Role of Connective Tissue Growth Factor in Extracellular Matrix Degradation in Renal Tubular Epithelial Cells

In order to investigate the effects of connective tissue growth factor （CTGF） antisense oligodeoxynucleotide （ODN） on plasminogen activator inhibitor-1 （PAI-1） expression in renal tubular cells induced by transforming growth factor β1 （TGF-β1） and to explore the role of CTGF in the degradation of renal extracellular matrix （ECM）, a human proximal tubular epithelial cell line （HKC） was cultured in vitro. Cationic lipid-mediated CTGF antisense ODN was transfected into HKC. After HKC were stimulated with TGF-β1 （5 μg/L）, the mRNA level of PAI-1 was detected by RT-PCR. Intracellular PAI-1 protein synthesis was assessed by flow cytometry. The secreted PAI-1 in the media was determined by Western blot. The results showed that TGF-β1 could induce tubular CTGF and PAI-1 mRNA expression. The PAI-1 mRNA expression induced by TGF-β1 was significantly inhibited by CTGF antisense ODN. CTGF antisense ODN also inhibited intracellular PAI-1 protein synthesis and lowered the levels of PAI-1 protein secreted into the media. It was concluded that CTGF might play a crucial role in the degradation of excessive ECM during tubulointerstitial fibrosis, and blocking the biological effect of CTGF may he a novel way in preventing renal fibrosis.

The Effect of Connective Tissue Growth Factor on Human Renal Tubular Epithelial Cell Transdifferentiation

To investigate the role of connective tissue growth factor (CTGF) in transdifferentiation of human renal tubular epithelial cell (HKC), in vitro cultured HKC cells were divided into 3 groups: negtive control, low dose CTGF-treated group (rh CTGF, 2.5 ng/ml) and high dose CTGF-treated (rhCTGF, 5.0 ng/ml). Then the expression of α-smooth muscle actin (α-SMA) were assessed by indirect immuno-fluorescence, and the percentage of α-SMA positive cells were assessed by flow cytometry. RT-PCR were also performed to examine the mRNA level of α-SMA. Upon the stimulation of different concentrations of rhCTGF, the expression of α-SMA were markedly stronger than that in negative controls. The percentages of α-SMA positive cells were significantly higher in the stimulated groups than that of negative controls (38.9 %, 65.5 % vs 2.4 %, P<0.01) .α-SMA mRNA levels were also up-regulated by the stimulation of rhCTGF (P<0.01). These results suggest that CTGF can promote the transdifferentiation of human renal tubular epithelial cells towards myofibroblast (Myo-F).

Experimental Study on Detached Renal Tubular Epithelial Cells in Urine of Nephropathia Epidemic Patients

To elucidate the pathogenesis of acute renal failure (ARF) with nephropathia epidemic (NE), provide experimental evidence for the new therapy to NE and observe the effects of Arg-Gly-Asp (RGD) peptides on adhesion of re-nal tubular epithelial cell (RTEC), urine specimens of patients were collected un-der sterile conditions. Detached RTECs were separated, cultured and identified.Hantan Virus antigen was determined by using indirect immunofluorescence method and effects of RGD on adhesion of RTECs was observed by subgroup counting as well as by flow cytometry. This study showed that: (1) sublethal RTECs existed in the urine of NE-ARF patients, which could be cultured in monolayer form; (2 ) there was NE antigen in RTECs; and (3) adhesion of RTECs could be inhibited by RGD.

