JAK2/STAT通路在人肾小球系膜细胞衰老过程中的作用研究
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摘要
目的
肾脏是机体衰老的主要器官,肾小球滤过率(glomerular filtration rate,GFR)随增龄而降低,进入45岁以后GFR下降速度加快。基于随着经济发展老龄人口逐渐增加及老年人是慢性肾脏病高危人群这两方面因素,在我国逐渐迈入老龄化社会的今天,研究肾脏衰老机制、探讨延缓肾脏衰老的方法,显得尤为重要。肾脏衰老的机制及防治是目前国内外学者关注的重要问题之一。
目前关于肾脏衰老的机制尚不十分明确。多数学者认为肾脏衰老主要是由于肾脏固有细胞功能减退所致。肾小球系膜细胞是肾脏的主要固有细胞,其表型和功能改变在肾脏衰老进程中发挥着重要的作用。目前的研究证实,高糖、血管紧张素Ⅱ(angiotensin, AngⅡ)、叔丁基过氧化氢(tert-butly hydroperoxide, tBHP)可促进细胞衰老。这些研究多见于脐静脉血管内皮细胞、肺成纤维细胞、腹膜间皮细胞、血管平滑肌细胞等,尚没有对人肾小球系膜细胞(Human Glomerular Mesangial Cell, HGMC)衰老的研究报道。JAK2／STAT通路是一条重要的信号转导途径,在心肌细胞、巨噬细胞、T细胞等多种细胞的衰老过程中发挥作用,但其与HGMC衰老之间的关系尚不清楚。本研究以AngⅡtBHP及高糖作为刺激因素,诱导HGMC发生衰老,检测衰老HGMC中JAK2/STAT通路活性及STAT蛋白表达的变化,观察洛沙坦、普罗布考及JAK2阻断剂AG490对JAK2/STAT通路活性变化及STAT蛋白表达的影响,探讨它们在延缓HGMC衰老过程中的作用。
方法
1、HGMC衰老诱导因素的筛选
(1)用10-8、10-7、10-6、10-5、104molL-1。AngⅡ刺激HGMC,刺激时间为24h、48 h、72 h、96h；(2)用10、30、50、70、100μmolL-1 tBHP刺激HGMC,每天1h,连续5天；(3)用10、20、30、40、50mmolL-1葡萄糖刺激HGMC,刺激时间为24 h、48 h、72 h、96h、120h。(4)对照组细胞在不含任何刺激因素的培养液中培养。HGMC在各因素刺激条件下培养,刺激结束后检测细胞增殖情况、衰老相关β半乳糖苷酶(senescence-associatedβ-galactosidase,SAP-gal)染色阳性细胞百分率、细胞周期变化,并将SAβ-gal阳性细胞率及Go-G1期细胞比例达80%作为判定细胞衰老的标准,明确三种因素诱导HGMC发生衰老的最适浓度和最适培养时间。观察衰老HGMC形态学及超微结构改变,进一步判定细胞进入衰老期的最佳刺激因素。
2、HGMC衰老过程中JAK2/STAT通路活性及STAT蛋白表达的变化
采用最佳促进HGMC衰老的刺激因素,诱导HGMC发生衰老。分组情况
(1)对照组(在不含任何刺激因素的培养液中培养)；
(2)衰老组
AngⅡ诱导组(10-6molL-1 AngⅡ刺激72h)；
tBHP诱导组(30μmolL-1tBHP每天刺激1h连续5天)；
高糖诱导组30mmolL-1葡萄糖刺激96h)。
应用Western Blot及细胞免疫荧光法检测衰老HGMC内STAT1、p-STAT1、STAT3、p-STAT3的表达变化,研究HGMC衰老过程中JAK2/STAT通路变化及STAT蛋白的表达变化。
3、洛沙坦、普罗布考及AG490对HGMC衰老过程中JAK2/STAT通路活性变化及STAT蛋白表达的影响
将洛沙坦、普罗布考、AG490提前加入HGMC培养液中,并在诱导细胞衰老的环境中培养,通过检测细胞增殖情况、细胞周期变化、SAβ-gal染色阳性细胞率、细胞形态和超微结构改变及STAT1、p-STAT1、STAT3、p-STAT3的变化,探讨洛沙坦、普罗布考及AG490对HGMC衰老过程中JAK2/STAT通路活性变化及STAT蛋白表达的影响。
(1) AngⅡ作用组对照组(不含AngⅡ的培养液中培养);AngⅡ诱导衰老组(10-6molL-1AngⅡ);洛沙坦(10-5mo1L-1洛沙坦预处理)+AngⅡ组;AG490(10μmolL-1AG490预处理)+AngⅡ组。
(2)tBHP作用组对照组(不含tBHP的培养液中培养)；tBHP诱导衰老组(30μmolL-1tBHP);普罗布考40μmolL-1普罗布考预处理)+tBHP组;AG490(10μmolL-1AG490预处理)+tBHP组。
结果
1、促HGMC衰老刺激因素的筛选
AngⅡ的促衰老作用：AngⅡ作用0-48h促进HGMC增殖,72h后抑制细胞增殖。10-7molL-1、10-6molL-1、10-5molL-1AngⅡ刺激HGMC 72h,存活的细胞数分别为对照组的94.34±5.16%、81.09±4.52%、59.13±3.12%；SAβ-gal染色阳性率分别为23.03±0.91%、81.23±6.71%、92.10±6.48%；G0-G1期细胞比例分别为72.33±3.83%、90.01±5.62%、95.32±6.03%。
tBHP的促衰老作用：10μmolL-1、30μmolL-1、50μmolL-1tBHP每天刺激HGMC1h、连续5天,存活的细胞数分别为对照组的96.03±5.38%、80.12±4.05%、62.16±3.01%; SAβ-gal染色阳性率分别为25.22±1.23%、84.52±5.86%、90.30±6.25%；G0-G1期细胞比例分别为72.33±3.83%、90.01±5.62%、95.32±6.03%。
葡萄糖的促衰老作用：高浓度葡萄糖作用0-72h促进HGMC增殖,96h后抑制细胞增殖。10mmolL-1、30mmolL-1、50mmolL-1葡萄糖刺激HGMC 96h,存活的细胞数分别为对照组的98.15±5.48%、82.84±3.45%、53.18±3.08%、SAβ-gal染色阳性率分别为22.48±1.03%、82.23±6.05%、90.13±6.13%；G0-G1期细胞比例分别为68.82±4.13%、89.06±5.46%、91.91±5.22%。
