| IgAN (IgA nephropathy) which is also been called Berger’s Disease, is a special type of glomerulonephritis. The pathological feature of IgAN is IgA antibody deposition in mesangial region. IgAN has various pathological changes from slight mesangial proliferation to glomerulosclerosis and cresent formation. IgAN’s typical clinical manifestation is repeated gross hematuria or microscopic hematuria, with or without proteinuria. The mechanism for this is that after antigen stimulation, the body produces large amount of IgA antibody, IgA and IgA immune complex deposite in the mesangial region, activating complement alternative pathway, which recruits inflammatory cells and inflammatory factors to cause renal damage. The etiology of IgAN is not very clear yet, the prognosis is also not very good. Until now, it still lackes effective therapy to cure this disease. Only10%of IgAN patients spontaneously relieve from their abnormal urinalysis,25%-30%patients progress to ESRD within10-20yrs after renal biopsy. Exploration of the renal damage mechanism and prolonging the process have developed to be key goals of IgAN research.Tonsillar mucosal immunity has close relationship with IgAN renal damage. IgAN patients always get worsen urinalysis after upper respiratory tract infection. Compared to pure methylprednisolone pulse treatment (MPT), tonsillectomy combined with MPT is much more effective at meliorating hematuria and proteinuria, lessening IgA deposition at mesangial region and mesangial proliferation, also protecting renal function. Various researches have proven that IgAN patients have lots of abnormal aspects in tonsils:Structurally, IgAN patients’tonsil crypts are abnormally reticulated epithelium; After exogenous antigen challenge, IgAN patients have much stronger tonsillar immuno-reaction, it not only has the immune cell subgroup imbalance but also the secretion is abnormal. IgAN tonsils have imbalance at immune cell types, secretion abnormality. After exogenous antigen stimulation as Hemophilus parainfluenzae, tonsillar T cell accumulated district and T cell nodule enlarged, tonsillar immune regulation overacted, leading to B cell over-stimulation, follicular outer proliferation, the number of IgA secreting tonsillar lymphocytes enlarged. Additionally, the number of tonsillar lymphocytes which secrete poly-IgA get enlarged. The IgA which has underglycosylated hinge region has the same character as IgA deposited in mesangial region. The above implications has proven the relationship between tonsil and renal damage.IgAN patients’ tonsillar mononuclear cells(TMCs) are main reactive cells during tonsillar abnormal immunity, their secretion products abnormality has been proven by various research groups. IgAN TMCs overact upon antigen, secrets like TGF-β1,IL-4,MCP-1more than normal, stimulates B cell over-proliferated, producing more IgA-producing plasma cells.Reactive Oxygen Species(ROS) are a type of chemical species which are oxygen-originated, radical components including oxygen in human body. The two main types of oxygen radicals are superoxide anion(O2·-) and hydroxyl radical(OH). ROS could damage biological structural materials through oxidizing DNA, protein, polyunsaturated fatty acid, especially the fatty acid oxidation. Modern researchers showed that various diseases including IgAN have relationship with ROS oxidation damage. Whether TMCs secretion could induce renal tubular epithelial microenvironment ROS level rising and oxidation reaction which induce apoptosis have not been published.We collect IgAN and chronic tonsillitis (CT) patients’right side tonsils after tonsillectomy, divide and culture TMCs, collect the supernatants and moderate on renal tubular epithelial cell-line HK-2cells, observe HK-2cells’ apoptotic level ans bcl-2, simultaneously we investigate HK-2’s ROS level, after which we moderate HK-2cells with TGF-β1to investigate its effect on HK-2apoptosis and ROS level. From these, we explored TMCs’ pathway to affect renal tubular epithelial cells. Our research is basis for further research on policy of protecting renal function. Our research has been separated into3parts. Part Ⅰ Exploration of effect of IgAN TMCs supernatants on tubular epithelial cell HK-2superoxide type ROS level and apoptosisObjective:To observe the effect of IgAN patients’TMCs supernatants on human tubular epithelial cell ROS level and apoptosis, these experiment are to explore specific ROS pathway for IgAN TMCs secretion to damage tubular epithelial.Methods:We collected10IgAN and10non-IgAN chronic tonsillitis (CT) patients’right palatine tonsils, separated TMCs for in vitro culture with or without PHA. After72hrs’culture, collect:(1) IgAN+group:PHA stimulated;(2) IgAN-group:PHA non-stimulated;(3) CT+group:PHA stimulated CT;(4) CT-group:PHA non-stimulated CT totally four groups of TMCs supernatants. Add20%supernatants to renal tubular epithelial cells HK-2cells for co-culture. After12hr, set negative control