Additive effect of alpha-tocopherol and ascorbic acid in combating ethanol-induced hepatic fibrosis

Figures & data

Table 1. Primer sequences used for RT-PCR analysis

Genes	Primer sequences	Melting temperature in (°C)
GAPDH (Glyceraldehyde-3 phosphatedehydrogenase)	Forward: 5′-TGA CAA CTC CCT CAA GAT TGT CA-3′	63.7
	Reverse: 5′-GGC ATG GAC TGT GGT CAT GA-3′	64.7
CYP2E1 (cytochrome P4502E1)	Forward 5′ -GCC ACC CTC CTC GTC ATA TC- 3′	59.4
	Reverse 5′- GCA GCC AAT CAG AAA TGT GG- 3′	60.0
NFkB (nuclear transcription factor kB)	Forward 5′CAC CAA AGA CCC ACC TCA CC-3′	58.9
	Reverse 5′ GGA CCG CAT TCA AGT CAT AGT- 3′	59.4
TNF-alpha (tumor necrosis factor alpha)	Forward: 5′-GAA CAA CCC TAC GAG CAC CT-3′	59.4
	Reverse: 5′-GGG TAG TTT GGC TGG GAT AA-3′	59.4
TGF-beta1	Forward: 5′-CCG CAA CAA CGC AAT CTA TG-3′	61.4
	Reverse: 5′-AGC CCT GTA TTC CGT CTC CTT-3′	62.4
Collagen type I	Forward: 5′-GAG CGG AGA GTA CTG GTA CG-3′	53.0
	Reverse: 5′-TAC TCG AAC GGG AAT CCA TC-3′	53.0

Table 2. Activity of toxicity marker enzymes in the liver and serum of rats

	ALT (μM of pyruvatelib/min/mg protein)		AST (μM of oxaloacetatelib/min/mg protein)		GGT (μM of p-nitroanilinelib/min/mg protein)
Groups	Liver	Serum	Liver	Serum	Serum
CN	15.12 ± 0.58^a	48.97 ± 1.91^a	19.16 ± 0.74^a	180.64 ± 7.07^a	16.98 ± 0.66^a
E	51.31 ± 2.00^d	105.54 ± 4.13^d	78.51 ± 3.00^d	346.78 ± 13.57^c	63.54 ± 2.48^d
AT	15.28 ± 0.59^a	50.39 ± 1.97^a	19.33 ± 0.75^a	180.99 ± 7.08^a	17.16 ± 0.67^a
E + AT	22.99 ± 0.89^b	63.71 ± 2.44^b	30.00 ± 1.16^b	209.72 ± 8.21^b	37.67 ± 1.47^b
AA	15.91 ± 0.61^a	50.44 ± 1.97^a	19.37 ± 0.75^a	181.66 ± 7.11^a	17.37 ± 0.67^a
E + AA	24.19 ± 0.94^b	66.46 ± 2.59^b	30.76 ± 1.19^b	211.86 ± 8.30^b	38.16 ± 1.48^b
AT + AA	16.35 ± 0.63^a	50.53 ± 1.97^a	19.62 ± 0.76 ^a	182.18 ± 7.13^a	17.46 ± 0.67^a
E + AT + AA	19.66 ± 0.76^c	53.81 ± 1.99^c	21.18 ± 0.78^c	186.97 ± 7.24^a	24.84 ± 0.96^c

Values are expressed as mean ± SEM of six rats in each group. Values not sharing a common superscript differ significantly at P < 0.05 within rows.

Table 3. Lipid peroxidation products, protein peroxidation products, total collagen, and hydroxyproline content in the liver

Groups	MDA (mmol/100 g wettissue)	HP (mmol/100 g wettissue)	CD (mmol/100 g wettissue)	Protein carbonyls(μmol/gwet tissue)	Total collagen(mg/100 gprotein)	Hydroxyproline(mg/100 gprotein)
CN	0.46 ± 0.01^a	8.76 ± 0.34^a	59.11 ± 2.31^a	7.24 ± 0.28^a	57.19 ± 2.23^a	8.99 ± 0.35^a
E	0.96 ± 0.03^c	18.15 ± 0.71^c	142.8 ± 5.59^d	24.98 ± 0.97^d	115.24 ± 4.51^c	16.73 ± 0.65^c
AT	0.43 ± 0.01^a	8.45 ± 0.33^a	57.39 ± 2.24^a	7.47 ± 0.29^a	58.80 ± 2.30^a	9.56 ± 0.37^a
E + AT	0.58 ± 0.02^b	10.76 ± 0.42^b	102.65 ± 4.01^c	16.95 ± 0.66^c	88.47 ± 3.45^b	11.00 ± 0.43^b
AA	0.44 ± 0.01^a	7.94 ± 0.31^a	58.24 ± 2.27^a	7.8 ± 0.30^a	65.77 ± 2.57^a	9.77 ± 0.38^a
E + AA	0.58 ± 0.04^b	11.82 ± 0.46^b	103.85 ± 4.06^c	17.40 ± 0.68^c	86.21 ± 3.75^b	11.66 ± 0.45^b
AT + AA	0.47 ± 0.01^a	8.64 ± 0.34^a	60.75 ± 2.29^a	9.75 ± 0.93^b	63.71 ± 2.48^a	9.45 ± 0.36^a
E + AT + AA	0.51 ± 0.02^a	9.30 ± 0.36^a	86.13 ± 3.28^b	12.8 ± 0.88^e	62.64 ± 2.45^a	9.97 ± 0.35^a

Values are expressed as mean ± SEM of six rats in each group. Values not sharing a common superscript differ significantly at P < 0.05 within rows.

