馬 強(qiáng)， 王洪飛， 包俊強(qiáng)， 吳業(yè)賓， 溫 靜， 齊 云， 龔美亮

(1. 解放軍總醫(yī)院老年腎科， 北京 100853； 2. 解放軍裝甲兵學(xué)院， 安徽 蚌埠 233032； 3. 解放軍總醫(yī)院南樓檢驗(yàn)科， 北京 100853)

微波輻射對(duì)腎小管上皮細(xì)胞的影響以及金雀異黃素對(duì)其保護(hù)作用*

馬 強(qiáng)1△， 王洪飛2， 包俊強(qiáng)2， 吳業(yè)賓2， 溫 靜1， 齊 云1， 龔美亮3

(1. 解放軍總醫(yī)院老年腎科， 北京 100853； 2. 解放軍裝甲兵學(xué)院， 安徽 蚌埠 233032； 3. 解放軍總醫(yī)院南樓檢驗(yàn)科， 北京 100853)

目的：觀察微波輻照對(duì)人近端腎小管上皮細(xì)胞(HKC)的影響及金雀異黃素對(duì)其的保護(hù)作用。方法：HKC分為對(duì)照組、微波輻照組、金雀異黃素組(n=6)。金雀異黃素組在輻照前2 h用含30 μmol/L金雀異黃素的DMEM培養(yǎng)基進(jìn)行預(yù)培養(yǎng)。輻照后24 h留取上清進(jìn)行乳酸脫氫酶(LDH)、β-N-乙酰氨基葡萄糖苷酶(NAG)活性及丙二醛(MDA)、超氧化物歧化酶(SOD)活性檢測(cè)。Hoechst 33258染色觀察細(xì)胞凋亡情況。結(jié)果：與對(duì)照組比較，微波輻照組上清NAG、LDH活性明顯增加(P<0.01)，金雀異黃素預(yù)處理組則較微波輻照組明顯下降(P<0.01)；微波輻照組上清活性也較對(duì)照組明顯增加(P<0.01)。Hoechst 33258染色顯示，微波輻照可導(dǎo)致較多量的細(xì)胞凋亡，而應(yīng)用金雀異黃素預(yù)處理后細(xì)胞凋亡的比例均大大減少。微波輻照可大大提高HKC細(xì)胞中的MDA含量，SOD活性降低(P<0.01)，應(yīng)用金雀異黃素預(yù)處理后MDA的含量無(wú)明顯降低，SOD的活性明顯增大(P<0.01)。結(jié)論：微波輻照可導(dǎo)致人近端腎小管上皮細(xì)胞出現(xiàn)功能損傷，金雀異黃素對(duì)其具有一定的保護(hù)作用，可能與其減輕氧化應(yīng)激損傷、減少細(xì)胞凋亡有關(guān)。

金雀異黃素；腎小管上皮細(xì)胞；電磁輻射

【DOI】 10.12047/j.cjap.5382.2017.028

電磁輻射作為當(dāng)今社會(huì)生存環(huán)境中最大的污染源，對(duì)人類健康的危害受到廣泛關(guān)注[1]。研究發(fā)現(xiàn)，電磁輻射可對(duì)腎臟組織學(xué)和功能均造成一定影響，其具體機(jī)制目前并不明確[2,3]，也缺乏必要的預(yù)防個(gè)干預(yù)措施。金雀異黃素對(duì)各種原因引起的腎臟損傷均有一定的保護(hù)作用[4,5]。因此本研究觀察微波輻照對(duì)培養(yǎng)的人近端腎小管上皮細(xì)胞的影響，并觀察金雀異黃素對(duì)其的保護(hù)作用。

1 材料與方法

1.1 材料

DMEM培養(yǎng)基干粉(GIBCO)；胎牛血清(北京四季青)；胰蛋白酶(Sigma)；某型合成信號(hào)發(fā)生器(頻率范圍：2～6.2 GHz)。

1.2 細(xì)胞實(shí)驗(yàn)

