Qingshen granule-medicated serum attenuates high glucose-induced epithelial-mesenchymal transition of HK-2 cells via inhibiting oxidative stress-mediated NF-κB signaling pathway*

Zhang Lei， Jin Hua， Wang Dong， Cheng Meng， Li Zhuo-ya， Wang Yi-ping△

（1Department of Nephrology，the First Affiliated Hospital of Anhui University of Chinese Medicine，Hefei 230031，China；2Department of Nephrology，Fanchang Hospital of Traditional Chinese Medicine，Wuhu 241200，China.E-mail：wypwyp54@aliyun.com）

［ABSTRACT］ AIM：To investigate whether Qingshen granules（QSG）-medicated serum inhibits oxidative stressmediated NF-κB signaling pathway and attenuates epithelial-mesenchymal transition（EMT）of human proximal tubule epithelial HK-2 cells induced by high glucose. METHODS：The active components in QSG were analyzed by HPLC. The HK-2 cells were randomly divided into control group，mannitol group，high glucose group，low-dose QSG group，medium-dose QSG group，high-dose QSG group and pyrrolidine dithiocarbamate（PDTC）group. The morphological changes of the cells were observed by inverted phase contrast microscopy. MTT assay was used to detect the cell viability. Flow cytometry was used to detect the content of reactive oxygen species（ROS）in HK-2 cells. ELISA was used to detect the content of malondial dehyde（MDA）and the activity of superoxide dismutase（SOD）. The DNA binding activity of nuclear factor-κB（NF-κB）p65 in HK-2 cells was detected by electrophoretic mobility-shift assay（EMSA）. The protein expression of NF-κB p65，phosphorylated inhibitor of kappa B alpha（p-IκBα），inhibitor of kappa B kinase alpha（IKKα），monocyte chemoattractant protein-1（MCP-1）and intercellular adhesion molecule-1（ICAM-1）in HK-2 cells was detected by Western blot. Immunofluorescence staining was used to detect NF-κB p65 and α-smooth mucle actin（α-SMA）protein expression in HK-2 cells.RESULTS：Chlorogenic acid，berberine hydrochloride，plantamajoside，6，7-dimethoxycoumarin，epiberberine，coptisine，lithospermicacid B，palmatine，leonurine hydrochloride，rheic acid and tanshinone IIA in QSG were preliminarily determined by HPLC. Compared with control group，the levels of ROS and MDA in HK-2 cells induced by high glucose increased（P＜0.05），while the activity of SOD decreased（P＜0.05）. The protein levels of NF-κB p65，p-IκBα，IKKα，MCP-1，ICAM-1 and α-SMA were increased（P＜0.05）. After intervened by QSG-medicated serum，the levels of ROS and MDA were decreased（P＜0.05），while the activity of SOD was increased（P＜0.05）. The protein levels of NF-κB p65，p-IκBα，IKKα，MCP-1，ICAM-1 and α-SMA were decreased（P＜0.05）. CONCLUSION：QSG-medicated serum inhibited oxidative stress-mediated NF-κB signaling pathway，thus attenuating the EMT of HK-2 cells induced by high glucose.

［KEY WORDS］ Qingshen granules；NF-κB signaling pathway；HK-2 cells；Epithelial-mesenchymal transition；Oxidative stress

Chronic kidney disease（CKD）has become a major threat to human health worldwide. It has high prevalence and mortality and imposes significant financial costs［1］. The incidence of CKD has been rising，and the prevalence of CKD was 10.8% in China in 2012［2］.The pathogenesis of CKD has been found to include renal fibrosis，which is the main pathological feature of glomerulosclerosis，renal interstitial fibrosis（RIF），and renal arterial sclerosis［3-4］. Renal tubular epithelialmesenchymal transition（EMT）is one of the key events for the incidence of RIF［5-6］. Nuclear factor-κB（NF-κB）signaling pathway is the main signaling pathway of immune and inflammatory responses. It is directly involved in the processes of fibrosis development and progression. Excessive reactive oxygen species （ROS）during oxidative stress in kidney activates NF-κB signaling and initiates inflammatory and immune responses，causing renal tubule EMT，which is followed by excessive deposition of extracellular matrix（ECM），eventually leading to RIF. Renal tubular epithelial cells undergo transition under high-glucose conditions，manifesting as the loss of the original phenotypes of epithelial cells and the acquisition of other new phenotypes that further convert into myofibroblasts and subsequently secrete ECM to promote fibrosis. Hence，this study used high glucose to induce oxidative stress in human proximal tubule epithelial HK-2 cells，thereby establishing an RIF model. A previous study has shown that Chinese herbal compound Qingshen granules（QSG）alleviates the clinical symptoms of CKD patients effectively［7］. Other studies have confirmed that QSG improve the progression of renal fibrosis in a rat model with unilateral ureteral obstruction by inhibiting the expression of NF-κB signaling mediated by oxidative stress［8］. This study further evaluated the effects of QSG on oxidation and the associated NF-κB signaling activation at the cellular level to provide a fully theoretical basis for its anti-renal fibrosis mechanism.