10-6molL-1AngⅡ刺激HGMC 72h、30μmolL-1tBHP每天刺激HGMC 1h,连续5天、30mmolL-1葡萄糖刺激HGMC 96h,均可抑制HGMC增殖,且80%以上的HGMC SAβ-gal染色阳性,82%以上的HGMC进入Go-G1期。而10-5molL-1AngⅡ50nmolL-1tBHP、50mmolL-1葡萄糖作用后细胞存活率明显降低,尽管80%以上的细胞也进入G0-G1期,SAβ-gal染色阳性率达到80%,但死亡细胞、碎屑细胞明显增多。
10-6molL-1AngⅡ作用72h、30μmolL-1tBHP每天刺激1h连续5天、30mmolL-1葡萄糖作用96h后诱导的衰老HGMC在光镜下表现为体积增大,胞体扁平,胞浆内颗粒增多,细胞排列不规则,多型核及双核细胞增多；电镜下显示核膜内陷,染色质凝集、边集,细胞质空泡形成,出现髓样溶酶体,粗面内质网扩张。结合SAβ-gal染色阳性率达80%及G0-G1期细胞比例达82%这两个指标,提示HGMC已达到细胞衰老的标准。
本研究选择10-6molL-1AngⅡ刺激HGMC72h、30μmolL-1tBH-1每天刺激HGMC1h,连续5天、30mmolL-1葡萄糖刺激HGMC 96h作为诱导HGMC发生衰老的最佳刺激因素。
2. HGMC衰老过程中JAK2/STAT通路活性及STAT蛋白的变化
通过对各衰老因素作用后HGMC衰老指标的检测,判定实验组HGMC已处于衰老状态。Western blot法检测STAT蛋白的变化,结果显示：衰老HGMC中STAT1、p-STAT1、STAT3、p-STAT3的表达较正常对照的HGMC增加,尤其是p-STAT1、P-STAT3增加更明显。细胞免疫荧光显示：正常对照的HGMC中, STAT1、p-STAT1、p-STAT3在细胞浆和细胞核均有表达；10-6molL-1 AngⅡ、30μmolL-1tBHP、30mmolL-1葡萄糖诱导的衰老HGMC中,STAT蛋白表达增加主要是在细胞核,其中以AngⅡ组作用最为明显。10-5molL-1AngⅡ、50μmolL-1tBHP、50mmolL-1葡萄糖作用后STAT1、p-STAT1、STAT3、p-STAT3的表达略下降；10-7moIL-1AngⅡ、10μmolL-1tBHP、10mmolL-1葡萄糖作用后STAT1、p-STAT1、STAT3、p-STAT3表达略增加。
10-6molL-1AngⅡ、30μmolL-1tBHP及30mmolL-1葡萄糖诱导的衰老细胞中STAT1、STAT3的磷酸化增加,提示JAK2/STAT通路的激活参与了HGMC的衰老过程。
3、三种干预因素对HGMC衰老过程中JAK2/STAT通路活性变化及STAT蛋白表达的影响
应用10-5molL-1洛沙坦及10μmolL"'AG490后细胞存活率由81.12±3.25%分别增至90.43±4.12%及87.82±4.63%;SAβ-gal阳性细胞率由83.10±6.71%分别降至42.13±3.61%及46.28±3.73%；G0-G1期细胞比例由91.36±6.45%分别降至71.56±4.28%及70.97±4.03%；衰老细胞内STAT1、p-STAT1、STAT3、p-STAT3的表达降低。提示洛沙坦及AG490可以改善AngⅡ所致的细胞增殖抑制作用,增加存活细胞数,降低衰老HGMC内STAT1、STAT3蛋白的表达,以磷酸化STAT1、STAT3的降低更明显。
应用40μmolL-1普罗布考及10μmolL-1 AG490后细胞存活率由80.12±4.05%分别增至92.68±4.13%及89.49±4.06%；SAβ-gal阳性细胞由81.03±5.86%分别降至45.32±4.13%及43.16±3.28%；G0-G1期细胞比例由90.76±6.34%分别降至72.96±4.32%及72.73±4.17%；衰老细胞内STAT1、p-STAT1、STAT3、p-STAT3的表达降低。提示普罗布考及AG490可以改善tBHP所致的细胞增殖抑制作用,增加存活细胞数,降低衰老HGMC内STAT1、STAT3蛋白的表达,以磷酸化STAT1、STAT3的降低更明显。
结论
1、AngⅡ、tBHP及高糖在体外可以诱导HMGC衰老。10-6molL-1AngⅡ刺激72h、30μmolL-1tBHP每天刺激1h,连续5天、30mmolL-1葡萄糖刺激96h,是促进HGMC衰老的最佳刺激因素。
2、AngⅡtBHP及高糖诱导的衰老HMGC中STAT1、STAT3磷酸化增加,JAK2/STAT通路的激活参与了HMGC衰老过程。
3、拮抗AT1受体、抗氧化及阻断JAK2/STAT通路,可使STAT1、p-STAT1、STAT3及P-STAT3的表达降低,改善衰老的病理改变,延缓HGMC衰老。
Objective
Kidney is a major senescent organ. Glomerular filtration rate falls progressively with increasing age,especially after 45 years old. Accompaniment that older people is the high risk group of chronic kidney disease and the morbidity rate increases gradually, it is much more important to investigate mechanisms responsible for the senescent kidney and to protect the function of kiney aging. Study of kidney aging mechanism and prevention methods are hot spots that scholars have paid close attension.