人腎小管上皮細(xì)胞系，保存于解放軍總醫(yī)院南樓腎科實(shí)驗(yàn)室，待生長(zhǎng)至90%以上融合后應(yīng)用于實(shí)驗(yàn)。分為對(duì)照組、輻照組、金雀異黃素組。應(yīng)用某型合成信號(hào)發(fā)生器(頻率范圍：2～6.2 GHz)平均功率密度90 mW/cm2在暗室中進(jìn)行輻照，輻照時(shí)將培養(yǎng)瓶或培養(yǎng)板置于可透射微波的37.5℃水浴專用輻照盒內(nèi)，連續(xù)輻照30 min，每組設(shè)3個(gè)副孔，輻照后24 h留取上清進(jìn)行乳酸脫氫酶(lattate dehydrogenase, LDH)、β-N-乙酰氨基葡萄糖苷酶(β-N-acetyl glucosaminidase, NAG)活性檢測(cè)。細(xì)胞用刮匙刮下，檢測(cè)丙二醛(malondialdehyde, MDA)水平和超氧化物歧化酶(superoxide dismutase, SOD)活性。對(duì)照組細(xì)胞與實(shí)驗(yàn)組作相同處理，只是不接受微波輻照。結(jié)合袁偉杰等[6]的研究結(jié)果，我們決定應(yīng)用中低劑量(30 μmol/L)的金雀異黃素進(jìn)行細(xì)胞預(yù)培養(yǎng)，金雀異黃素用二甲亞砜溶解，實(shí)驗(yàn)前配置成含金雀異黃素的DMEM培養(yǎng)基，金雀異黃素組在輻照前2 h用含30 μmol/L金雀異黃素的DMEM培養(yǎng)基進(jìn)行預(yù)培養(yǎng)。

1.3 NAG、LDH活性的檢測(cè)

應(yīng)用日本東芝 TBR-120FR全自動(dòng)生化分析儀檢測(cè)NAG活性，應(yīng)用Sysmex UF 1000i分析儀檢測(cè)LDH活性，所有標(biāo)本檢測(cè)均在我院檢驗(yàn)科進(jìn)行。

1.4 Hoechst 33258熒光染色檢測(cè)細(xì)胞凋亡

收集對(duì)數(shù)期細(xì)胞，調(diào)整細(xì)胞懸液濃度為2×104cells/ml，接種于6孔培養(yǎng)板中，1 ml/well，細(xì)胞在6孔板中爬片，37℃，5%CO2培養(yǎng)箱培養(yǎng)使細(xì)胞貼壁，待生長(zhǎng)至90%融合后，進(jìn)行微波輻照，分組和實(shí)驗(yàn)步驟同前，每組各設(shè)3個(gè)復(fù)孔，輻照結(jié)束后應(yīng)用預(yù)溫至37℃的PBS洗滌1～2次，放入37℃孵箱中孵育20 min，37℃的PBS洗滌3次，按Hoechst 33258熒光染色試劑盒(江蘇碧云天生物技術(shù)公司)說(shuō)明書操作，倒置熒光顯微鏡下觀察、拍照。

1.5 MDA含量和SOD活性檢測(cè)

采用MDA、SOD試劑盒(南京建成生物工程研究所)進(jìn)行檢測(cè)，根據(jù)試劑盒說(shuō)明操作。

1.6 統(tǒng)計(jì)學(xué)方法

2 結(jié)果

2.1 3組細(xì)胞上清NAG、LDH酶活性的比較

培養(yǎng)液中LDH和NAG活性反映腎小管上皮細(xì)胞的受損情況。結(jié)果顯示，與對(duì)照組比較，微波輻射組培養(yǎng)液中的NAG、LDH活性明顯增加(P<0.01)，金雀異黃素預(yù)處理組NAG、LDH活性較輻照組明顯降低(P<0.01)，但仍較對(duì)照組明顯增高(P<0.01，表1)。

GroupNAG(U/L)LDH(U/L)Control48.37±2.38200.15±12.13Microwaveirradia-tion274.93±38.44**720.74±56.88**Genisteinprecondi-tioning195.84±7.99##574.36±43.92##

NAG: β-N-acety1 glucosaminidase; LDH: Lactate dehydrogenase

**P<0.01vscontrol group;?##P<0.01vsmicrowave irradiation group

2.2 金雀異黃素對(duì)微波輻照HKC凋亡的影響

Hoechst 33258 熒光染色顯示，對(duì)照組細(xì)胞核呈均質(zhì)淡藍(lán)熒光，僅見個(gè)別凋亡形態(tài)細(xì)胞(3.91±0.47)%，輻照組可見細(xì)胞核呈不同程度的縮小，熒光強(qiáng)度增加，典型凋亡形態(tài)細(xì)胞明顯增多，金雀異黃素組凋亡細(xì)胞數(shù)較輻照組明顯減少(11.07±0.62)%vs(24.33±1.56)%(P<0.01，圖1)。

Fig. 1 Hoechst 33258 fluorescein stain of control group, microwave irradiation group and genistein preconditioning group A: Control group; B: Microwave irradiation group; C: Genistein preconditioning group

2.3 金雀異黃素對(duì)微波輻照HKC氧化應(yīng)激的影響

與對(duì)照組比較，微波輻照組腎小管上皮細(xì)胞中MDA含量顯著升高(P<0.01)，金雀異黃素預(yù)培養(yǎng)可有效降低微波輻照所導(dǎo)致的MDA水平(P<0.01)，但不能降至對(duì)照組的正常水平(P<0.01)。微波輻照可顯著降低腎小管上皮細(xì)胞中SOD活性(P<0.01)，金雀異黃素則可有效增加微波輻照所導(dǎo)致的SOD活性(P<0.01)，但與對(duì)照組比較仍有顯著差異(P<0.01，表2)。

