The Role Of Nf-κB Signaling Pathway And Fcγ RIIB/C In Diabetic Nephropathy And Baicalein Intervention Study

The impact of NF-kB inflammatory signaling pathway and immune receptor Fcγ RIIB/C on the development and progression of diabetic nephropathy were observed respectively from experimental and clinical research.Experimental research—The role of baicalein in high glucose-i nduced human renal proximal tubular epithelial cell(HK-2) injuryNF-kB-mediated inflammatory response, as a key factor of an inflammatory signaling pathway, plays a very important role in the pathological process of diabetic nephropathy, but the role of NF-kB in high glucose-induced human renal proximal tubule epithelial cell injury is not clear. In vitro study, we demonstrated that NF-kB activation took part in renal tubular epithelial cell injury induced by high glucose, and baicalein could inhibit NF-kB-mediated inflammatory response in high glucose (HG)-induced HK-2cell injury.Objective:1. To investigate the toxic effects of high glucose on human renal tubular epithelial cell line (HK-2) and related signaling pathway.2. To investigate the role of baicalein in HG-induced HK-2cell injury.Methods:Different glucose concentrations (5.5mM-60mM) and mannitol concentrations (30mM, 45mM, respectively, corresponding to the osmotic pressure of the high glucose) stimulated HK-2cells for24h, cell viability was observed. Different glucose concentrations (5.5mM-45mM) stimulated HK-2cells for24h, the expression of IκBα and p65were observed.Baicalein (25-200uM) stimulated HK-2cells for36h with any treatment, cell viability was observed. HG (45mM) was selected to stimulate HK-2cells for24h, the NF-kappaB inhibitor PDTC was used as a positive control.To observe the inhibitory effect of different doses of baicalein (25-100uM) on NF-kappaB pathway activation and its downstream inflammatory molecules and extracellular matrix in HG-induced HK-2cells. Cell viability was detected by MTT assay. The key proteins of NF-kappaB signaling pathway (IκBa, p65, p-p65, IKKa), and its downstream inflammatory molecules (MCP-1, ICAM-1) and an important component of the extracellular matrix (Collagen IV) were measured by Western blot analysis.Result:1. The toxic effect of high glucose on HK-2cellsDifferent glucose concentrations (5.5mM,15mM,45mM,60mM) were incubated for HK-2for24h, cell viability was detected by MTT assay, normal glucose (5.5mM) as control group (NG). Found that the different glucose concentrations had an inhibitory effect on cell viability of HK-2cells in a dose-dependent manner.compared with NG, HG (30mM,45mM,60mM) had significant difference(P<0.05,P<0.01); compared with HG (60mM), HG(30mM,45mM) had significant difference (P<0.05);compared with HG(30mM), HG(45mM) had no significant difference (P>0.05). Mannitol concentrations (30mM,45mM, corresponding to the osmotic pressure of high glucose, respectively) were incubated for HK-2cells for24h. Found that HM(30mM,45mM) had no significant inhibitory effect on cell viability of HK-2cells compared with NG (P>0.05); compared with HM(30mM), HM(45mM) had no significant difference (P>0.05).These results suggested that the toxic effect of high glucose on HK-2cells was dose-dependant, which had no significant correlation with the osmotic pressure. According to domestic and foreign research and dead cells too much will affect the experimental results, glucose concentrations(55mM to45mM) were subjected to further analysis.2. The role of NF-κB activation in HG-induced HK-2cell injuryExpression of IκBa and p65in HK-2cells following exposure to different glucose concentrations (5.5-45mM) for24h and were measured by Western blot. The results showed that hκBa protein expression decreased with increasing glucose concentration, compared with NG, HG(30mM,45mM) had significant difference (P<0.05, P<0.01) compared with HG(30mM),HG(45mM) had no significant difference (P>0.05).p65protein expression increased with increasing glucose concentration, compared with NG, HG(30mM,45mM) had significant difference (P<0.05,P<0.01); compared with HG(30mM), HG(45mM) had no significant difference (P>0.05).The results demonstrated that down-regulation of IκBα protein and up-regulation of p65protein in HG-induced