Inhibition of Ubiquitin-specific Protease 4 Attenuates Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells via Transforming Growth Factor Beta Receptor Type Ⅰ

Objective Ubiquitin-specific protease 4(USP4)facilitates the development of transforming growth factor-beta 1(TGF-β1)-induced epithelial-mesenchymal transition(EMT)in various cancer cells.Moreover,EMT of renal tubular epithelial cells(RTECs)is required for the progression of renal interstitial fibrosis.However,the role of USP4 in EMT of RTECs remains unknown.The present study aimed to explore the effect of USP4 on the EMT of RTECs as well as the involved mechanism.Methods In established unilateral ureteral obstruction(UUO)rats and NRK-52E cells,immunohistochemistry and Western blot assays were performed.Results USP4 expression was increased significantly with obstruction time.In NRK-52E cells stimulated by TGF-β1,USP4 expression was increased in a time-dependent manner.In addition,USP4 silencing with specific siRNA indicated that USP4 protein was suppressed effectively.Meanwhile,USP4 siRNA treatment restored E-cadherin and weakened alpha smooth muscle actin(α-SMA)expression,indicating that USP4 may promote EMT.After treatment with USP4 siRNA and TGF-β1 for 24 h,the expression of TGF-β1 receptor type I(TβRI)was decreased.Conclusion USP4 promotes the EMT of RTECs through upregulating TβRI,thereby facilitating renal interstitial fibrosis.These findings may provide a potential target of USP4 in the treatment of renal fibrosis.

Comparative proteomic analysis of renal tubular epithelial cell injury caused by oxalic acid and calcium oxalate monohydrate

Objective To analyze and identify the differentially expressed proteins in human renal tubular epithelial ceils ( HK-2) after injury caused by oxalic acid and calcium oxalate monohydrate ( COM ) crystal,and to explore the potential role of renal tubular cell injury in kidney stone formation. Methods Normal HK-2 cells

Effect and mechanism of adrenomedullin on apoptosis of renal tubular epithelial cell in rats induced by renal ischemia reperfusion injury

Objective To investigate the effect and mechanism of adrenomedullin ( AM ) on apoptosis of renal tubular epithelial cell in rats induced by renal ischemia reperfusion injury. Methods Thirty-two Wistar rats were randomly divided into 4 groups: control group,IRI group, empty plasmid group and AM group. One week after re-

Decreased TRPM7 alleviates high glucose-induced renal tubular epithelial cell injury by inhibiting the HMGB1/TLR4 signaling pathway

Objective:To explore the regulatory mechanism of transient receptor potential melastatin-7(TRPM7)in high glucose-induced renal tubular epithelial cell injury.Methods:The expression of TRPM7 in the serum of diabetic nephropathy patients and high glucose-induced HK-2 cells was detected by RT-qPCR.Then,the TRPM7 interference vector was constructed,and the downstream high mobility group box 1(HMGB1)/Toll-like receptor 4(TLR4)signaling pathway proteins were detected.Next,in addition to interference with TRPM7 expression,overexpression of HMGB1 in high glucose-induced HK-2 cells was performed.Cell activity,apoptosis,oxidative stress levels,and inflammation levels were determined by CCK8,TUNEL,Western blotting,immunofluorescence and related kits.Results:TRPM7 expression was upregulated in the serum of diabetic nephropathy patients and high glucose-induced HK-2 cells.Interference with TRPM7 reduced cell damage,epithelial-mesenchymal transition,oxidative stress,and inflammatory response in high glucose-induced HK-2 cells via inhibiting the HMGB1/TLR4 signaling pathway.However,the effects induced by TRPM7 silencing were abrogated by HMGB1 overexpression.Conclusions:Decreased TRPM7 alleviates high glucose-induced renal tubular epithelial cell injury by inhibiting the HMGB1/TLR4 signaling pathway.Further animal experiments and clinical trials are warranted to verify its effect.