GroupMDA(nmol/mgprot)SOD(U/mgprot)Control0.24±0.03105.29±20.37Microwaveirradia-tion3.77±0.52**23.16±1.31**Genisteinprecondi-tioning3.55±0.5048.77±5.29##

MDA: Malondialdehyde; SOD: Superoxide dismutase

**P<0.01vscontrol group;?##P<0.01vsmicrowave irradiation group

3 討論

電磁波的危害程度與場(chǎng)強(qiáng)、作用時(shí)間以及生物體接受輻射的總劑量有關(guān)，低劑量的電磁輻射即可對(duì)新西蘭兔腎功能產(chǎn)生影響，高能量的電磁輻射，還可導(dǎo)致放射性腎炎的發(fā)生[2,3]。

研究發(fā)現(xiàn)，微波輻射對(duì)接觸人員和實(shí)驗(yàn)動(dòng)物消化系統(tǒng)、免疫系統(tǒng)、神經(jīng)系統(tǒng)等有一定影響[7-9]，并可導(dǎo)致腎臟細(xì)胞DNA損傷[10]。當(dāng)出現(xiàn)腎小管間質(zhì)損傷時(shí)，尿NAG活性異常，是早期腎小管功能損害的可靠、敏感指標(biāo)[11]。LDH是一種糖酵解酶。乳酸脫氫酶存在于機(jī)體所有組織細(xì)胞的胞質(zhì)內(nèi)，其中以腎臟含量較高，當(dāng)腎臟細(xì)胞損傷時(shí)，釋放到細(xì)胞外而被檢測(cè)到。

本研究發(fā)現(xiàn)小劑量微波輻照HKC可使培養(yǎng)液上清液中NAG、LDH活性增加，而應(yīng)用金雀異黃素預(yù)培養(yǎng)可保護(hù)HKC免于輻射損傷。金雀異黃素具有抗氧化、抗腫瘤等廣泛的生物學(xué)效應(yīng)，可保護(hù)多種細(xì)胞免于放射損傷[12,13]。電磁輻照的腎損害機(jī)制目前尚不清楚，研究發(fā)現(xiàn)與輻射導(dǎo)致的氧化應(yīng)激有關(guān)[14]，金雀異黃素對(duì)腎臟和心臟的氧化應(yīng)激損傷有一定的保護(hù)作用[15]，其保護(hù)機(jī)制可能與抑制Ape1/Ref-1表達(dá)[16]以及NF-kappaBJNKERK信號(hào)通路[17]有關(guān)，但具體的作用機(jī)制目前尚不清楚。

本研究發(fā)現(xiàn)，微波輻照可導(dǎo)致人近端腎小管上皮細(xì)胞出現(xiàn)氧化應(yīng)激損傷和細(xì)胞凋亡，金雀異黃素可保護(hù)微波輻照的人近端腎小管上皮細(xì)胞，其保護(hù)機(jī)制可能與減輕氧化應(yīng)激損傷、減少細(xì)胞凋亡有關(guān)。

[1] 孫云娟. 電磁輻射的原理及危害[J]. 科技情報(bào)開發(fā)與經(jīng)濟(jì), 2008, 8(32): 136-138.

[2] 趙洪雯， 張廣斌， 王 源， 等． 電磁輻射對(duì)大鼠內(nèi)皮祖細(xì)胞和腎臟組織學(xué)的影響[J]． 現(xiàn)代生物醫(yī)學(xué)進(jìn)展， 2011, 11(2): 262-266

[3] 周傳艷, 府偉靈, 羅 陽(yáng). 低強(qiáng)度電磁輻射對(duì)新西蘭兔腎臟功能的影響[J]. 國(guó)際檢驗(yàn)醫(yī)學(xué)雜志, 2013, 34(15): 1921-1922.

[4] Zhu G, Xiang X, Chen X,etal. Renal dysfunction induced by long-term exposure to depleted uranium in rats[J].ArchToxicol, 2009, 83(1): 37-46.

[5] Kin MJ, Lim Y. Protective effect of short-term genistein supplementation on the early stage in diabetes-induced renal damage[J].MediatorsInflamm, 2013, 510212.

[6] 袁偉杰, 彭 燕, 張 威, 等. 高磷飲食對(duì)5/6腎切除大鼠繼發(fā)性甲狀旁腺功能亢進(jìn)及腎臟纖維化的影響及金雀異黃素干預(yù)作用[J]. 上海醫(yī)學(xué), 2010, 33(11): 1043-1045.

