促红细胞生成素对牦牛肾间质成纤维细胞凋亡因子表达的影响

doi:10.11843/j.issn.0366-6964.2024.08.019

摘要/Abstract

摘要：

旨在探讨促红细胞生成素(EPO)对牦牛肾间质成纤维细胞(RIFs)中凋亡因子的影响。本研究首先分别采集3头4岁龄的健康牦牛和黄牛的肾脏组织，采用免疫组织化学方法和Western blot检测牦牛和黄牛肾脏中EPO的准确分布及其表达量。同时对牦牛RIFs进行原代培养鉴定，并对其进行不同药物处理来调控RIFs中EPO表达水平，检测EPO对凋亡相关基因Bax，Bcl-2，PCNA在mRNA和蛋白表达水平的影响。免疫组化学检测结果显示，EPO在牦牛和黄牛肾脏中集合管上皮细胞、肾小管上皮细胞和小管周间质成纤维样细胞中均呈阳性表达。qRT-PCR和Western blot结果显示，EPO在牦牛肾脏中的表达量显著高于黄牛(P < 0.001)。随后对生成EPO的RIFs进行原代培养并调控EPO的表达，结果表明：激活组显著升高RIFs中Bcl-2和PCNA的表达，降低Bax的表达(P < 0.001)。EPO抑制组显著降低RIFs中Bcl-2、PCNA的表达，上调Bax的表达(P < 0.001)。综上表明，EPO在牦牛和黄牛肾脏中集合管上皮细胞、肾小管上皮细胞和小管周间质成纤维样细胞中均呈阳性表达，牦牛肾脏中的EPO高表达能够促进RIFs中Bcl-2和PCNA的表达，并降低Bax的表达，说明EPO可以在牦牛肾脏中起到抗凋亡的作用。

关键词: 牦牛, 促红细胞生成素(EPO), 肾间质成纤维细胞(RIFs), 凋亡

Abstract:

The purpose of this study was to investigate the effect of erythropoietin (EPO) on apoptosis factors in yak renal interstitial fibroblasts (RIFs). Firstly, the kidney tissues of 3 healthy yaks and cattle aged 4 years were collected respectively, immunohistochemistry and Western blot were used to detect the exact distribution of EPO and its expression in the kidneys of yaks and calves. Meanwhile, the yak RIFs were identified by primary culture, and different drug treatments were applied to regulate the EPO expression level in the RIFs, and the effects of EPO on the apoptosis-related genes Bax, Bcl-2, and PCNA at mRNA and protein expression levels were detected. Immunohistochemical results showed that EPO was positively expressed in collecting duct epithelial cells, renal tubular epithelial cells, and peritubular mesenchymal fibroblast-like cells in the kidneys of both yaks and calves. qRT-PCR and Western blot results showed that the expression of EPO in the kidneys of yaks was significantly higher than that in the calves (P < 0.001). Subsequently, the EPO-generating RIFs were cultured in primary culture and the expression of EPO was regulated, and the results showed that the activation group significantly elevated the expression of Bcl-2 and PCNA and decreased the expression of Bax in the RIFs (P < 0.001).The EPO-inhibition group significantly decreased the expression of Bcl-2, PCNA and up-regulated the expression of Bax in the RIFs (P < 0.001). In conclusion, it was showed that EPO was positively expressed in collecting duct epithelial cells, renal tubular epithelial cells and peritubular mesenchymal fibroblast-like cells in the kidney of yak and calf, and the high expression of EPO in the kidney of yak was able to promote the expression of Bcl-2 and PCNA and decrease the expression of Bax in the RIFs, which indicated that EPO could play an anti-apoptotic role in the kidney of yak.

Key words: yak, erythropoietin (EPO), renal interstitial fibroblasts (RIFs), apoptosis

中图分类号:

S823.85

卢增华, 崔燕, 余四九, 白雪峰, 卢鸿琴, 何俊峰, 卢凯, 翟国亮, 祁正满. 促红细胞生成素对牦牛肾间质成纤维细胞凋亡因子表达的影响[J]. 畜牧兽医学报, 2024, 55(8): 3460-3471.

Zenghua LU, Yan CUI, Sijiu YU, Xuefeng BAI, Hongqin LU, Junfeng HE, Kai LU, Guoliang ZHAI, Zhengman QI. Effect of Erythropoietin on the Expression of Apoptotic Factor in Yak Renal Interstitial Fibroblasts[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3460-3471.