HK-2cell injury was dose-dependant. Therefore, HG (45mM) was selected for future baicalein study.3. The effect of baicalein on cell viability of HK-2cells without any treatmentMTT assay showed that baicalein (25uM,50uM,100uM) had no significant inhibitory effect on cell viability of HK-2cells without any treatment compared with control group (P>0.05).Baicalein (200uM) had an significant inhibitory effect on cell viability of HK-2cells without any treatment compared with control group (P<0.05).The results suggested that baicalein (200uM) had toxicity to HK-2cells. Based on the experimental results, baicalein (25uM,50uM,100uM) was selected initially for further study.4. The effect of baicalein on NF-κB activation in HG-induced HK-2cells1) The inhibitory effect of baicalein on down-regulation of IκBα protein in HG-induced HK-2cells.Western blot assay showed that NF-κB specific inhibitor PDTC(100uM) had an significant inhibitory effect on down-regulation of IκBα protein compared with HG (P<0.01).Baicalein suppressed the down-regulation of IκBα protein in a dose-dependant manner, Baicalein(50uM,100uM) were significantly different compared with HG(P<0.05, P<0.01),Baicalein (25uM) showed no significant difference compared with HG (P>0.05)2) The inhibitory effect of baicalein on p65activation in HG-induced HK-2cells.Western blot assay showed that the p-p65/p65ratio was raised in HG-induced HK-2cells compared with NG (P<0.01); Baicalein(50uM,100uM) and PDTC(100uM) down-regulated the p-p65/p65ratio compared with HG (P<0.05,P<0.01); Baicalein (25uM) showed no significant difference compared with HG(P>0.05).The results implied that high glucose could activate p65to promote its translocation to nucleus through phosphorylation of p65; and baicalein inhibited p65activation in a dose-dependant manner.3) Baicalein inhibited HG-induced protein expression of IKKa in HK-2cells.Western blot assay showed HG treatment for24h markedly increased IKKa protein expression compared with NG (P<0.01);Baicalein(50uM,100uM) decreased IKKa protein expression compared with HG(P<0.05,P<0.01).The combination of these results implied that baicalein inhibited NF-κB pathway activation through IKKa-mediated IκBα degradation and p65phosphorylation in HG-induced HK-2cells.5. The effect of baicalein on inflammatory molecules (MCP-1, ICAM-1) in HG-induced HK-2cells.Western blot assay showed that HG treatment for24h markedly increased the MCP-1and ICAM-1protein expression compared with NG (P<0.01).Baicalein(50uM,100uM) and PDTC(100uM) decreased MCP-1and ICAM-1protein expression compared with HG (P<0.05, P<0.01)6. The effect of baicalein on extracellular matrix component Collagen Ⅳ in HG-induced HK-2cells.Western blot assay showed that HG treatment for24h markedly increased the Collagen Ⅳ protein expression compared with NG (P<0.01); Baicalein(50uM,100uM) and PDTC(100OuM) decreased Collagen IV protein expression compared with HG(P<0.05, P<0.01)Conclusion:1. The toxic effect of high glucose on HK-2cells was dose-dependant, which had no significant correlation with the osmotic pressure.IKKα/IκBα/p65signaling pathway was activated, its downstream inflammatory molecules (MCP-1, ICAM-1) were up-regulated, and extracellular matrix component Collagen Ⅳ was increased in HG-induced HK-2cell injury.2. Baicalein could suppress IKKα/IκBα/p65signaling pathway, down-regulated the protein expression of MCP-1、ICAM-1and Collagen Ⅳ in HG-induced HK-2injury. The results suggested that NF-κB activation took part in renal tubular epithelial cell injury induced by high glucose, and baicalein could inhibit NF-κB-mediated inflammatory response in HG-induced HK-2cell injury. Experimental research—the change of serum Fcγ RIIB/C in diabetic nephropathy and Analysis of TCM syndromesIn the first part of the study, we demonstrated that NF-kB signaling pathway involved in the development of DN. Previous studies had found that the proinflammatory immune receptors Fcγ Rs (Fc gamma receptors) could stimulate NF-kB activation in peripheral blood neutrophils, which implied that Fcγ Rs and NF-kB were closely related.Fcγ Rs is a constant region of immunoglobulin G (IgG) Fc receptors. In the latest study, Virginia proposed that activating FcyRs is required