group as0.5%FBS low-glucose DMEM cultured HK-2, test DHE level using flow cytometry for each group.Add PHA to IgAN patients TMCs, culture for72hrs, collect supernatants. Divide HK-2cells into3groups:(1) Con group:0.5%FBS low glucose DMEM culture;(2) IgAN+group:20%PHA stimulated IgAN TMCs supernatants culture;(3) IgAN+&SOD group:20%PHA stimulated TMCs supernatants and Cu/Zn SOD(100u/mL) for culture.24hrs later, use flow cytometry for AnnexinV, Propodium Iodide(reflect apoptotic level) intensity, trypan blue for cell viability. Results:Compared to negative control, PHA non--stimulated IgAN-group, PHA stimulated CT+group, PHA non-stimulated CT groups, PHA stimulated IgAN TMCs supernatant significantly rise HK-2cell DHE level(p<0.01). compared to negative control, PHA stimulated CT+and non-stimulated CT-groups, Unstimulated IgAN-group TMCs supernatant has higher ROS level(P<0.01). PHA stimulated IgAN TMCs supernatant increase cell apoptosis which could be inhibited41%by SOD, cell viability also could be rised partially by SOD.Conclusions:1. IgAN TMCs, either PHA stimulated or non-stimulated, could rise tubular epithelial cells’superoxide type ROS level, this implicates that IgAN TMCs secretion as an stressor could induce renal tubular epithelial cell react upon oxidative stress;2. PHA stimulated IgAN TMCs supernatant induced HK-2apoptosis, which could be inhibited by SOD. Part Ⅱ Time variation Effect of IgAN TMCs supernatants on HK-2’s superoxide type ROS and bcl-2and p53Objectives:To observe TMCs’supernatant’s time-course effect on renal tubular epithelial cell’s superoxide type ROS level and the relationship of HK-2’s bcl-2and p53and IgAN TMCs supernatants.Methods:We collect72hrs’PHA stimulated IgAN TMCs supernatants and coculture the supernatants with TMC cell line HK-2cells. In0,1,3,6, and12hrs of co-culture, evaluate HK-2cell ROS levels. In Ohr,3hr and12hr, extract HK-2protein, using western blooting for bcl-2and p53level.Divide HK-2cells into3groups:(1) Con group:0.5%FBS low-glucose DMEM culture;(2) IgAN+group:PHA stimulated IgAN TMCs supernatants20%add to culture;(3) IgAN+&SOD group:PHA stimulated IgAN TMCs supernatant20%and Cu/Zn SOD (100u/mL) add to culture. After12hr, extract protein, use western blotting to test bcl-2and p53level.Results:HK-2cells increase its DHE level during0-3hr culture,3hr is the time for DHE peak, after which the DHE level dropped down.12hr’s DHE level is only55%of peak time. PHA stimulated IgAN TMCs supernatant didn’t change HK-2cell bcl-2at3hr’s time, but bcl-2shortened after12hrs’culture; Add SOD could shorten IgAN TMCs’ negative regulation on HK-2bcl-2. P53have no changes during the whole process.Conclusions:1.PHA stimulated IgAN TMCs supernatants increase HK-2cells’02-type ROS level during0-3hr coculture, at3hrs’ coculture time, the ROS rises to peak level, after that it drops down;2.IgAN TMCs supernatants could decrease HK-2cells’Bcl-2level12hr after coculture while there’s no change during3hrs timepoint. Additionally, SOD could inhibit this bcl-2regulation;3.p53has no obvious change neither during3hr or12hr, adding SOD could neither change p53level. Part Ⅲ Abnormal expressed TGF-β1after IgAN patients’TMCs supernatants modulation on renal tubular epithelial cells HK-2and TGF-β’s effect on HK-2Objectives:Through modulating IgAN patients’TMCs supernatants on HK-2cells to observe HK-2’s TGF-β1level and TGF-β1’s effect on HK-2ROS level and apoptotic ratio.Methods:Divide renal tubular epithelial cell-line HK-2cells into3groups:(1) Con group:one group add0.5%FBS’s low glucose DMEM media;(2) IgAN+group:add20%PHA stimulated IgAN patients’ TMCs supernatants;(3) CT+group:add20%chronic tonsillitis’ patients’ TMCs supernatants after PHA stimulation. After24hrs’cultivation, using double-antibody sandwich ELISA to test each group’s media TGF-β1level.Divide HK-2cells into4groups:(1) Con group:add0.5%FBS’s low glucose DMEM media;(2) IgAN+group:add20%IgAN patients’ TMCs supernatants after PHA stimulation;(3) TGF-β1group:add TGF-β1(5ng/mL);(4) TGF-β1&SOD group:add TGF-β1(5ng/mL) and Cu/ZnSOD(100units/mL). After12hrs’cultivation, using Flow Cytometry to test cells’DHE level and24hrs%Annexin V(+) apoptotic cells in each group.Results:HK-2cells increase its TGF-β1level after cultured with PHA stimulated IgAN+group supernatants and CT+group supernatants, however, IgAN+group increases HK-2cells’TGF-β1level more than CT+group(1162.76±162.13vs314.81±43.79). TGF-β1increase HK-2cells’ apoptosis and DHE level, it increases DHE level as IgAN+TMCs20%supernatant, but the apoptotic level is less than IgAN+group. Additionally, the effect could be partially inhibited by SOD.Conclusions:1. PHA stimulated.IgAN TMCs supernatants adding to HK-2cells could obviously increase co-culture media’s TGF-β1level;2. TGF-β1, when adding to HK-2cells, could increase HK-2ROS level and apoptotic ratio, and TGF-β1’s effect could be partly inhibited by SOD. |
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