Chronic hepatitis B serum promotes apoptotic damage in human renal tubular cells

AIM： To investigate the effect of the serum of patients with chronic hepatitis B （CHB） on apoptosis of renal tubular epithelial cells in vitro and to study the role of hepatitis B virus （HBV） and transforming growth factor-β1 （TGF-β1） in the pathogenesis of hepatitis B virus associated glomerulonephritis （HBV-GN）. METHODS： The levels of serum TGF-β1 were measured by specific enzyme linked immunosorbent assay （ELISA） and HBV DNA was tested by polymerase chain reaction （PCR） in 44 patients with CHB ,and 20 healthy persons as the control. The normal human kidney proximal tubular cell （HK-2） was cultured together with the sera of healthy persons, CHB patients with HBV-DNA negative（20 cases） and HBV-DNA positive （24 cases） for up to 72 h. Apoptosis and Fas expression of the HK-2 were detected by flow cytometer. RESULTS： The apoptosis rate and Fas expression of HK-2 cells were significantly higher in HBV DNA positive serum group 19.01±5.85% and 17.58±8.35%, HBV DNA negative serum group 8.12±2.80% and 6.96 ± 2.76% than those in control group 4.25±0.65% and 2.33 ± 1.09%, respectively （P 〈 0.01）. The apoptosis rate and Fas expression of HK-2 in HBV DNA positive serum group was significantly higher than those in HBV DNA negative serum （P 〈 0.01）. Apoptosis rate of HK-2 cells in HBV DNA positive serum group was positively correlated with the level of HBV-DNA （r = 0.657）. The level of serum TGF-β1 in CHB group was 163.05 ± 91.35 μg/L, signifi- cantly higher as compared with 81.40 ± 40.75 μg/L in the control group （P 〈 0.01）.CONCLUSION： The serum of patients with chronic hepatitis B promotes apoptotic damage in human renal tubular cells by triggering a pathway of Fas up-regulation. HBV and TGF-β1 may play important roles in the mechanism of hepatitis B virus associated glomerulonephritis.

Construction of Bioartificial Renal Tubule Assist Device In Vitro and Its Function of Transporting Sodium and Glucose

To explore a new way of constructing bioartificial renal tubule assist device （RAD） in vitro and its function of transporting sodium （Na^＋） and glucose and to evaluate the application of atomic force microscope in the RAD construction, rat renal tubular epithelial cell line NRK-52E was cultured in vitro, seeded onto the outer surfaces of hollow fibers in a bioreactor, and then cultured for two weeks to construct RAD. Bioreactor hollow fibers without NRK-52E cells were used as control. The morphologies of attached cells were observed with scanning electron microscope, and the junctions of cells and polysulfone membrane were observed with atomic force microscope. Transportation of Na＋ and glucose was measured. Oubaine and phlorizin were used to inhibit the transporting property. The results showed that NRK-52E cells and polysulfone membrane were closely linked, as observed under atomic force microscope. After exposure to oubaine and phlorizin, transporting rates of Na^＋ and glucose were decreased significantly in the RAD group as compared with that in the control group （P〈0.01）. Furthermore, when the inhibitors were removed, transportation of Na^＋ and glucose was restored. It is concluded that a new RAD was constructed successfully in vitro, and it is able to selectively transport Na^＋ and glucose.

TRPC6 Knockout Alleviates Renal Fibrosis through PI3K/AKT/GSK3B Pathway

Objective Renal fibrosis is the ultimate pathway of various forms of acute and chronic kidney damage.Notably,the knockout of transient receptor potential channel 6(TRPC6)has shown promise in alleviating renal fibrosis.However,the regulatory impact of TRPC6 on renal fibrosis remains unclear.Methods In vivo,TRPC6 knockout(TRPC6−/−)mice and age-matched 129 SvEv(WT)mice underwent unilateral renal ischemia-reperfusion(uIR)injury surgery on the left renal pedicle or sham operation.Kidneys and serum were collected on days 7,14,21,and 28 after euthanasia.In vitro,primary tubular epithelial cells(PTECs)were isolated from TRPC6−/−and WT mice,followed by treatment with transforming growth factorβ1(TGFβ1)for 72 h.The anti-fibrotic effect of TRPC6−/−and the underlying mechanisms were assessed through hematoxylin-eosin staining,Masson staining,immunostaining,qRT-PCR,and Western blotting.Results Increased TRPC6 expression was observed in uIR mice and PTECs treated with TGFβ1.TRPC6−/−alleviated renal fibrosis by reducing the expression of fibrotic markers(Col-1,α-SMA,and vimentin),as well as decreasing the apoptosis and inflammation of PTECs during fibrotic progression both in vivo and in vitro.Additionally,we found that the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/glycogen synthase kinase 3 beta(GSK3β)signaling pathway,a pivotal player in renal fibrosis,was down-regulated following TRPC6 deletion.Conclusion These results suggest that the ablation of TRPC6 may mitigate renal fibrosis by inhibiting the apoptosis and inflammation of PTECs through down-regulation of the PI3K/AKT/GSK3βpathway.Targeting TRPC6 could be a novel therapeutic strategy for preventing chronic kidney disease.

