Oxidative stress disturbs energy metabolism of mitochondria in ethanol-induced gastric mucosa injury

Table 1 Reacting system of RT

Component	Amount
25 mmol/L MgCl2	4.00 μL
Reverse transcription 10 × buffer	2.00 μL
10 mmol/L dNTP	2.00 μL
Recombinant ribonuclease inhibitor	0.50 μL
AMV reverse transcriptase	15.00 U
Oligo (dT) 15 primer	0.50 μg
Total RNA	1.00 μg
Nuclease-rree water	Proper volume
Reacting volume	20.00 μL

Table 2 Reacting system of PCR

Component	Amount
cDNA	4.00 μL
10 × PCR buffer solution	1.96 μL
MgCl2 (25 mmol/L)	2.40 μL
Upper primer (20 pmol/L)	1.00 μL
Down primer (20 pmol/L)	1.00 μL
dNTP (10 mmol/L)	0.36 μL
Taq DNA polymerase (5 IU/μL)	0.10 μL
Deionized water	10.08 μL
Reacting volume	20.00 μL

Table 3 Primers used for PCR amplification

Primers	GenBank (No.)	Sequence
β-actin	NM_031144 (nt 1-1296)	Up: 5′-TCACCCACACTGTGCCCATCTATGA-3′
		Down: 5′-CATCGGAACCGCTCATTGCCGATAG-3′
ATPase subunit6	X14848 (nt 7904-8584)	Up: 5′-ACA CCAAAAGGA CGAACCTG-3′
		Down: 5′-CGGTGAGAAGTGGGCTAAAG-3′
ATPase subunit8	X14848 (nt 7743-7946)	Up: 5′-TGCCACAACTAGACACATCCA-3′
		Down: 5′-TGTGGGGGTAATGAAAGAGG-3′

Table 4 PCR amplification conditions (mean ± SD)

Target sequence	Amplification conditions
β-actin	94.0°C 30 s, 59.0°C 30 s, 72.0°C 30 s, 35cycles
ATPase subunit 6	94.0°C 30 s, 53.0°C 30 s, 72.0°C 30 s, 33cycles
ATPase subunit 8	94.0°C 30 s, 52.0°C 30 s, 72.0°C 30 s, 32cycles

Table 5 LI and MDA content in acute gastric mucosa injury treated with different concentrations of ethanol (mean ± SD)

Group	LI	MDA (nmol/mL)
Control	0.000 ± 0.000	0.968 ± 0.136
40% (W/W) ethanol	38.375 ± 6.368a	1.363 ± 0.152a
70% (W/W) ethanol	70.000 ± 8.018ab	1.703 ± 0.124ab
100% (W/W) ethanol	118.750 ± 14.820abc	2.083 ± 0.153abc

^aP < 0.01 vs control group;

^bP < 0.01 vs 40% (W/W) ethanol group;

^cP < 0.01 vs 70% (W/W) ethanol group.

Table 6 Integral indices and pathological integrals for chronic gastric mucosa injury (mean ± SD)

Group	3 wk		6 wk		9 wk
	Integral indices	Pathological integrals	Integral indices	Pathological integrals	Integral indices	Pathological integrals
Normal sodium	0.000 ± 0.000	0.200 ± 0.422	0.000 ± 0.000	0.500 ± 0.707	0.000 ± 0.000	0.600 ± 0.699
25% (W/W) alcohol	0.200 ± 0.422	0.700 ± 0.675	1.100 ± 0.568	2.400 ± 0.516	1.700 ± 0.483	4.500 ± 1.080
45% (W/W) alcohol	0.600 ± 0.699	1.500 ± 0.527	3.300 ± 1.059	6.000 ± 0.817	8.900 ± 0.994	11.400 ± 0.966

Table 7 Mitochondria, ATPase subunits 6 and 8 mRNA in ethanol-induced acute gastric mucosal injury (mean ± SD)

Group	Mitochondria (n)	ATPase subunit 6 mRNA	ATPase subunit 8 mRNA
Normal sodium	74.800 ± 15.399	0.935 ± 0.162	0.768 ± 0.145
40% (W/W) ethanol	69.200 ± 16.262	1.113 ± 0.135a	0.945 ± 0.139a
70% (W/W) ethanol	63.500 ± 14.246	0.868 ± 0.089b	0.677 ± 0.106b
100% (W/W) ethanol	51.500 ± 18.739	0.691 ± 0.053abc	0.433 ± 0.055abc

^aP < 0.01 vs control group;

^bP < 0.01 vs 40% (W/W) ethanol group;

^cP < 0.01 vs 70% (W/W) ethanol group.

Table 8 Mitochondria and MDA content in ethanol-induced chronic gastric mucosa injury (mean ± SD)

Group	3 wk		6 wk		9 wk
	Mitochondria (n)	MDA content	Mitochondria (n)	MDA content	Mitochondria (n)	MDA content
Normal sodium	123.000 ± 23.613	0.717 ± 0.057	109.400 ± 30.303	0.757 ± 0.064	104.600 ± 20.571	0.728 ± 0.044
25% (W/W) alcohol	111.000 ± 18.514	0.820 ± 0.051	106.500 ± 17.031	1.029 ± 0.102	86.300 ± 26.833	1.156 ± 0.058
45% (W/W) alcohol	101.100 ± 13.186	0.969 ± 0.126	75.700 ± 17.639	1.225 ± 0.053	59.400 ± 21.767	1.477 ± 0.072

Table 9 Expression of mtDNA ATPase subunits 6 and 8 mRNA in ethanol-induced chronic gastric mucosa injury¹ (mean ± SD)

Group	3 wk		6 wk		9 wk
	mtDNA ATPase 6 mRNA	mtDNA ATPase 8 mRNA	mtDNA ATPase 6 mRNA	mtDNA ATPase 8 mRNA	mtDNA ATPase 6 mRNA	mtDNA ATPase 8 mRNA
Normal sodium	0.907 ± 0.079	0.678 ± 0.033	0.876 ± 0.045	0.671 ± 0.038	0.894 ± 0.059	0.659 ± 0.037
25% (W/W) alcohol	0.857 ± 0.062	0.655 ± 0.029	0.737 ± 0.060	0.557 ± 0.028	0.664 ± 0.039	0.512 ± 0.043
45% (W/W) alcohol	0.756 ± 0.051	0.617 ± 0.026	0.607 ± 0.039	0.474 ± 0.031	0.501 ± 0.043	0.366 ± 0.028

¹Magnitude relative to β-actin from respective specimens.