[7] 丁朝陽(yáng), 呂志忠. 微波輻射對(duì)作業(yè)人員某些生理功能的影響[J]. 解放軍預(yù)防醫(yī)學(xué)雜志, 1994, 12(6): 454-456.

[8] Santini R, Deschaux P. Incidence of low-level microwave irradiation on intestinal myoelectrical activity in the rat [J].HealthPhys, 1983, 45(3): 775-778.

[9] D Andrea JA, Chou CK, Johnston SA,etal. Microwave effects on the nervous system [J].Bioelectromagnetics, 2003, 24(S6): S107-147.

[11]馬 強(qiáng), 王洪飛, 邢長(zhǎng)江, 等. 坦克作業(yè)對(duì)乘員腎臟功能的影響[J]. 中國(guó)應(yīng)用生理學(xué)雜志, 2014, 30(5): 475-477.

[12]Terra VA, Souza-Neto FP, Frade MA,etal. Genistein prevents ultraviolet B radiation-induced nitrosative skin injury and promotes cell proliferation[J].JPhotochemPhotobiolB, 2015, 144: 20-27.

[13]Song L, Ma L, Cong F,etal. Radioprotective effects of genistein on HL-7702 cells via the inhibition of apoptosis and DNA damage[J].CancerLett, 2015, 366(1): 100-110.

[14]Xu S, Zhou Z, Zhang L,etal. Exposure to 1800 MHz radiofrequency radiation induces oxidative damage to mitochondrial DNA in primary cultured neurons[J].BrainRes, 2010, 1311: 189-196.

[15]Gupta SK, Dongare S, Mathur R,etal. Genistein ameliorates cardiac inflammation and oxidative stress in streptozotocin-induced diabetic cardiomyopathy in rats[J].MolCellBiochem, 2015, 408(1-2): 63-72.

[16]Liu GD, Xia L, Zhu JW,etal. Genistein alleviates radiation-induced pneumonitis by depressing Ape1/Ref-1 expression to down-regulate inflammatory cytokines[J].CellBiochemBiophys, 2014, 69(3): 725-733.

[17]Qian Y, Cao L, Guan T,etal. Protection by genistein on cortical neurons against oxidative stress injury via inhibition of NF-kappaB, JNK and ERK signaling pathway[J].PharmBiol, 2015, 26: 1-9.

Protective effects of Genistein on human renal tubular epithelial cells damage of microwave radiation

MA Qiang1△, WANG Hong-fei2, BAO Jun-qiang2, WU Ye-bin2, WEN Jing1, QI Yun1, GONG Mei-liang3

(1. Department of Geriatric Nephrology, PLA General Hospital, Beijing 100853; 2. College of Armored Forces, Bengbu 233010;3. Department of Clinical Laboratory, General Hospital of PLA, Beijing 100853, China)

Objective: To observe the effects of microwave irradiation on human proximal renal tubular epithelial cells (HKC) and protective effects of genistein. Methods: HKC cells were divided into control group, microwave irradiation group and genistein group(n=6 ) respectively. The genistein group cells were pre-incubated with 30 μmol/L genistein in DMEM for 2 hours. After irradiation for 24 hours, the concentrations of lactate dehydrogenase(LDH) and β-N-acetyl glucosaminidase(NAG) in culture solution were measured to evaluate cell injury. Cells were curetted to measure the levels of malondialdehyde (MDA) and superoxide dismutase (SOD). Cell apoptosis and necrosis were detected with Hoechst 33258 stain. Results: Compared with the control group, the NAG activity of the microwave irradiation group was significantly increased(P<0.01), and NAG activity of genistein pre-incubated group was significantly decreased(P<0.01). The levels of LDH in microwave irradiation group were also increased significantly (P<0.01vscontrol group). LDH levels could be decreased obviously (P<0.01vsmicrowave irradiation group) after genistein pre-incubate. Hoechst 33258 fluorescent staining revealed that the nucleus crimpled, crescent liked and chromatin condensed, even nucleus disintegrated. Our research showed that microwave irradiation could lead to large amount of cell apoptosis and necrosis, and genistein pre-treatment could reduce the ratio of apoptosis and necrosis than that in microwave irradiation group(P<0.01). The concentration of MDA in microwave irradiation group was higher than that in control group (P<0.01). At the same time, the activity of SOD was significantly reduced (P<0.01). Pre-incubated with genistein could not decrease the MDA levels, but could increase the activities of SOD (P<0.01vsmicrowave irradiation group). Conclusion: microwave irradiation can induce human proximal renal tubular epithelial cells injury. The protective effects of genistein may partly correlated with decreasing oxidative stress damage and cell apoptosis in HKC cells.

Genistein; renal tubular epithelial cell; microwave radiation

2015-11-20

2016-10-24

R827

A

1000-6834(2017)02-109-03

△【通訊作者】Tel: 15810393581； E-mail： mq78448@163.com