Role of connective growth factor in plasminogen activator inhibitor-1 and fibronectin expression induced by transforming growth factor β1 in renal tubular cells

Background Connective tissue growth factor (CTGF) contributes greatly to renal tubulointerstitial fibrosis, which is the final event leading to end-stage renal failure. This study was designed to investigate the effects of CTGF antisense oligodeoxynucleotides (ODNs) on the expressions of plasminogen activator inhibitor-1 (PAI-1) and fibronectin in renal tubular cells induced by transforming growth factor β1 (TGF-β1) in addition to the role of CTGF in the accumulation and degradation of renal extracellular matrix (ECM).Methods A human proximal tubular epithelial cell line (HKC) was cultured in vitro. Cationic lipid-mediated CTGF antisense ODNs were transfected into HKC cells. After HKC cells were stimulated with TGF-β1 (5 μg/L), the mRNA levels of PAI-1 and fibronectin were measured by RT-PCR. Intracellular PAI-1 protein synthesis was assessed by flow cytometry. The secreted PAI-1 and fibronectin in the medium were determined by Western blot and ELISA, respectively.Results TGF-β1 was found to induce tubular CTGF, PAI-1, and fibronectin mRNA expression. PAI-1 and fibronectin mRNA expression induced by TGF-β1 was significantly inhibited by CTGF antisense ODNs. CTGF antisense ODNs also inhibited intracellular PAI-1 protein synthesis and lowered the levels of PAI-1 and fibronectin protein secreted into the medium.Conclusions CTGF may play a crucial role in the accumulation and degradation of excessive ECM during tubulointerstitial fibrosis, and transfecting CTGF antisense ODNs may be an effective way to prevent renal fibrosis.

Preventive effect of Shenkang injection against high glucose-induced senescence of renal tubular cells

Shenkang injection (SKI) is a classic prescription composed of Radix Astragali, rhubarb, Astragalus, Safflower, and Salvia. This treatment was approved by the State Food and Drug Administration of China in 1999 for treatment of chronic kidney diseases based on good efficacy and safety. This study aimed to investigate the protective effect of SKI against high glucose (HG)-induced renal tubular cell senescence and its underlying mechanism. Primary renal proximal tubule epithelial cells were cultured in (1) control medium (control group), medium containing 5 mmol/L glucose;(2) mannitol medium (mannitol group), medium containing 5 mmol/L glucose, and 25 mmol/L mannitol;(3) HG medium (HG group) containing 30 mmol/L glucose;(4) SKI treatment at high (200 mg/L), medium (100 mg/L), or low (50 mg/L) concentration in HG medium (HG+ SKI group);or (5) 200 mg/L SKI treatment in control medium (control+ SKI group) for 72 h. HG-induced senescent cells showed the emergence of senescence associated heterochromatin foci, up-regulation of P16INK4 and cyclin D1, increased senescence-associated β-galactosidase activity, and elevated expression of membrane decoy receptor 2. SKI treatment potently prevented these changes in a dose-independent manner. SKI treatment prevented HG-induced up-regulation of pro-senescence molecule mammalian target of rapamycin and p66Shc and down-regulation of anti-senescence molecules klotho, sirt1, and peroxisome proliferator-activated receptor-g in renal tubular epithelial cells. SKI may be a novel strategy for protecting against HG-induced renal tubular cell senescence in treatment of diabetic nephropathy.