The molecular basis of age-related changes in the kidney is unknown. However, organ aging may reflect aspects of cellular senescence. Glomerular mesangial cells (GMCs) are the most important cells of the kidney.Changes in the phenotype and func-tion of GMCs must produce considerable effect. Studies have revealed that angiotensinⅡ(AngⅡ), tert-butly hydroperoxide(tBHP) and high glucose can induce cells aging, especially focused on umbilical vein vascular endothelial cells,lung fibroblast cells, peritoneal mesothelium cells and vascular smooth muscle cells.But there is no report about their relationship with human glomerular mesangial cells(HGMC). The signal transducers and activators of transcription (STATs) are proteins that play a pivotal role in transmitting cytokine signals. It palys role in myocardial cells, macrophage,T cells aging process.To date, nothing is known about the relationship between aging human GMCs and STAT protein. AngⅡ, tBHP and high glucose were used to stimulate HGMC to induce cells aging.Activity of JAK2/STAT pathway and STAT proteins were examined in aging HGMC. Activity of JAK2/STAT pathway and STAT proteins were also examined after losartan,probucol and AG490 used to investigate their function in delaying GMC aging.
Methods
1. Selection of factors inducing HGMC aging
(1)Cells were stimulated with 10-8,10-7,10-6,10-5,10-4molL-1AngⅡfor 24,48,72,96 h; (2) Cells were submitted to 1h stress per day for 5 day under 10,30,50,70, 100μmolL-1 tBHP; (3)Cells were exposed to10,20,30,40,50mmolL-1 glucose for 24,48, 72,96,120h;(4) Cells in control group were cultured in mesangial cell medium without stimulating factor. Then senescence associatedβ-galactosidase(SAP-gal) staining, cell viability and cell cycle analysis were investigated.That 80% cells were SAP-gal positive and 80% cells were in Go-G1 phase was used to assess cells aging. Then the most suitable concentration and times that they can induce cells aging were identified. Morphologic and transmission electron microscopy changes of aging HGMCs were also examined to test whether cells were in senescent stage.
2. Examination of activity of JAK2/STAT pathway and STAT proteins during HGMC aging
Optimization factors were used to induce HGMC aging. Groups including control group and aging groups. Control group (without stimulating factor). Aging groups: AngⅡ-induced group(10-6molL-1AngⅡfor 72h), tBHP-induced group(30μmolL-1 tBHP 1h stress per day for 5 day), high glucose-induced group (30mmolL-1 glucose for 96h).Expression of STAT1, pSTATl,STAT3 and pSTAT3 was analyzed by Western blot analysis and immunofluorescence staining to investigate changes and function of STAT proteins during HGMC aging.
3.Effect of losartan, probucol and AG490 on the activity of JAK2/STAT pathway and STAT proteins during HGMC aging
Losartan, probucol, AG490 were added ahead,then HGMCs were stimulated by factors to induce cells aging. Cell viability, SAβ-gal staining,cell cycle analysis, morphologic and transmission electron microscopy,expression of STAT1, pSTATl,STAT3 and pSTAT3 were examined to investigate their function in delaying GMCs senescence. Groups including AngⅡ-related groups and tBHP-related groups (1)AngⅡ-related groups:Control group(without AngⅡ); AngⅡ-induced group(10-6 molL-1AngⅡ),Losartan(10-5molL-1)+AngⅡ-stimulated group, AG490(10μmolL-1)+ AngⅡ-stimulated group. (2)tBHP-related groups:Control group (without tBHP), tBHP-induced group (30μmolL-1tBHP), Probucol(40μmolL-1probucol)+tBHP-stimul-ated, AG490(10μmolL-1)+tBHP-simulated group.
Results
1.Selection of factors inducing HGMC aging
Promoting aging function of AngⅡ:AngⅡstimulated HGMC proliferation during 0-48h,but suppressed cells proliferation after 72h. After 72h of incubation with 10-7,10-6,10-5molL-1AngⅡ, cell survival rate was 94.34±5.16%,81.09±4.52%, 59.13±3.12%;SAP-gal staining was 23.03±0.91%,81.23±6.71%,92.10±6.48%; G0-G1 phase percent was 72.33±3.83%,90.01±5.62%,95.32±6.03%.
