miRNA-138-5p过表达对香烟烟雾暴露所致大鼠睾丸支持细胞损伤的调控作用

doi:10.13481/j.1671-587X.20220318

Abstract

Abstract: Objective

To investigate the damage effect of cigarette smoking exposure on the testicular sertoli cells of the rats， and to clarify the protective effect of miR-138-5p in this process.

Methods

A total of 200 SPF male SD rats were randomly divided into control group and low， middle， and high doses of cigarette exposure groups （given 10， 20， and 30 cigarettes per rat per day）. The rats were anesthetized at 2rd， 4th， 6th， 8th， and 12th weeks， respectively. The plasma inhibin B levels of the rats in various groups were detected， the expression levels of miR-138-5p in testicular tissue of the rats in various groups were detected by real-time fluorescence quantitative PCR（RT-qPCR） method， and the expression levels of vimentin in testicular TM4 cells of the rats in various groups were detected by immunohistochemistry. The testicular TM4 cells （control group） were cultured in vitro. The cigarette smoke extract （CSE） stock solution was diluted to 5% and 10% and to treat the TM4 cells，and used as 5% CSE group and 10% CSE group. The survival rates of the testicular TM4 cells and lactate dehydrogenase （LDH） activities in the testicular TM4 cells in various groups were detected by MTT method， and the apoptotic rates of the cells in various groups were detected by flow cytometry and TUNEL method. After transfected with miR-138-5p plasmid，the testicular TM4 cells were divided into control group， 5% CSE group， 5% CSE+Pri-mirna-138-5p Ctrl group， 5% CSE+Pri-mirna-138-5p group， 10% CSE group， 10% CSE+Pri-mirna-138-5p Ctrl group， and 10% CSE+Pri-mirna-138-5p group. The survival rates of the cells， LDH activities in the cells and apoptotic rates of the cells in various groups were detected by the above methods.

Results

Compared with control group， the plasma inhibin B levels of the rats in different doses of cigarette exposure groups were decreased； compared with control group， the plasma inhibin levels of the rats in high dose of cigarette exposure group at the 8 weeks and in middle and high doses of cigarette expasure groups at the 12 th week were decreased （P<0.05）. The light microscope detection results showed that the cytoplasm of the testicular cells in control group showed positive yellow staining， and the expression levels of vimentin in the testicular TM4 cells of the rats in cigarette exposure groups were decreased gradually； compared with control group， the expression levels of vimentin in testicular TM4 cells of the rats in high dose of cigarette exposure group at different time points were decreased （P<0.05）. Compared with control group， the expression levels of miR-138-5p in testicular tissue of the rats in middle and high doses of cigarette exposure groups at the 8 th week and in different doses of cigarette exposure groups at the 12th week were decreased （P<0.05）. The survival rates of the cells in 5% CSE group and 10% CSE group were significantly lower than that in control group （P<0.01）. Compared with control group， the activities of LDH in testicular TM4 cells of the rats in 5% CSE group and 10% CSE group were decreased （P<0.05 or P<0.01）. After over-expression of miR-138-5p， compared with control group， the proliferation rates of testicular TM4 cells in 5% CSE group， 5% CSE+Pri-mirna-138-5p Ctrl group， 5% CSE+Pri-mirna-138-5p group， 10% CSE group， 10% CSE+Pri-mirna-138-5p Ctrl group，and 10% CSE+Pri-mirna-138-5p group were decreased（P<0.05 or P<0.01）； compared with 5% CSE group， the proliferation rate of testicular TM4 cells in 5% CSE+Pri-mirna-138-5p group was increased （P<0.05 or P<0.01）， and the apoptotic rate was decreased （P<0.05 or P<0.01）； compared with 10% CSE group， the proliferation rate of testicular TM4 cells in 10% CSE+Pri-mirna-138-5p group was increased （P<0.05 or P<0.01）， and the apoptotic rate was decreased （P<0.05）.