Vitamin C Attenuates Hemorrhagic Shock-induced Dendritic Cell-specific Intercellular Adhesion Molecule 3-grabbing Nonintegrin Expression in Tubular Epithelial Cells and Renal Injury in Rats

Background： The expression of dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin （DC-SIGN） in renal tubular epithelial cells has been thought to be highly correlated with the occurrence of several kidney diseases, but whether it takes place in renal tissues during hemorrhagic shock （HS） is unknown. The present study airned to investigate this phenomenon and the inhibitory effect of Vitamin C （VitC）. Methods： A Sprague Dawley rat HS model was established in vivo in this study. The expression level and location of DC-SIGN were observed in kidneys. Also, the degree of histological damage, the concentrations of tumor necrosis factor-or and interleukin-6 in the renal tissues, and the serum concentration of blood urea nitrogen and creatinine at different times （2-24 h） alter HS （six rats in each group）, with or without VitC treatment belbre resuscitation, were evaluated. Results： HS induced DC-SIGN expression in rat tubular epithelial cells. The proinflarnmatory cytokine concentration, histological damage scores, and functional injury of kidneys had increased. All these phenornena induced by HS were relieved when the rats were treated with VitC before resuscitation. Conclusions： The results of the present study illustrated that HS could induce tubular epithelial cells expressing DC-SIGN, and the levels of proinflarnmatory cytokines in the kidney tissues improved correspondingly. The results also indicated that VitC could suppress the DC-SIGN expression in the tubular epithelial cells induced by HS and alleviate the inflammation and functional injury in the kidney.

Renal progenitors: Roles in kidney disease and regeneration

Kidney disease is a devastating condition that affects millions of people worldwide, and its prevalence predicted to significantly increase. The kidney is complex organ encompassing many diverse cell type organized in a elaborate tissue architecture, makin regeneration a challenging feat. In recent years, there ha been a surge in the field of stem cell research to develo regenerative therapies for various organ systems. Here we review some recent progressions in characterizing th role of renal progenitors in development, regeneration and kidney disease in mammals. We also discuss how the zebrafish provides a unique experimental anima model that can provide a greater molecular and genet understanding of renal progenitors, which may contribut to the development of potential regenerative therapies fo human renal afflictions.

枸杞多糖通过激活Nrf2/HO-1通路保护HK-2细胞氧化损伤的作用

目的分析枸杞多糖(LBP)干预对氧化损伤的人肾小管上皮细胞(HK-2)内的核因子E2相关因子2(Nrf2)/血红素氧合酶-1(HO-1)通路蛋白表达的变化,探索LBP拮抗HK-2细胞氧化损伤的分子机制。方法采用过氧化氢诱导HK-2细胞制备氧化损伤细胞模型,HK-2被分成4组:正常组、LBP组、氧化损伤组及LBP干预组。观察4组细胞的长势和形态变化;细胞活力测定法比较每组细胞的存活率;比色法分析氧化产物丙二醛(malondialdehyde,MDA)含量以及超氧化物歧化酶(superoxide dismutase,SOD)和谷胱甘肽过氧化酶(GSH-Px)的水平;免疫印迹技术检测细胞内Nrf2/HO-1通路蛋白Nrf2、HO-1的相对水平。结果正常组和LBP组相比,两组的细胞长势、形态和存活率,细胞产生MDA的量、SOD和GSH-Px的水平,通路蛋白Nrf2、HO-1的相对水平均无明显差异(均P>0.05);氧化损伤组和正常组相比,细胞皱缩变圆并脱落、贴壁细胞变少,存活率减少、MDA量增加、SOD和GSH-Px降低,通路蛋白Nrf2、HO-1的相对值下降(均P<0.05);LBP干预组和氧化损伤组相比,细胞长势、形状恢复、贴壁细胞较多,存活率增加、MDA量减少、SOD和GSH-Px升高,通路蛋白Nrf2、HO-1的相对值升高(均P<0.05)。结论枸杞多糖能通过激活Nrf2/HO-1通路提高HK-2细胞的抗氧化酶活性达到减轻细胞氧化损伤的目的。