Promoting aging function of tBHP:After stimulated with 1 stress per day for 5 day under10,30,50μmolL-1tBHP, cell survival rate was 96.03±5.38%,80.12±4.05 %,62.16±3.01%;SAβ-gal staining was 25.22±1.23%,84.52±5.86%,90.30±6.25%; G0-G1 phase percent was72.33±3.83%,90.01±5.62%,95.32±6.03%.
Promoting aging function glucose:High glucose stimulated HGMC proliferation during 0-72h,but suppressed cells proliferation after 96h. After 96h of incubation with 10, 30,50mmolL"'glucose, cell survival rate was 99.15±5.48%,82.84±3.45%,53.18±3.08 %; SAβ-gal staining was 22.48±1.03%,82.23±6.05%,90.13±6.13%; G0-G1 phase percent was 68.82±4.13%,89.06±5.46%,91.91±5.22%.
After 72h of incubation with 10-6molL-1 AngⅡ,5 successive stresses of 30μmolL-1 tBHP,96h with 30mmolL-1glucose, cellular proliferation was permanently inhibited in the aging groups, with an OD value significantly lower than that of the control group;SAβ-gal staining was significantly increased;The cell cycle was arrested at the Go-G1 phase. Cellular proliferation was obviously inhibited after stimulated with 10-5molL-1Angll,50μmolL-1tBHP,50mmolL-1glucose.Althoug 80 percent cells were arrest at G0-G1 phase and SAP-gal staining positive, death and debris cells increased. Aging cells induced by 10-6molL-1AngⅡ,30μmolL-1tBHP,30mmolL-1glucose were characterized by enlarged cell morphology, polymorphic nuclei, chromatin condensation at the nuclear margin, invagination of the nuclear membrane, the disappearance of mitochondria, dictyosome, tigroid body and pulp-lysosome identified.The results indicated that HGMCs have achieved aging standard.
10-6molL-1AngⅡ,30μmolL-1tBHP,30mmolL-1 glucose were used to induce HGMC aging in our studies.
2. Changes of JAK2/STAT pathway and STAT proteins during HGMC aging
After stimulated by the aging-induced factors, aging indexes were examined. HGMCs were tested to be aging cells. Western blot analysis indicated that expressions of STAT1, pSTAT1, STAT3 and pSTAT3 were increased in aging cells compared to the control cells, especially of pSTAT1 and pSTAT3. Using immunofluorescence detection, we found that STAT1, pSTATl, STAT3 and pSTAT3 were expressed in cytoplasm and cytoblast in normal HGMCs, and their expressions were increased in aging cells, in particular in the cytoblast.It is most obvious in AngⅡ-induced group. Expressions of STAT1, pSTATl, STAT3 and pSTAT3 were slightly decresed after stimulated with 10-5 molL-1 AngⅡ,50μmolL-1tBHP, 50mmolL-1 gluccose,but there is no statistics difference. However their expressions were slightly increased after stimulated with 10-7molL-1 AngⅡ,10μmolL-1tBHP,10mmolL-1 glucose,and there is also no statistics difference.. The results indicated that activation of JAK2/STAT pathway involved in HGMC aging through increasing phosphorylation of STAT1,STAT3 induced by 10-6molL-1 AngⅡ, 30μmolL-1tBHP,30mmol-1 glucose.
3. Effect of the three intervention factors on JAK2/STAT pathway and STAT proteins during HGMC aging
After using 10-5molL-1losartan,10μmolL-1AG490,living cells increased with cell survival percent raised from81.12±3.25% to 90.43±4.12% and 87.82±4.63%; SAβ-gal positive rate falled from 83.10±6.71% to 42.13±3.61% and 46.28+3.73%; proportion of G0-G1 phase reduced from 91.36±6.45% to 71.56±4.28% and 70.97±4.03%; expressions of STAT1,pSTAT1,STAT3 and pSTAT3 decreased.These results indicated that after using losartan and AG490, depressant effect of cell proliferation were improved and living cells were increased,expressions of STAT protein were decreased in AngⅡ-induced aging HGMC.
After using 40μmolL-1 probucol,10μmolL-'AG490, living cells increased with cell survival percent raised from 80.12±4.05% to 92.68±4.13% and 89.49±4.06%; SAβ-gal positive rate falled from 81.03±5.86% to 45.32±4.13% and 43.16±3.28%; proportion of G0-G1 phase reduced from 90.76±6.34% to 72.96±4.32% and 72.73±4.17%;expressions of STAT1, pSTAT1, STAT3 and pSTAT3 decreased.These results indicated that after using probucol and AG490, depressant effect of cell proliferation were improved and living cells were increased,expressions of STAT protein were decreased in tBHP-induced aging HGMC.
Conclusion
1.Ang II,tBHP,high glucose induced HGMC aging in vitro. Conditions of incubation with 10-6molL-1AngⅡfor 72h,5 successive stresses of 1 h per day with 30μmolL-1tBHP,30mmolL-1 glucose for 96h were the most suitable factors to induce HGMC aging.
2. Phosphorylation of STAT1,STAT3 were increased in aging HMGC induced by Ang II,tBHP,high glucose,and activation of JAK2/STAT involved in HGMC aging. 3. Antagonizing AT1 acceptor, antioxidation and blocking JAK2/STAT pathway can decrease expressions of STAT1,pSTAT1,STAT3 and pSTAT3 and improve aging HGMCs patho-state to delay HGMC aging.
