Prophylactic role of Enhydra fluctuans against arsenic-induced hepatotoxicity via anti-apoptotic and antioxidant mechanisms

Figures & data

Figure 1 (A) Effect of NaAsO₂ on isolated mouse hepatocytes. (B) Time- and dose-dependent effect on cell viability in absence (NaAsO₂) and presence of AEEF in isolated murine hepatocytes. Values are expressed as mean ± SE (n = 3). (C) Hoechst staining of murine hepatocytes in absence (NaAsO₂) and presence of AEEF.

Table 1 ROS production, lipid peroxidation, protein carbonylation, and antioxidant parameters in the absence (NaAsO₂) and presence of AEEF (AEEF + NaAsO₂) in isolated mouse hepatocytes

	Parameters
Groups	TBARS (µg/g of protein)	Protein carbonyl (nmol/mg of protein)	CAT (U/mg of protein)	SOD (U/mg of protein)	GST (µmol/h/mg protein)	GR (nmol/min/mg of protein)	G6PD (nmol/min/mg of protein)	GSH (nmol/mg of protein)
Control	2.36 ± 0.25	38.53 ± 2.65	197.03 ± 16.48	74.45 ± 2.25	1.36 ± 0.11	75.78 ± 3.46	106.42 ± 4.26	5.82 ± 0.51
NaAsO2 (10 µM)	5.31 ± 0.32*	71.08 ± 2.05*	109.19 ± 12.92*	55.53 ± 2.68*	0.91 ± 0.07*	37.66 ± 4.85*	70.83 ± 6.00*	3.29 ± 0.38*
NaAsO2 (10 µM) +  AEEF (400 µg/ml)	3.64 ± 0.29^‡	45.46 ± 2.54^‡	178.24 ± 16.01^‡	68.77 ± 3.30^‡	1.23 ± 0.09^†	62.54 ± 3.04^‡	92.91 ± 3.73^‡	5.05 ± 0.56^†

Values are expressed as mean ± SE (n = 3).

*Values differ significantly from normal control (P < 0.01).

^†Values differ significantly from NaAsO₂ control (P < 0.05).

^‡Values differ significantly from NaAsO₂ control (P < 0.01).

CAT unit, ‘U’, is defined as micromoles of H₂O₂ consumed per minute. SOD unit, ‘U’, is defined as the micromoles inhibition of nitroblue tetrazolium reduction per minute.

Figure 2 Respective Western blot analysis of the intrinsic transcription proteins (A) viz. Bad, Bcl-2, cleaved caspase-9, cleaved caspase-3, and the extrinsic transcription proteins (B) viz. Fas, Bid, and cleaved caspase-8 in absence (NaAsO²) and presence of AEEF followed by densitometric analysis of the respective protein levels and the normal control band was given an arbitrary value of 1. beta-Actin was used as a loading protein. Values are expressed as mean ± SE (n=3). ^$Values differ significantly (P < 0.05) from normal control. ^#Values differ significantly (P < 0.01) from normal control.

*Values differ significantly (P < 0.05) from toxic control. **Values differ significantly (P < 0.01) from toxic control.

Table 2 Hematological and serum biochemical parameters in the absence (NaAsO₂) and presence of AEEF (AEEF + NaAsO₂) in mice

	Parameters
					Cholesterol (mg/dl)
Groups	Total erythrocyte count (×10^6/mm^3)	Hemoglobin (g/dl)	ALT (IU/l)	AST (IU/l)	Total	HDL	Triglyceride (mg/dl)
Normal control	5.77 ± 0.28	9.25 ± 0.33	71.27 ± 3.70	53.33 ± 2.30	118.88 ± 5.62	55.76 ± 4.28	111.38 ± 6.50
Toxic control (NaAsO2)	3.21 ± 0.36*	4.44 ± 0.64*	120.38 ± 7.04*	78.39 ± 4.59*	161.93 ± 7.04*	31.44 ± 4.24*	213.22 ± 8.77*
NaAsO2 (5 mg/kg) + AEEF (50 mg/kg)	4.35 ± 0.28^†	7.13 ± 0.48^‡	89.50 ± 5.21^†	64.51 ± 2.52^†	138.20 ± 7.34	47.73 ± 5.35^†	149.47 ± 5.08^‡
NaAsO2 (5 mg/kg) + AEEF (100 mg/kg)	4.29 ± 0.32^‡	7.96 ± 0.64^‡	81.56 ± 4.52^‡	59.49 ± 4.16^‡	127.13 ± 4.95^‡	49.41 ± 3.30^†	133.20 ± 9.54^‡

Values are expressed as mean ± SE, for six animals in each group.