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参考文献 37

1	JELKMANN W . Physiology and pharmacology of erythropoietin[J]. Transfus Med Hemother, 2013, 40 (5): 302- 309. doi: 10.1159/000356193
2	李慧. 低氧条件下成年牦牛肾间质成纤维样细胞中EPO的适应性调控研究[D]. 兰州: 甘肃农业大学, 2020.
	LI H. Adaptive regulation of EPO in adult yak renal interstitial fibroblasts-like cells under hypoxia[D]. Lanzhou: Gansu Agricultural University, 2020. (in Chinese)
3	SUZUKI N . Erythropoietin gene expression: developmental-stage specificity, cell-type specificity, and hypoxia inducibility[J]. Tohoku J Exp Med, 2015, 235 (3): 233- 240. doi: 10.1620/tjem.235.233
4	REN C C , ZHU W , WANG Q W , et al. The renal protect function of erythropoietin after release of bilateral ureteral obstruction in a rat model[J]. Clin Sci (Lond), 2018, 132 (18): 2071- 2085. doi: 10.1042/CS20180178
5	HAN X P , ZHANG F Q , TAN X S , et al. EPO modified MSCs can inhibit asthmatic airway remodeling in an animal model[J]. J Cell Biochem, 2018, 119 (1): 1008- 1016. doi: 10.1002/jcb.26268
6	朱国萍, 娄阳, 徐冲. Bcl-2蛋白家族与细胞凋亡[J]. 细胞生物学杂志, 2001, 23 (1): 20- 23.
	ZHU G P , LOU Y , XU C . Bcl-2 protein family and apoptosis[J]. Journal of Cell Biology, 2001, 23 (1): 20- 23.
7	庞睿. 重组人促红细胞生成素抑制内皮细胞凋亡的实验研究[J]. 科学技术创新, 2019, 17 (10): 66- 67.
	PANG R . Experimental study on the inhibition of endothelial cell apoptosis by recombinant human erythropoietin[J]. Scientific and Technological Innovation, 2019, 17 (10): 66- 67.
8	NEKOUI A , DEL CARMEN ESCALANTE TRESIERRA V , ABDOLMOHAMMADI S , et al. Neuroprotective effect of erythropoietin in postoperation cervical spinal cord injury: case report and review[J]. Anesthesiol Pain Med, 2015, 5 (6): e28849.
9	SINGH A K , KOLLIGUNDLA L P , FRANCIS J , et al. Detrimental effects of hypoxia on glomerular podocytes[J]. J Physiol Biochem, 2021, 77 (2): 193- 203. doi: 10.1007/s13105-021-00788-y
10	CIANCI R , SIMEONI M , CIANCI E , et al. Stem cells in kidney ischemia: from inflammation and fibrosis to renal tissue regeneration[J]. Int J Mol Sci, 2023, 24 (5): 4631. doi: 10.3390/ijms24054631
11	NASU K , KAWAKAMI T , SHINOHARA A , et al. Munc18-1-interacting protein 3 mitigates renal fibrosis through protection of tubular epithelial cells from apoptosis[J]. Nephrol Dial Transplant, 2020, 35 (4): 576- 586. doi: 10.1093/ndt/gfz177
12	ADAMS J M , CORY S . The Bcl-2 protein family: arbiters of cell survival[J]. Science, 1998, 281 (5381): 1322- 1326. doi: 10.1126/science.281.5381.1322
13	罗文捷, 张启芳, 孟云超, 等. 骨髓间充质干细胞对溃疡性结肠炎小鼠受损肠黏膜修复作用的探究[J]. 安徽医科大学学报, 2019, 54 (12): 1882- 1887.
	LUO W J , ZHANG Q F , MENG Y C , et al. Effect of bone marrow mesenchymal stem cells on the repair of damaged intestinal mucosa in mice with ulcerative colitis[J]. Acta Universitatis Medicinalis Anhui, 2019, 54 (12): 1882- 1887.
14	宋楠萌, 桑建利, 徐恒. 增殖细胞核抗原(PCNA)的分子结构及其生物学功能研究进展[J]. 自然科学进展, 2006, 16 (10): 1201- 1209. doi: 10.3321/j.issn:1002-008X.2006.10.001