肾脏是机体衰老的主要器官,肾小球滤过率(glomerular filtration rate,GFR)随增龄而降低,进入45岁以后GFR下降速度加快。基于随着经济发展老龄人口逐渐增加及老年人是慢性肾脏病高危人群这两方面因素,在我国逐渐迈入老龄化社会的今天,研究肾脏衰老机制、探讨延缓肾脏衰老的方法,显得尤为重要。肾脏衰老的机制及防治是目前国内外学者关注的重要问题之一。
目前关于肾脏衰老的机制尚不十分明确。多数学者认为肾脏衰老主要是由于肾脏固有细胞功能减退所致。肾小球系膜细胞是肾脏的主要固有细胞,其表型和功能改变在肾脏衰老进程中发挥着重要的作用。目前的研究证实,高糖、血管紧张素Ⅱ(angiotensin, AngⅡ)、叔丁基过氧化氢(tert-butly hydroperoxide, tBHP)可促进细胞衰老。这些研究多见于脐静脉血管内皮细胞、肺成纤维细胞、腹膜间皮细胞、血管平滑肌细胞等,尚没有对人肾小球系膜细胞(Human Glomerular Mesangial Cell, HGMC)衰老的研究报道。JAK2／STAT通路是一条重要的信号转导途径,在心肌细胞、巨噬细胞、T细胞等多种细胞的衰老过程中发挥作用,但其与HGMC衰老之间的关系尚不清楚。本研究以AngⅡtBHP及高糖作为刺激因素,诱导HGMC发生衰老,检测衰老HGMC中JAK2/STAT通路活性及STAT蛋白表达的变化,观察洛沙坦、普罗布考及JAK2阻断剂AG490对JAK2/STAT通路活性变化及STAT蛋白表达的影响,探讨它们在延缓HGMC衰老过程中的作用。
方法
1、HGMC衰老诱导因素的筛选
(1)用10-8、10-7、10-6、10-5、104molL-1。AngⅡ刺激HGMC,刺激时间为24h、48 h、72 h、96h；(2)用10、30、50、70、100μmolL-1 tBHP刺激HGMC,每天1h,连续5天；(3)用10、20、30、40、50mmolL-1葡萄糖刺激HGMC,刺激时间为24 h、48 h、72 h、96h、120h。(4)对照组细胞在不含任何刺激因素的培养液中培养。HGMC在各因素刺激条件下培养,刺激结束后检测细胞增殖情况、衰老相关β半乳糖苷酶(senescence-associatedβ-galactosidase,SAP-gal)染色阳性细胞百分率、细胞周期变化,并将SAβ-gal阳性细胞率及Go-G1期细胞比例达80%作为判定细胞衰老的标准,明确三种因素诱导HGMC发生衰老的最适浓度和最适培养时间。观察衰老HGMC形态学及超微结构改变,进一步判定细胞进入衰老期的最佳刺激因素。
2、HGMC衰老过程中JAK2/STAT通路活性及STAT蛋白表达的变化
采用最佳促进HGMC衰老的刺激因素,诱导HGMC发生衰老。分组情况
(1)对照组(在不含任何刺激因素的培养液中培养)；
(2)衰老组
AngⅡ诱导组(10-6molL-1 AngⅡ刺激72h)；
tBHP诱导组(30μmolL-1tBHP每天刺激1h连续5天)；
高糖诱导组30mmolL-1葡萄糖刺激96h)。
应用Western Blot及细胞免疫荧光法检测衰老HGMC内STAT1、p-STAT1、STAT3、p-STAT3的表达变化,研究HGMC衰老过程中JAK2/STAT通路变化及STAT蛋白的表达变化。
3、洛沙坦、普罗布考及AG490对HGMC衰老过程中JAK2/STAT通路活性变化及STAT蛋白表达的影响
将洛沙坦、普罗布考、AG490提前加入HGMC培养液中,并在诱导细胞衰老的环境中培养,通过检测细胞增殖情况、细胞周期变化、SAβ-gal染色阳性细胞率、细胞形态和超微结构改变及STAT1、p-STAT1、STAT3、p-STAT3的变化,探讨洛沙坦、普罗布考及AG490对HGMC衰老过程中JAK2/STAT通路活性变化及STAT蛋白表达的影响。
(1) AngⅡ作用组对照组(不含AngⅡ的培养液中培养);AngⅡ诱导衰老组(10-6molL-1AngⅡ);洛沙坦(10-5mo1L-1洛沙坦预处理)+AngⅡ组;AG490(10μmolL-1AG490预处理)+AngⅡ组。
(2)tBHP作用组对照组(不含tBHP的培养液中培养)；tBHP诱导衰老组(30μmolL-1tBHP);普罗布考40μmolL-1普罗布考预处理)+tBHP组;AG490(10μmolL-1AG490预处理)+tBHP组。
结果
1、促HGMC衰老刺激因素的筛选
AngⅡ的促衰老作用：AngⅡ作用0-48h促进HGMC增殖,72h后抑制细胞增殖。10-7molL-1、10-6molL-1、10-5molL-1AngⅡ刺激HGMC 72h,存活的细胞数分别为对照组的94.34±5.16%、81.09±4.52%、59.13±3.12%；SAβ-gal染色阳性率分别为23.03±0.91%、81.23±6.71%、92.10±6.48%；G0-G1期细胞比例分别为72.33±3.83%、90.01±5.62%、95.32±6.03%。
tBHP的促衰老作用：10μmolL-1、30μmolL-1、50μmolL-1tBHP每天刺激HGMC1h、连续5天,存活的细胞数分别为对照组的96.03±5.38%、80.12±4.05%、62.16±3.01%; SAβ-gal染色阳性率分别为25.22±1.23%、84.52±5.86%、90.30±6.25%；G0-G1期细胞比例分别为72.33±3.83%、90.01±5.62%、95.32±6.03%。
葡萄糖的促衰老作用：高浓度葡萄糖作用0-72h促进HGMC增殖,96h后抑制细胞增殖。10mmolL-1、30mmolL-1、50mmolL-1葡萄糖刺激HGMC 96h,存活的细胞数分别为对照组的98.15±5.48%、82.84±3.45%、53.18±3.08%、SAβ-gal染色阳性率分别为22.48±1.03%、82.23±6.05%、90.13±6.13%；G0-G1期细胞比例分别为68.82±4.13%、89.06±5.46%、91.91±5.22%。