*Values differ significantly from normal control (P < 0.01).

^†Values differ significantly from NaAsO₂ control (P < 0.05).

^‡Values differ significantly from NaAsO₂ control (P < 0.01).

Table 3 ROS production, lipid peroxidation, protein carbonylation, and antioxidant markers in the absence (NaAsO₂) and presence of AEEF (AEEF + NaAsO₂) in mouse liver

Groups	Parameters	Values	Parameters	Values
Normal control	ROS production (nmol min/mg of protein)	31.42 ± 1.48	CAT (U/mg of protein)	264.00 ± 18.05
Toxic control (NaAsO2)		88.43 ± 2.97*		177.83 ± 24.10*
NaAsO2 (5 mg/kg) + AEEF (50 mg/kg)		50.45 ± 2.83^‡		234.67 ± 13.51
NaAsO2 (5 mg/kg) + AEEF (100 mg/kg)		43.57 ± 2.64^‡		246.83 ± 12.55^†
Normal control	Lipid peroxidation (TBARS level in μg/g of protein)	4.76 ± 0.40	SOD (U/mg of protein)	66.25 ± 1.79
Toxic control (NaAsO2)		8.92 ± 0.67*		35.87 ± 2.64*
NaAsO2 (5 mg/kg) + AEEF (50 mg/kg)		7.21 ± 0.62		54.35 ± 4.25^‡
NaAsO2 (5 mg/kg)  + AEEF (100 mg/kg)		5.85 ± 0.37^‡		58.67 ± 3.95^‡
Normal control	Protein carbonylation (nmol/mg of protein)	33.56 ± 2.89	GST (µmol/h/mg protein)	2.25 ± 0.16
Toxic control (NaAsO2)		60.31 ± 4.38*		1.37 ± 0.14*
NaAsO2 (5 mg/kg) + AEEF (50 mg/kg)		40.09 ± 3.09^‡		1.81 ± 0.13
NaAsO2 (5 mg/kg) + AEEF (100 mg/kg)		37.11 ± 4.68^‡		2.03 ± 0.11^‡
Normal control	Total coenzyme Q9 (nmol/g of wet tissue)	184.31 ± 7.31	GR (nmol/min/mg protein)	82.60 ± 4.78
Toxic control (NaAsO2)		133.85 ± 5.45*		47.71 ± 6.68*
NaAsO2 (5 mg/kg) + AEEF (50 mg/kg)		155.63 ± 4.67^†		73.89 ± 6.09^†
NaAsO2 (5 mg/kg) + AEEF (100 mg/kg)		161.32 ± 4.89^‡		76.97 ± 9.86^†
Normal control	Total coenzyme Q10 (nmol/g of wet tissue)	33.42 ± 2.55	G6PD (nmol/min/mg protein)	92.88 ± 3.62
Toxic control (NaAsO2)		19.46 ± 1.98*		64.08 ± 4.57*
NaAsO2 (5 mg/kg) + AEEF (50 mg/kg)		25.79 ± 2.67		78.46 ± 3.02^†
NaAsO2 (5 mg/kg) + AEEF (100 mg/kg)		28.43 ± 2.32^‡		81.12 ± 4.52^†
Normal control	NADPH oxidase (pmol/min/mg of protein)	0.42 ± 0.03	GSH (nmol/mg protein)	20.48 ± 1.88
Toxic control (NaAsO2)		3.42 ± 0.52*		10.50 ± 0.82*
NaAsO2 (5 mg/kg) + AEEF (50 mg/kg)		2.41 ± 0.28^†		15.44 ± 1.11^†
NaAsO2 (5 mg/kg) + AEEF (100 mg/kg)		2.48 ± 0.25^†		17.64 ± 1.42^‡

Values are expressed as mean ± SE, for six animals in each group. CAT unit, ‘U’, is defined as micromoles of H₂O₂ consumed per minute. SOD unit, ‘U’, is defined as the micromoles inhibition of NBT reduction per minute.

*Values differ significantly from normal control (P < 0.01).

^†Values differ significantly from NaAsO₂ control (P < 0.05).

^‡Values differ significantly from NaAsO₂ control (P < 0.01).

Figure 3 Histological sections of livers of normal mice (A), NaAsO₂-treated mice (B), mice pretreated with AEEF (50 mg/kg) followed by NaAsO₂ (C), and mice pretreated with AEEF (100 mg/kg) followed by NaAsO₂ (D). Blue arrows represent normal hepatocytes and yellow arrow represents central vein; red arrow represents dilated portal vein; green arrows represent enlarged sinusoids between the plates of hepatocytes; and black arrow represents infiltrating leukocytes.