Table 4. Activities of scavenging enzymes Catalase, SOD, GPx, GR, and GSH in the liver of rats

Groups	Catalase (U*/mgprotein)	SOD (U**/mgprotein)	GPx (1 µmol of NADPHoxidized/min/mg protein)	GR (1 µmol of NADPHoxidized/min/mg protein)	GSH (mM/100 gwet tissue)
CN	63.54 ± 2.48^a	79.14 ± 3.09^a	13.22 ± 0.51^a	21.08 ± 0.82^a	497.77 ± 19.49^a
E	41.76 ± 1.63^d	25.66 ± 1.00^d	5.01 ± 0.19^d	4.93 ± 0.19^d	375.64 ± 14.71^d
AT	63.81 ± 2.49^a	79.78 ± 3.12^a	13.39 ± 0.51^a	21.30 ± 0.83^a	500.33 ± 19.59^a
E + AT	54.82 ± 2.14^b	66.31 ± 2.59^b	10.93 ± 0.42^b	18.16 ± 0.71^b	429.30 ± 16.81^b
AA	64.49 ± 2.52^a	80.59 ± 3.15^a	13.47 ± 0.52^a	21.43 ± 0.83^a	502.48 ± 19.67^a
E + AA	55.76 ± 2.17^b	65.88 ± 2.57^b	10.58 ± 0.41^b	17.75 ± 0.69^b	412.18 ± 13.32^b
AT + AA	64.57 ± 2.52^a	80.88 ± 3.16^a	13.58 ± 0.52^a	21.65 ± 0.84^a	510.69 ± 15.96^a
E + AT + AA	60.03 ± 2.34^a	76.56 ± 2.99^a	12.08 ± 0.46^c	20.26 ± 0.78^a	468.16 ± 18.11^c

*Velocity constants.

**Enzyme concentration required to inhibit chromogen produced by 50% in 1 minute.

Values are expressed as mean ± SEM of six rats in each group. Values not sharing a common superscript differ significantlyat P < 0.05 within rows.

Figure 1. mRNA expression of transcription factors. (A) Expression of NF kappa B. (B) Intensity of TNFalpha using gel doc. Expression of TNF-alpha was analyzed in the cytoplasmic fraction of liver by agarose gel electrophoresis and the intensities of the bands were compared with that of the intensities of GAPDH bands expressed in the samples. Intensities of the bands were quantified using Bio-Rad gel doc and plotted. The results presented are average of quadruplicate experiments ± SEM statistically significant at P < 0.05. (C) Intensity of CYP2E1 using gel doc. Expression of CYP2E1 was analyzed in the cytoplasmic fraction of liver by agarose gel electrophoresis and the intensities of the bands were compared with that of the intensities of GAPDH bands expressed in the samples. Intensities of the bands were quantified using Bio-Rad gel doc and plotted. The results presented are average of quadruplicate experiments ± SEM statistically significant at P < 0.05. (D) Intensity of TGF beta1 using gel doc. Expression of TGF beta1 was analyzed in the cytoplasmic fraction of liver by agarose gel electrophoresis and the intensities of the bands were compared with that of the intensities of GAPDH bands expressed in the samples. Intensities of the bands were quantified using Bio-Rad gel doc and plotted. The results presented are average of quadruplicate experiments ± SEM statistically significant at P < 0.05. (E) Intensity of collagen type I using gel doc. Expression of collagen was analyzed in the cytoplasmic fraction of liver by agarose gel electrophoresis and the intensities of the bands were compared with that of the intensities of GAPDH bands expressed in the samples. Intensities of the bands were quantified using Bio-Rad gel doc and plotted. The results presented are average of quadruplicate experiments ± SEM statistically significant at P < 0.05.

Figure 2. Graphical representation of alpha-tocopherol content in serum of CN, E, AT, E + AT, AA, E + AA, AT + AA, and E + AT + AA groups. Values are expressed as mean ± SEM of six rats in each group. Values not sharing a common superscript letter differ significantly at P < 0.05.

Figure 3. Graphical representation of ascorbic acid content in serum of CN, E, AT, E + AT, AA, E + AA, AT + AA and E + AT + AA groups. Values are expressed as mean ± SEM of six rats in each group. Values not sharing a common superscript letter differ significantly at P < 0.05.

Figure 4. Histopathological analysis of liver (magnification 40×). (A) Control group showed normal liver architecture and cell structure. (B) Ethanol group showed extensive hepatocellular damage as evidenced by steatosis, vacuolization, and dilation of sinusoids. (C) AT group exhibited normal cell architecture as almost that of control. (D) E + AT group exhibited slight recovery from steatosis and inflammation. (E) AA group showed cells that were almost similar to the control. (F) E + AA group exhibited slight recovery from steatosis and inflammation. (G) AT + AA group showed cells almost similar to the control. (H) E + AT + AA group exhibited almost near normal cell architecture of CN group as evidenced by reduction in steatosis, vacuolization, dilation of sinusoids, and inflammation.