for initiating immuncomplex-mediated inflammation in the diabetic kidney, but limited in its response by inhibitory Fcγ RIIB.In addition, C-reactive protein (CRP) involved in the Fcγ Rs and NF-kB activation. Therefore, it might be some valuable to observe the change of Fcγ RIIB/C in diabetic nephropathy and its correlation with related indicators.Objective:1. To observe the change of serum Fcγ RIIB/C in diabetic nephropathy;2. To observe the change of serum hs-CRP in diabetic nephropathy;3. To observe the correlation of Fcγ RIIB/C with hs-CRP and other related indicators;4. To analyze syndrome distribution of traditional Chinese medicine (TCM);5. To analyze the correlation of TCM syndromes with hs-CRP.Methods:53cases of patients were divided into three groups according to the urinary albumin/creatinine ratio:①diabetes with normal albuminuria group (DM), urinary albumin/creatinine ratio<40mg/g, including8males,10females, aged55.44±14.51years;②diabetes with microalbuminuria group (DN1), urinary albumin/creatinine ratio of40to300mg/g including10males and8females, aged59.15±13.45years;③diabetes with clinical albuminuria group (DN2),urinary albumin/creatinine ratio>300mg/g, including8males and9females, aged62.60±8.73years. In addition,10cases of healthy subjects as normal control group (NC), including4males and6females, aged52.00±13.58years.The basic clinical information, such as age, height, weight, blood pressure, HbAlc, hs-CRP,HOMA-IR,TC,TG,LDL,HDL,Scr and so on,were recorded. At the same time, TCM syndrome types were analyzed. Serum Fcγ RIIB/C level was measured by enzyme-linked immunosorbent assay.Results:1. DN2was the oldest compared with NC (P<0.05).The duration of DN2and DN2were longer compared with DM (P<0.05).TC increased in DN2compared with DM (P<0.05). LDL decreased in NC,DM,DN2compared with DN1(P<0.05).HOMA-IR increased in DM,DN2compared with NC (P<0.05).There was not any difference of blood pressure,BMI,TG, HDL,Scr,HbA1c among three diabetic groups and NC (P>0.05).2. Serum Fey RIIB/C decreased in DM (18.06±5.34) compared with NC (25.28±8.96),DN1(26.75±9.15),DN2(24.09±8.39)(P<0.05, P<0.01),but there was no difference in NC,DN1,DN2(P>0.05).3. Serum hs-CRP increased in DM (2.06±1.60),DN1(3.21±2.20),DN2(1.90±2.14) compared with NC (P<0.05, P<0.01),but there was no difference in DM,DN1,DN2.4. Serum Fey RIIB/C had a weak negative correlation with Serum hs-CRP(r=-0.276, P=0.048). Serum Fcγ RIIB/C had no correlation with age, duration, blood pressure, BMI, TC, TG, LDL, HDL, Scr, HOMA-IR, urinary albumin/creatinine ratio.5. In the four deficiency syndromes of DN, syndrome of deficiency of Yin and Yang was absent.the proportion of syndrome of deficiency of Qi and Yin was noticeably higher than the other two (P<0.01).In the four accompanied symptoms of DN, blood stasis and damp-heat symptom took the initiative, blood stasis symptom was significantly more than the dampness-heat symptom (P<0.01).6. In the three deficiency syndromes of DN, Serum hs-CRP increased in syndrome of deficiency of Qi and Yin(3.84±1.93), syndrome of Yin deficiency and dry-heat(2.40±1.06), syndrome of Qi deficiency of spleen and kidney (2.13±0.90) compared with NC (0.86±0.94)(P<0.05, P<0.01). Serum hs-CRP increased in syndrome of deficiency of Qi and Yin compared with syndrome of Yin deficiency and dry-heat, and syndrome of Qi deficiency of spleen and kidney (P<0.05).in the accompanied symptoms of DN, Serum hs-CRP increased in blood stasis (1.83±1.65) and dampness-heat symptom (4.28±2.12) compared with NC (P<0.05, P<0.01), Serum hs-CRP increased in blood stasis symptom compared with dampness-heat symptom (P<0.05)Conelusion:1. Serum Fcγ RIIB/C did not change significantly in DN, which could not provide the basis for the diagnosis and treatment of DN;2. Patients of diabetic nephropathy were in micro-inflammatory state; 3. Serum Fcγ RIIB/C had a weak negative correlation with micro-inflammatory state;4. Qi and Yin deficiency, accompanied with blood stasis inhibition and dampness-heat was the basic pathogenesis in the early and middle stages of diabetic nephropathy;5. Serum hs-CRP was related with TCM syndromes, to some extent, which provided an objective basis for the Chinese medicine dialectical type.