Protective mechanism of ginkgo leaf extract and dipyridamole injection on hepatic ischemia reperfusion injury in rats

doi:10.3877/cma.j.issn.2095-655X.2022.04.009

Abstract

Abstract:

Objective

To investigate the mechanism of protective effect of ginkgo leaf extract and dipyridamole injection (Gin) on hepatic ischemia reperfusion injury (HIRI) in rats.

Methods

A total of 36 Wistar rats were selected and divided into normal group (N group), hepatic ischemia reperfusion model group (M group) and Gin intervention group (T group) according to the random number table, with 12 rats in each group. Group N was the control group, and group M and T were simulated by clamping the proper hepatic artery for 1 h follawing 1 h reperfusion. The T group was intraperitoneally injected with Gin at 3.6 ml/kg daily 1 week before modeling, and the N and M groups were intraperitoneally injected with the same amount of normal saline 1 week before modeling. Hematoxylin-eosin (HE) staining was used to detect the cell morphology of liver tissue. Masson and Sirius red staining were used to detect the collagen fibers. Western blot was used to detect the protein expression levels of B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-associated X protein (Bax), Smad and protein kinase B (Akt) in liver tissue. The mRNA expression levels of Bax, Bcl-2, Smad and Akt in liver tissues were detected by polymerase chain reaction (PCR). The expressions of Bax and Bcl-2, interleukin-6 (IL-6) and IL-10 in liver tissues were detected by immunohistochemistry, and the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in serum were detected.

Results

HE staining showed that the liver tissue cells in group M were seriously damaged, with more inflammatory cells and obvious edema. Masson and Sirius red staining showed that the accumulation of collagen fibers in group M was more obvious than that in group N. Moreover, the three staining results showed that the degree of hepatic inflammatory infiltration and collagen fiber in group T was lighter than that in group M. Using β-actin as internal reference, the relative expression levels of Bax and Bcl-2 in group M (0.91±0.28, 0.68±0.24) were higher than those in group N (0.22±0.19, 0.23±0.12) (all P<0.01). The relative expression level of Bax protein in T group (0.48±0.11) was lower than that in group M (P<0.01), and the relative expression level of Bcl-2 protein in group T (0.89±0.25) was higher than that in group M (P<0.01). Using GAPDH as internal reference, the relative expression levels of Akt and Smad protein in group M (0.72±0.34, 1.03±0.13) were higher than those in group N (0.17±0.09, 0.57±0.26) (all P<0.01). The relative expression level of Akt in group T (1.47±0.89) was higher than that in group M (P<0.01), and the relative expression level of Smad in group T (0.62±0.42) was lower than that in group M (P<0.01). PCR results showed that the mRNA expression levels of Bax, Bcl-2, Akt and Smad in group M (0.76±0.03, 0.55±0.06, 0.96±0.09, 1.58±0.16) were higher than those in group N (0.29±0.04, 0.36±0.05, 0.64±0.06, 0.53±0.14) (all P<0.01). The mRNA expression levels of Bax and Smad in group T (0.36±0.04, 1.05±0.26) were lower than those in group M (all P<0.01). The mRNA expression levels of Bcl-2 and Akt in group T (0.85±0.04, 1.46±0.19) were lower than those in group M (all P<0.01). Immunohistochemical results showed that the positive expression area of Bax, Bcl-2, IL-6 and IL-10 in group M [(39.52±0.78)%, (4.62±0.94)%, (38.04±3.11)%, (6.48±1.14)%] was higher than that in group N [(0.99±0.13)%, (0.96±0.12)%, (0.46±0.06)%, (0.47±0.17)%] (all P<0.01). The positive expression area of Bax and IL-6 in T group [(8.18±1.22)%, (6.05±0.92)%] was lower than that in group M (all P<0.01), and the positive expression area of Bcl-2 and IL-10 [(48.13±2.65)%, (31.91±4.86)%] was higher than that in group M (all P<0.01). SOD and MDA levels were (274.81±10.42)U/ml and (2.25±0.51)nmol/ml in the N group, respectively. In comparison, the SOD level in group M [(113.24±8.52)U/ml] was decreased, the MDA level in group M [(5.19±0.99)nmol/ml] was increased (all P<0.01). The SOD level in group T [(221.51±6.25)U/ml] was higher than that in group M (P<0.01), and the MDA level in group T [(3.91±0.86)nmol/ml] was lower than that in group M (P<0.01).