10-6molL-1AngⅡ刺激HGMC 72h、30μmolL-1tBHP每天刺激HGMC 1h,连续5天、30mmolL-1葡萄糖刺激HGMC 96h,均可抑制HGMC增殖,且80%以上的HGMC SAβ-gal染色阳性,82%以上的HGMC进入Go-G1期。而10-5molL-1AngⅡ50nmolL-1tBHP、50mmolL-1葡萄糖作用后细胞存活率明显降低,尽管80%以上的细胞也进入G0-G1期,SAβ-gal染色阳性率达到80%,但死亡细胞、碎屑细胞明显增多。
10-6molL-1AngⅡ作用72h、30μmolL-1tBHP每天刺激1h连续5天、30mmolL-1葡萄糖作用96h后诱导的衰老HGMC在光镜下表现为体积增大,胞体扁平,胞浆内颗粒增多,细胞排列不规则,多型核及双核细胞增多；电镜下显示核膜内陷,染色质凝集、边集,细胞质空泡形成,出现髓样溶酶体,粗面内质网扩张。结合SAβ-gal染色阳性率达80%及G0-G1期细胞比例达82%这两个指标,提示HGMC已达到细胞衰老的标准。
本研究选择10-6molL-1AngⅡ刺激HGMC72h、30μmolL-1tBH-1每天刺激HGMC1h,连续5天、30mmolL-1葡萄糖刺激HGMC 96h作为诱导HGMC发生衰老的最佳刺激因素。
2. HGMC衰老过程中JAK2/STAT通路活性及STAT蛋白的变化
通过对各衰老因素作用后HGMC衰老指标的检测,判定实验组HGMC已处于衰老状态。Western blot法检测STAT蛋白的变化,结果显示：衰老HGMC中STAT1、p-STAT1、STAT3、p-STAT3的表达较正常对照的HGMC增加,尤其是p-STAT1、P-STAT3增加更明显。细胞免疫荧光显示：正常对照的HGMC中, STAT1、p-STAT1、p-STAT3在细胞浆和细胞核均有表达；10-6molL-1 AngⅡ、30μmolL-1tBHP、30mmolL-1葡萄糖诱导的衰老HGMC中,STAT蛋白表达增加主要是在细胞核,其中以AngⅡ组作用最为明显。10-5molL-1AngⅡ、50μmolL-1tBHP、50mmolL-1葡萄糖作用后STAT1、p-STAT1、STAT3、p-STAT3的表达略下降；10-7moIL-1AngⅡ、10μmolL-1tBHP、10mmolL-1葡萄糖作用后STAT1、p-STAT1、STAT3、p-STAT3表达略增加。
10-6molL-1AngⅡ、30μmolL-1tBHP及30mmolL-1葡萄糖诱导的衰老细胞中STAT1、STAT3的磷酸化增加,提示JAK2/STAT通路的激活参与了HGMC的衰老过程。
3、三种干预因素对HGMC衰老过程中JAK2/STAT通路活性变化及STAT蛋白表达的影响
应用10-5molL-1洛沙坦及10μmolL"'AG490后细胞存活率由81.12±3.25%分别增至90.43±4.12%及87.82±4.63%;SAβ-gal阳性细胞率由83.10±6.71%分别降至42.13±3.61%及46.28±3.73%；G0-G1期细胞比例由91.36±6.45%分别降至71.56±4.28%及70.97±4.03%；衰老细胞内STAT1、p-STAT1、STAT3、p-STAT3的表达降低。提示洛沙坦及AG490可以改善AngⅡ所致的细胞增殖抑制作用,增加存活细胞数,降低衰老HGMC内STAT1、STAT3蛋白的表达,以磷酸化STAT1、STAT3的降低更明显。
应用40μmolL-1普罗布考及10μmolL-1 AG490后细胞存活率由80.12±4.05%分别增至92.68±4.13%及89.49±4.06%；SAβ-gal阳性细胞由81.03±5.86%分别降至45.32±4.13%及43.16±3.28%；G0-G1期细胞比例由90.76±6.34%分别降至72.96±4.32%及72.73±4.17%；衰老细胞内STAT1、p-STAT1、STAT3、p-STAT3的表达降低。提示普罗布考及AG490可以改善tBHP所致的细胞增殖抑制作用,增加存活细胞数,降低衰老HGMC内STAT1、STAT3蛋白的表达,以磷酸化STAT1、STAT3的降低更明显。
结论
1、AngⅡ、tBHP及高糖在体外可以诱导HMGC衰老。10-6molL-1AngⅡ刺激72h、30μmolL-1tBHP每天刺激1h,连续5天、30mmolL-1葡萄糖刺激96h,是促进HGMC衰老的最佳刺激因素。
2、AngⅡtBHP及高糖诱导的衰老HMGC中STAT1、STAT3磷酸化增加,JAK2/STAT通路的激活参与了HMGC衰老过程。
3、拮抗AT1受体、抗氧化及阻断JAK2/STAT通路,可使STAT1、p-STAT1、STAT3及P-STAT3的表达降低,改善衰老的病理改变,延缓HGMC衰老。
Objective
Kidney is a major senescent organ. Glomerular filtration rate falls progressively with increasing age,especially after 45 years old. Accompaniment that older people is the high risk group of chronic kidney disease and the morbidity rate increases gradually, it is much more important to investigate mechanisms responsible for the senescent kidney and to protect the function of kiney aging. Study of kidney aging mechanism and prevention methods are hot spots that scholars have paid close attension.