	SONG N M , SANG J L , XU H . Advances in molecular structure and biologicalfunction of proliferating cell nuclear antigen (PCNA)[J]. Progress in Natural Science, 2006, 16 (10): 1201- 1209. doi: 10.3321/j.issn:1002-008X.2006.10.001
15	PALIEGE A , ROSENBERGER C , BONDKE A , et al. Hypoxia-inducible factor-2α-expressing interstitial fibroblasts are the only renal cells that express erythropoietin under hypoxia-inducible factor stabilization[J]. Kidney Int, 2010, 77 (4): 312- 318. doi: 10.1038/ki.2009.460
16	钱庆元, 潘锦超, 杨军, 等. 不同缺氧干预对低压低氧模型鼠血气及红细胞相关指标的影响[J]. 浙江大学学报(医学版), 2023, 52 (6): 777- 784.
	QIAN Q Y , PAN J C , YANG J , et al. Effect of different hypoxic and hypobaric interventions on blood gas and erythrocyte-related indicators in rats[J]. Journal of Zhejiang University (Medical Sciences), 2023, 52 (6): 777- 784.
17	HAASE V H . HIF-prolyl hydroxylases as therapeutic targets in erythropoiesis and iron metabolism[J]. Hemodial Int, 2017, 21 (S1): S110- S124.
18	SUZUKI N , YAMAMOTO M . Roles of renal erythropoietin-producing (REP) cells in the maintenance of systemic oxygen homeostasis[J]. Pflugers Arch Eur J Physiol, 2016, 468 (1): 3- 12. doi: 10.1007/s00424-015-1740-2
19	SANKARAN V G , WEISS M J . Anemia: progress in molecular mechanisms and therapies[J]. Nat Med, 2015, 21 (3): 221- 230. doi: 10.1038/nm.3814
20	VICTOR S , ROCHA-FERREIRA E , RAHIM A , et al. New possibilities for neuroprotection in neonatal hypoxic-ischemic encephalopathy[J]. Eur J Pediatr, 2022, 181 (3): 875- 887. doi: 10.1007/s00431-021-04320-8
21	SEMENZA G L . Involvement of oxygen-sensing pathways in physiologic and pathologic erythropoiesis[J]. Blood, 2009, 114 (10): 2015- 2019. doi: 10.1182/blood-2009-05-189985
22	杨欢, 石玉红, 冉海凤, 等. 促红细胞生成素的生理功能及生成来源[J]. 中国临床药理学与治疗学, 2021, 26 (4): 434- 443.
	YANG H , SHI Y H , RAN H F , et al. Erythropoietin's biological function and source[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2021, 26 (4): 434- 443.
23	姚一凡, 张伊阳, 董仕慧, 等. 低氧下牦牛、黑白花牛肾间质成纤维细胞PDK1、Smad2、Caspase3等因子表达差异研究[J]. 核农学报, 2023, 37 (7): 1335- 1343.
	YAO Y F , ZHANG Y Y , DONG S H , et al. Differential expression of PDK1, Smad2 and Caspase3 in renal interstitial fibroblasts of Yak and Holstein under hypoxia[J]. Journal of Nuclear Agricultural Sciences, 2023, 37 (7): 1335- 1343.
24	张晶, 张承英. 抗EPO抗体特性、分类及其相关纯红细胞再生障碍性贫血的发病机制及诊治进展[J]. 武警医学, 2018, 29 (12): 1169- 1172.
	ZHANG J , ZHANG C Y . Latest diagnosis of pure red cell aplastic anemia related to anti-EPO antibodies[J]. Medical Journal of the Chinese Peoples Armed Police Forces, 2018, 29 (12): 1169- 1172.
25	李晶晶, 伍超, 柯慧, 等. 肾虚免疫低下大鼠EPO的变化及右归饮与外源性EPO的逆转作用[J]. 中国中药杂志, 2019, 44 (6): 1246- 1257.
	LI J J , WU C , KE H , et al. Changes of EPO in rats with chronic renal failure and low immunity and reversal effects of Yougui Yin and exogenous EPO[J]. China Journal of Chinese Materia Medica, 2019, 44 (6): 1246- 1257.
26	董雅洁, 高维娟. bcl-2、bax、caspase-3在细胞凋亡中的作用及其关系[J]. 中国老年学杂志, 2012, 32 (21): 4828- 4830.