姜黄素调控TLR4/NF-κB和NRF2/HO-1信号通路改善草酸钙晶体诱导的小鼠肾损伤

目的探讨姜黄素(curcumin,CUR)对乙醛酸诱导的小鼠肾结石形成模型中肾损伤的保护作用及其机制。方法通过连续腹腔注射乙醛酸,建立小鼠肾结石形成模型。以一水草酸钙(calcium oxalate monohydrate,COM)诱导HK-2细胞作为体外模型。小鼠模型经CUR作用后,测定肾小管损伤、炎症细胞因子水平,研究CUR对小鼠肾结石的保护作用;CUR对COM诱导HK-2作用后,检测细胞活力及炎症因子;Western blot检测小鼠肾组织和HK-2细胞Toll样受体4(TLR4)/核因子κB(NF-κB)和核因子红血球相关因子2(NRF2)/血红素加氧酶1(HO-1)通路相关蛋白;为进一步探讨CUR对TLR4/NF-κB和NRF2/HO-1通路的调控作用,采用NRF2抑制剂ML385和TLR4激动剂CCL-34分别作用于COM诱导的HK-2细胞,以进行功能增益和功能丧失检测。结果CUR改善小鼠肾结石形成模型损伤,抑制炎症和抗氧化作用;促进COM诱导HK-2细胞的活力,抑制炎症因子的表达。CUR抑制TLR4/NF-κB通路中蛋白的表达,促使NRF2从细胞质转移到细胞核,并促进HO-1的表达。ML385和CCL-34分别抵消CUR对COM诱导HK-2细胞抗炎作用的影响。结论CUR通过调控小鼠肾结石形成模型TLR4/NF-κB和NRF2/HO-1通路改善肾损伤。

过表达线粒体蛋白磷酸酶2C对人肾小管上皮细胞转录组的影响

背景:课题组前期研究发现相较于野生型小鼠,线粒体蛋白磷酸酶2C(protein phosphatase 2Cm,PP2Cm)基因缺失小鼠明显发生肾功能衰竭的症状,因此推测PP2Cm可能在肾脏纤维化发展过程中发挥重要保护作用,然而其分子机制尚不明确。目的:探究PP2Cm基因对于人肾小管上皮细胞转录组的影响。方法:培养人肾小管上皮细胞,用质粒将PP2Cm基因转染进入人肾小管上皮细胞,荧光定量PCR实验和Western blot实验检测细胞中PP2Cm的表达,随后分别提取细胞RNA进行转录组测序,寻找转染组和对照组之间的差异性表达基因,利用生物信息学方法进一步对所得的差异基因进行GO分析和KEGG分析。结果与结论:通过测序分析,与未转染空白细胞相比,在转染PP2Cm基因的人肾小管上皮细胞中存在796个差异性表达基因,其中553个下调基因,243个上调基因,GO分析结果显示,上调表达的基因显著富集在细胞生物合成过程、蛋白质翻译、内在凋亡信号通路等;下调表达的基因显著富集在内皮细胞增殖、细胞黏附等信号通路;KEGG分析结果显示,显著上调表达的基因富集在氨基酸代谢、生物合成等代谢相关信号通路;下调表达的基因显著富集在泛酸和辅酶A的生物合成等信号通路。结果表明,PP2Cm过表达可以影响肾小管上皮细胞的一系列生物学过程相关的多条信号通路,可能在氨基酸代谢、生物合成等代谢相关信号通路中起重要作用。