The molecular basis of age-related changes in the kidney is unknown. However, organ aging may reflect aspects of cellular senescence. Glomerular mesangial cells (GMCs) are the most important cells of the kidney.Changes in the phenotype and func-tion of GMCs must produce considerable effect. Studies have revealed that angiotensinⅡ(AngⅡ), tert-butly hydroperoxide(tBHP) and high glucose can induce cells aging, especially focused on umbilical vein vascular endothelial cells,lung fibroblast cells, peritoneal mesothelium cells and vascular smooth muscle cells.But there is no report about their relationship with human glomerular mesangial cells(HGMC). The signal transducers and activators of transcription (STATs) are proteins that play a pivotal role in transmitting cytokine signals. It palys role in myocardial cells, macrophage,T cells aging process.To date, nothing is known about the relationship between aging human GMCs and STAT protein. AngⅡ, tBHP and high glucose were used to stimulate HGMC to induce cells aging.Activity of JAK2/STAT pathway and STAT proteins were examined in aging HGMC. Activity of JAK2/STAT pathway and STAT proteins were also examined after losartan,probucol and AG490 used to investigate their function in delaying GMC aging.
Methods
1. Selection of factors inducing HGMC aging
(1)Cells were stimulated with 10-8,10-7,10-6,10-5,10-4molL-1AngⅡfor 24,48,72,96 h; (2) Cells were submitted to 1h stress per day for 5 day under 10,30,50,70, 100μmolL-1 tBHP; (3)Cells were exposed to10,20,30,40,50mmolL-1 glucose for 24,48, 72,96,120h;(4) Cells in control group were cultured in mesangial cell medium without stimulating factor. Then senescence associatedβ-galactosidase(SAP-gal) staining, cell viability and cell cycle analysis were investigated.That 80% cells were SAP-gal positive and 80% cells were in Go-G1 phase was used to assess cells aging. Then the most suitable concentration and times that they can induce cells aging were identified. Morphologic and transmission electron microscopy changes of aging HGMCs were also examined to test whether cells were in senescent stage.
2. Examination of activity of JAK2/STAT pathway and STAT proteins during HGMC aging
Optimization factors were used to induce HGMC aging. Groups including control group and aging groups. Control group (without stimulating factor). Aging groups: AngⅡ-induced group(10-6molL-1AngⅡfor 72h), tBHP-induced group(30μmolL-1 tBHP 1h stress per day for 5 day), high glucose-induced group (30mmolL-1 glucose for 96h).Expression of STAT1, pSTATl,STAT3 and pSTAT3 was analyzed by Western blot analysis and immunofluorescence staining to investigate changes and function of STAT proteins during HGMC aging.
3.Effect of losartan, probucol and AG490 on the activity of JAK2/STAT pathway and STAT proteins during HGMC aging
Losartan, probucol, AG490 were added ahead,then HGMCs were stimulated by factors to induce cells aging. Cell viability, SAβ-gal staining,cell cycle analysis, morphologic and transmission electron microscopy,expression of STAT1, pSTATl,STAT3 and pSTAT3 were examined to investigate their function in delaying GMCs senescence. Groups including AngⅡ-related groups and tBHP-related groups (1)AngⅡ-related groups:Control group(without AngⅡ); AngⅡ-induced group(10-6 molL-1AngⅡ),Losartan(10-5molL-1)+AngⅡ-stimulated group, AG490(10μmolL-1)+ AngⅡ-stimulated group. (2)tBHP-related groups:Control group (without tBHP), tBHP-induced group (30μmolL-1tBHP), Probucol(40μmolL-1probucol)+tBHP-stimul-ated, AG490(10μmolL-1)+tBHP-simulated group.
Results
1.Selection of factors inducing HGMC aging
Promoting aging function of AngⅡ:AngⅡstimulated HGMC proliferation during 0-48h,but suppressed cells proliferation after 72h. After 72h of incubation with 10-7,10-6,10-5molL-1AngⅡ, cell survival rate was 94.34±5.16%,81.09±4.52%, 59.13±3.12%;SAP-gal staining was 23.03±0.91%,81.23±6.71%,92.10±6.48%; G0-G1 phase percent was 72.33±3.83%,90.01±5.62%,95.32±6.03%.