Conclusion

Long-term exposure to heavy cigarette smoking can induce the irreversible damage of testicular sertoli cells， and miR-138-5p plays an important protective role in the process.

Key words: Cigarette smoke exposure, Testis, Sertoli cells, Cell damage, MicroRNA-138-5p

CLC Number:

Chunxue ZHONG,Hua XU,Chen ZHANG,Lijuan HE. Regulatory effect of over-expression of miRNA-138-5p on testicular sertoli cell injury of rats induced by cigarette smoking exposure[J].Journal of Jilin University(Medicine Edition), 2022, 48(3): 692-701.

Figures/Tables 9

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References 25

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Group	Level of inhibin B
Group	(weak) 2	4	6	8	12
Control	25.64±1.06	24.67±1.23	25.12±0.98	24.45±1.12	25.64±1.06
Cigarette exposure Low dose	27.05±2.26	27.43±1.69	24.85±1.14	23.63±1.58	20.19±1.99^*
Middle dose	25.69±1.82	24.34±1.70	23.87±0.76	19.30±0.77	20.34±1.43^*
High dose	23.37±2.99	21.82±1.27	22.80±1.32	19.88±0.94^*	19.14±1.02^**

Group	Expression level of vimentin in testicular TM4 cells
Group	(weak) 2	4	6	8	12
Control	27.90±6.27	27.13±3.76	26.12±6.12	28.12±4.93	25.12±6.12
Cigarette exposure Low dose	34.56±6.65	23.19±9.70	28.19±1.32	21.50±5.23	18.89±2.54
Middle dose	19.62±3.01^*	14.14±6.89^*	14.46±8.23^*	20.42±4.68^*	12.76±2.85^**
High dose	15.02±7.00^*	13.90±5.48^*	10.88±5.55^**	10.64±3.24^**	10.48±1.28^**

Group	Expression level of miR-138-5p
Group	(weak) 2	4	6	8	12
Control	1.02±0.21	1.01±0.33	1.01±0.12	1.01±0.17	1.01±0.17
Cigarette exposure Low dose	1.12±0.23	0.97±0.19	0.94±0.19	0.70±0.23	0.63±0.12^*
Middle dose	0.98±0.10	1.09±0.20	0.79±0.10	0.81±0.07^*	0.53±0.14^*
High dose	0.89±0.37	0.88±0.33	0.80±0.12	0.67±0.20^*	0.48±0.14^*

Group	Survival rate of TM4 cells (η/%)	LDH activity[λ_B/(U·L^－1)]
Control	100.00±0.00	187.27±10.62
5%CSE	79.18±1.92^*	244.19±19.11^*
10%CSE	68.34±4.09^**	573.78±17.02^**

Group	Proliferation rate of TM4 cells
Group	(t/h) 24	48	72	96
Control	100.00±0.00	100.00±0.00	100.00±0.00	100.00±0.00
5%CSE	87.85±0.70^*	88.95±1.08^*	90.96±1.19^*	83.54±0.91^*
5%CSE+Pri-miRNA-138-5p Ctrl	86.44±0.80^*	91.56±1.62^*	91.71±1.19^*	83.78±1.03^*
5%CSE+Pri-miRNA-138-5p	92.22±1.57^**△△	97.28±0.95^*△△	95.82±1.18^*△△	87.42±0.68^*△
10%CSE	77.92±1.31^*	70.51±2.79^*	66.46±0.83^*	65.38±3.90^*
10%CSE+Pri-miRNA-138-5p Ctrl	75.26±1.56^*	68.04±2.52^*	67.00±0.86^*	63.69±2.89^*
10%CSE+Pri-miRNA-138-5p	81.65±1.05^*#	86.88±0.92^*##	84.52±1.04^*#	84.47±0.77^*##

Regulatory effect of over-expression of miRNA-138-5p on testicular sertoli cell injury of rats induced by cigarette smoking exposure

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