MCP-1基因对脓毒症急性肾损伤发生的作用研究

目的研究单核细胞趋化蛋白-1(MCP-1)基因对脓毒症急性肾损伤(AKI)发生的作用。方法选取2022年9月-2023年9月新疆维吾尔自治区人民医院重症医学科收治的50例脓毒症AKI患者作为AKI组,纳入同期50例健康受试者作为对照组。分别采集全部受试者清晨空腹静脉血5 mL,采用ELISA法测定AKI患者及健康人群血清中MCP-1的表达情况;通过原代培养肾小管上皮细胞,利用CK14和CK18抗体进行细胞免疫荧光鉴定,过表达和干扰MCP-1基因,利用CCK8细胞增殖检测试剂盒和RT-qPCR检测肾小管上皮细胞增殖情况和肿瘤坏死因子α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素6(IL-6)、白细胞介素10(IL-10)、γ-干扰素(IFN-γ)炎性因子表达的变化。结果与对照组相比,AKI组患者外周血中和尿液中的MCP-1表达量显著升高(P均<0.05);通过细胞免疫荧光鉴定,选择上皮细胞标志物CK14和CK18,原代培养24 h,90%以上的细胞表达细胞标志物CK18,约84%的细胞表达CK14;与NC组相比,siRNA组在24、48 h细胞数量增加(P均<0.05);与MCP-1组相比,siRNA组在24、48、72 h的细胞数量增加(P均<0.05);NC组的IL-1β、IL-6、INF-γ因子的表达水平随时间推移逐渐升高,MCP-1组的IL-1β、IL-6、IL-10、INF-γ和TNF-α因子的表达水平随时间推移逐渐升高,siRNA组的IL-1β、IL-6、INF-γ因子表达水平随时间推移无明显升高趋势。结论MCP-1基因在脓毒症急性肾损伤的发病机制中可能发挥重要作用,该基因可能通过调节IL-1β等炎性细胞因子的表达来抑制肾小管上皮细胞的生长和增殖,从而参与脓毒症患者的AKI过程。

山柰酚通过改善肾小管上皮细胞的氧化应激与炎症反应减轻1型糖尿病小鼠肾损伤

背景糖尿病肾病是糖尿病最严重的并发症之一,长期高血糖会导致全身性的氧化应激和低度炎症状态。山柰酚是一种天然的黄酮类化合物,具有出众的抗炎和抗氧化的能力,可能会对糖尿病肾病的治疗有一定作用。目的研究山柰酚对糖尿病肾病小鼠肾损伤的治疗作用及其机制。方法18只6~8周龄FVB小鼠随机分为对照组、糖尿病模型组和山柰酚灌胃组,每组6只,并通过对模型组和山柰酚组腹腔注射链脲佐菌素构建1型糖尿病小鼠模型。模型建立后,治疗组灌胃山柰酚[70 mg/(kg·d)],对照组与模型组灌胃等量对照溶剂CMC-Na,16周后处死小鼠。病理染色观察小鼠肾病理损伤;qPCR和Western blot检测小鼠肾组织内炎症因子mRNA和氧化应激相关蛋白的表达;通过流式细胞术检测小鼠肾内巨噬细胞的数量和种类。体外建立高糖诱导肾小管上皮细胞(HK2)损伤模型,使用CCK-8试剂盒检测不同浓度山柰酚对HK2的活性影响,检测肾小管上皮细胞活性氧生成;qPCR和Western blot检测HK2内炎症因子mRNA和氧化应激相关蛋白以及炎症激活通路蛋白p38的表达。结果动物实验结果显示,与糖尿病模型组相比,经过灌胃山柰酚治疗后,肾病理损伤得到改善,肾小球系膜增生减少,足突融合减少;肾组织内白细胞介素(interleukin,IL)-1β、IL-6、肿瘤坏死因子α等炎症因子转录水平降低(P<0.01);NADPH氧化酶4表达降低(P<0.01);肾内炎性巨噬细胞数量减少,肾炎症微环境得到改善。细胞实验结果显示,山柰酚能够抑制高糖诱导下HK2的活性氧产生,改善氧化应激相关蛋白酶的表达(P<0.05);降低相关炎症因子mRNA的表达(P<0.01),且降低了p38的磷酸化(P<0.05)。结论山柰酚可能通过HO-1/p38通路减轻糖尿病肾小管上皮细胞炎症因子的分泌,增加相关氧化还原酶的表达,进而减轻糖尿病导致的肾损伤,保护肾功能。