Conclusion

Gin may play a protective role against HIRI in rats through inhibition of apoptosis and fibrosis mediated by PI3K/Akt and TGF-β/Smad signaling pathways.

Key words: Ginkgo leaf extract and dipyridamole injection, Hepatic ischemia reperfusion injury, Hepatic fibrosis, PI3K/Akt, TGF-β/Smad

Zhiqiang Liu, Xiangjie Dou, Bailu Liu, Xiaomeng Dong, Junyu Bao. Protective mechanism of ginkgo leaf extract and dipyridamole injection on hepatic ischemia reperfusion injury in rats[J]. Chinese Journal of Diagnostics(Electronic Edition), 2022, 10(04): 259-265.

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Figures/Tables 9

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基因	引物序列
Bcl-2	5′-GGTGAACTGGGGGAGGATTG-3′
	5′-AGAGCGATGTTGTCCACCAG-3′
Bax	5′-AGACACCTGAGCTGACCTTG-3′
	5′-AAGTTGCCATCAGCAAACAT-3′
β-actin	5′-CCTCACTGTCCACCTTCCA-3′
	5′-GGGTGTAAAACGCAGCTCA-3′
Akt	5′-AGTCCCCACTCAACAACTTCT-3′
	5′-AAGTAGGGAGGCATCTCG-3′
Smad	5′-CCATCTCCTACTACGAGCTGAA-3′
	5′-CACTGCTGCATTCCTGTTGAC-3′
GAPDH	5′-CAACGGGAAACCCATCACCA-3′
	5′-ACGCCAGTAGACTCCACGACAT-3′

基因	引物序列
Bcl-2	5′-GGTGAACTGGGGGAGGATTG-3′
	5′-AGAGCGATGTTGTCCACCAG-3′
Bax	5′-AGACACCTGAGCTGACCTTG-3′
	5′-AAGTTGCCATCAGCAAACAT-3′
β-actin	5′-CCTCACTGTCCACCTTCCA-3′
	5′-GGGTGTAAAACGCAGCTCA-3′
Akt	5′-AGTCCCCACTCAACAACTTCT-3′
	5′-AAGTAGGGAGGCATCTCG-3′
Smad	5′-CCATCTCCTACTACGAGCTGAA-3′
	5′-CACTGCTGCATTCCTGTTGAC-3′
GAPDH	5′-CAACGGGAAACCCATCACCA-3′
	5′-ACGCCAGTAGACTCCACGACAT-3′

组别	例数	Masson染色	天狼星红染色
N组	12	2.85±0.74	3.26±1.24
M组	12	27.11±1.62^a	32.79±5.13^a
T组	12	9.06±0.69^b	16.58±4.06^b
F值		1 424.05	162.75
P值		<0.01	<0.01

组别	例数	Masson染色	天狼星红染色
N组	12	2.85±0.74	3.26±1.24
M组	12	27.11±1.62^a	32.79±5.13^a
T组	12	9.06±0.69^b	16.58±4.06^b
F值		1 424.05	162.75
P值		<0.01	<0.01

组别	例数	Bax	Bcl-2	Akt	Smad
N组	12	0.22±0.19	0.23±0.12	0.17±0.09	0.57±0.26
M组	12	0.91±0.28^a	0.68±0.24^a	0.72±0.34^a	1.03±0.13^a
T组	12	0.48±0.11^b	0.89±0.25^b	1.47±0.89^b	0.62±0.42^b
F值		636.84	562.75	329.04	293.31
P值		<0.01	<0.01	<0.01	<0.01

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