Promoting aging function of tBHP:After stimulated with 1 stress per day for 5 day under10,30,50μmolL-1tBHP, cell survival rate was 96.03±5.38%,80.12±4.05 %,62.16±3.01%;SAβ-gal staining was 25.22±1.23%,84.52±5.86%,90.30±6.25%; G0-G1 phase percent was72.33±3.83%,90.01±5.62%,95.32±6.03%.
Promoting aging function glucose:High glucose stimulated HGMC proliferation during 0-72h,but suppressed cells proliferation after 96h. After 96h of incubation with 10, 30,50mmolL"'glucose, cell survival rate was 99.15±5.48%,82.84±3.45%,53.18±3.08 %; SAβ-gal staining was 22.48±1.03%,82.23±6.05%,90.13±6.13%; G0-G1 phase percent was 68.82±4.13%,89.06±5.46%,91.91±5.22%.
After 72h of incubation with 10-6molL-1 AngⅡ,5 successive stresses of 30μmolL-1 tBHP,96h with 30mmolL-1glucose, cellular proliferation was permanently inhibited in the aging groups, with an OD value significantly lower than that of the control group;SAβ-gal staining was significantly increased;The cell cycle was arrested at the Go-G1 phase. Cellular proliferation was obviously inhibited after stimulated with 10-5molL-1Angll,50μmolL-1tBHP,50mmolL-1glucose.Althoug 80 percent cells were arrest at G0-G1 phase and SAP-gal staining positive, death and debris cells increased. Aging cells induced by 10-6molL-1AngⅡ,30μmolL-1tBHP,30mmolL-1glucose were characterized by enlarged cell morphology, polymorphic nuclei, chromatin condensation at the nuclear margin, invagination of the nuclear membrane, the disappearance of mitochondria, dictyosome, tigroid body and pulp-lysosome identified.The results indicated that HGMCs have achieved aging standard.
10-6molL-1AngⅡ,30μmolL-1tBHP,30mmolL-1 glucose were used to induce HGMC aging in our studies.
2. Changes of JAK2/STAT pathway and STAT proteins during HGMC aging
After stimulated by the aging-induced factors, aging indexes were examined. HGMCs were tested to be aging cells. Western blot analysis indicated that expressions of STAT1, pSTAT1, STAT3 and pSTAT3 were increased in aging cells compared to the control cells, especially of pSTAT1 and pSTAT3. Using immunofluorescence detection, we found that STAT1, pSTATl, STAT3 and pSTAT3 were expressed in cytoplasm and cytoblast in normal HGMCs, and their expressions were increased in aging cells, in particular in the cytoblast.It is most obvious in AngⅡ-induced group. Expressions of STAT1, pSTATl, STAT3 and pSTAT3 were slightly decresed after stimulated with 10-5 molL-1 AngⅡ,50μmolL-1tBHP, 50mmolL-1 gluccose,but there is no statistics difference. However their expressions were slightly increased after stimulated with 10-7molL-1 AngⅡ,10μmolL-1tBHP,10mmolL-1 glucose,and there is also no statistics difference.. The results indicated that activation of JAK2/STAT pathway involved in HGMC aging through increasing phosphorylation of STAT1,STAT3 induced by 10-6molL-1 AngⅡ, 30μmolL-1tBHP,30mmol-1 glucose.
3. Effect of the three intervention factors on JAK2/STAT pathway and STAT proteins during HGMC aging
After using 10-5molL-1losartan,10μmolL-1AG490,living cells increased with cell survival percent raised from81.12±3.25% to 90.43±4.12% and 87.82±4.63%; SAβ-gal positive rate falled from 83.10±6.71% to 42.13±3.61% and 46.28+3.73%; proportion of G0-G1 phase reduced from 91.36±6.45% to 71.56±4.28% and 70.97±4.03%; expressions of STAT1,pSTAT1,STAT3 and pSTAT3 decreased.These results indicated that after using losartan and AG490, depressant effect of cell proliferation were improved and living cells were increased,expressions of STAT protein were decreased in AngⅡ-induced aging HGMC.
After using 40μmolL-1 probucol,10μmolL-'AG490, living cells increased with cell survival percent raised from 80.12±4.05% to 92.68±4.13% and 89.49±4.06%; SAβ-gal positive rate falled from 81.03±5.86% to 45.32±4.13% and 43.16±3.28%; proportion of G0-G1 phase reduced from 90.76±6.34% to 72.96±4.32% and 72.73±4.17%;expressions of STAT1, pSTAT1, STAT3 and pSTAT3 decreased.These results indicated that after using probucol and AG490, depressant effect of cell proliferation were improved and living cells were increased,expressions of STAT protein were decreased in tBHP-induced aging HGMC.
Conclusion
1.Ang II,tBHP,high glucose induced HGMC aging in vitro. Conditions of incubation with 10-6molL-1AngⅡfor 72h,5 successive stresses of 1 h per day with 30μmolL-1tBHP,30mmolL-1 glucose for 96h were the most suitable factors to induce HGMC aging.
2. Phosphorylation of STAT1,STAT3 were increased in aging HMGC induced by Ang II,tBHP,high glucose,and activation of JAK2/STAT involved in HGMC aging. 3. Antagonizing AT1 acceptor, antioxidation and blocking JAK2/STAT pathway can decrease expressions of STAT1,pSTAT1,STAT3 and pSTAT3 and improve aging HGMCs patho-state to delay HGMC aging.
引文
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