.53^a
1.00 mg/ml	7.00 ± 1.00	7.33 ± 0.58	12.33 ± 0.58^a	13.33 ± 1.53^a	16.68 ± 2.31^a	27.00 ± 4.36^a