	DONG Y J , GAO W J . The role of bcl-2, bax, caspase-3 in apoptosis and their relationship[J]. Chinese Journal of Gerontology, 2012, 32 (21): 4828- 4830.
27	TSUKAHARA S , YAMAMOTO S , TIN-TIN-WIN-SHWE , et al. Inhalation of low-level formaldehyde increases the Bcl-2/Bax expression ratio in the hippocampus of immunologically sensitized mice[J]. Neuroimmunomodulation, 2006, 13 (2): 63- 68.
28	刘大锐, 李报春, 李怀东. 细胞凋亡核心基因Caspase家族的研究进展[J]. 中国医药导刊, 2020, 22 (11): 800- 805.
	LIU D R , LI B C , LI H D . Research progress in Caspase family of apoptosis core gene[J]. Chinese Journal of Medicinal Guide, 2020, 22 (11): 800- 805.
29	AYMONNIER K , BOSETTA E , LEBORGNE N G F , et al. G-CSF reshapes the cytosolic PCNA scaffold and modulates glycolysis in neutrophils[J]. J Leukocyte Biol, 2024, 115 (2): 205- 221.
30	VÅTSVEEN T K , SPONAAS A M , TIAN E M , et al. Erythropoietin (EPO)-receptor signaling induces cell death of primary myeloma cells in vitro[J]. J Hematol Oncol, 2016, 9 (1): 75.
31	牟云鸿. 促红细胞生成素对高糖环境下系膜细胞凋亡的影响及其可能机制[D]. 南昌: 南昌大学, 2019.
	MOU Y H. Effect of EPO on high glucose-cultured apoptosis of human glomerular mesangial cells and its possible mechanisms[D]. Nanchang: Nanchang University, 2019. (in Chinese)
32	YI X Y , YAN W H , GUO T L , et al. Erythropoietin mitigates diabetic nephropathy by restoring PINK1/Parkin-mediated Mitophagy[J]. Front Pharmacol, 2022, 13, 883057.
33	WARREN J S , ZHAO Y , YUNG R , et al. Recombinant human erythropoietin suppresses endothelial cell apoptosis and reduces the ratio of Bax to Bcl-2 proteins in the aortas of apolipoprotein E-deficient mice[J]. J Cardiovasc Pharmacol, 2011, 57 (4): 424- 433.
34	潘广艳. 重组人促红细胞生成素对脑出血大鼠认知功能及海马组织凋亡相关因子Bcl-2、Bax表达的影响[D]. 遵义: 遵义医科大学, 2020.
	PAN G Y. Effects of recombinant human erythropoietin on cognitive function and expression of apoptosis-related factors Bcl-2 and Bax in hippocampus of rats with intracerebral hemorrhage[D]. Zunyi: Zunyi Medical University, 2020. (in Chinese)
35	CHOI D , SCHROER S A , LU S Y , et al. Erythropoietin protects against diabetes through direct effects on pancreatic β cells[J]. J Exp Med, 2010, 207 (13): 2831- 2842.
36	PAN X X , GONG X Q , PAN L L , et al. Erythropoietin relieves neuronal apoptosis in epilepsy rats via TGF-β/Smad signaling pathway[J]. Cell Mol Biol (Noisy-le-grand), 2023, 69 (10): 239- 243.
37	殷亮亮, 李春年. EPO对低氧状态下牙髓细胞增殖和保护的实验研究[J]. 河北医科大学学报, 2021, 42 (7): 765- 769.
	YIN L L , LI C N . Experimental study of EPO on proliferation and protection of dental pulp cells under hypoxia[J]. Journal of Hebei Medical University, 2021, 42 (7): 765- 769.

基因名称 Gene name	引物序列(5′→3′) Primer sequence	产物大小/bp Product size
EPO	F: AGGGGCTAGAGGAACTGTCC R: GCTGCCCCCAAGTTTAGGTT	111
Bax	F: TTTGCTTCAGGGTTTCATC R: CAGCTGCGATCATCCTCT	174
Bcl-2	F: CCTGTGGATGACCGAGTACC R: TGAGCAGTGCCTTCAGAGAC	143
PCNA	F: CTCGTCTCATGTCTCCTTGGT R: TGTCTTCATTGCCAGCACATT	137
β-actin	F: TCCTGCGGCATTCACGAAACTAC R: GTGTTGGCGTAGAGGTCCTTGC	80

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