MATERIALS AND METHODS

1 Experimental animals and cells

Six healthy male Japanese white rabbits weighing（2.5±0.2）kg were purchased from the Experimental Animal Center of Anhui Medical University，Hefei Province，China，and produced by Yizheng Anlimao Biotechnology， Jiangsu， China ［permit number：SCXK（Jiangsu）2016-0005］. Experimental cells were normal human proximal tubular epithelial cell line（HK-2）purchased from the cell bank of China Center for Type Culture Collection（Wuhan，China）and the second to third passages for the experiments.

2 Experimental medications

The QSG used here contained raw rhubarb，Oldenlandia diffusa，Coptis chinensis，oriental wormwood，Poria cocos，Polyporus umbellate，Rhizoma alismatis，Rhizoma atractylodis，hyacinth bean，coxi seed，white cardamom，Plantago asiatica，Leonurus japonicas，andSalvia miltiorrhiza（red sage）. The medicinal materials were from approved origins and passed quality control，and the QSG was prepared at Anhui Provincial Hospital of Traditional Chinese Medicine，China. Each package contained 10 g granules and contained approximately 34 g raw herbs（Anhui medicine production number：BZ20080011；product batch number：20141023）. Pyrrolidine dithiocarbamate（PDTC；1 g per bottle）was purchased from Beyotime Biotechnology.

3 Experimental methods

3.1 Analysis of QSG by UPLC-PDAWaters Acquity ultra-performance liquid chromatography（UPLC）H-Class system consisting an autosampler and aquaternary pump，thermostatted column compartment and PDA（Waters，Milford，MA）was used for acquiring chromatograms. Luna Omega 1.6 μm C18（100 mm×2.1 mm）analytical column coupled with acolumn filter were used with column temperature set at 30 ℃. The mobile phase consists of acetonitrile（A）and 0.05%phosphoric acid water（B）. The gradient elution from 5% to 30% A in 0～14 min，30%～45% A in 14～18 min，45%～70%A in 18～19 min，70%～100%A in 19～20 min，100% A in 20～21 min，100-5% A in 21～22 min，5% A in 22～25 min. The flow rate was 0.25 mL/min and the injection volume was 1 μL. The PDA detector wavelength was set at 320 nm for acquiring chromatograms. Sample preparation：a total of 0.3 g QSG was dissolved into 10 mL with 75% ethanol. After 30 min of ultrasonic concussion，the filtrate which was used to detect，was filtered through 0.22 μm filtration membrane. Eleven reference substances were used for qualitative analysis，including chlorogenic acid（Batch No. DST180504-21），berberine hydrochloride（Batch No. DST180105-028），plantamajoside（Batch No.DST160930-007），6，7-dimethoxycoumarin（Batch No.DST180313-031），epiberberine（Batch No. DST170711-109），coptisine（Batch No. DST170711-003），lithospermic acid B（Batch No. DST180128-009），palmatine（Batch No. DST170711-047），leonurine hydrochloride（Batch No. DST170301-111），rheic acid（Batch No. DST170805-29），and tanshinone IIA（Batch No. DST180105-011），which were purchased from Desite Biotech Co.，Ltd. The compounds were vertified based on comparing individual peak retention times with that of the reference substances（Figure 1）.

Figure 1. HPLC fingerprint profile of QSG extract at 320 nm. A：names and chemical structure formulas of active ingredients；B：HPLC fingerprint profile of QSG extract at 320 nm. a：the chromatograms of mixed standard compounds；b：the chromatograms of chemical structure of the main active ingredients of QSG and others unknown.

3.2 Preparation of drug-containing serum in rabbitsThe experimental and feeding protocols were in accordance with National Health guidelines and were approved by the Anhui University of Chinese Medicine Institutional Animal Care and Use Committee. Rabbits were given QSG （10 g·kg-1·d-1） by gavage once per day for 10 consecutive days. In the absence of anesthesia，blood samples were collected from the heart of each rabbit using a syringe and stood at room temperature for two hours，followed by centrifuging at 6 000×gfor 10 min to collect the drug-containing serum from each rabbit. All sera were mixed thoroughly and immediately filtered using a 0.22-μm disposable microfilter on an ultra-clean bench to remove bacteria，followed by incubating at 56 ℃water bath for 30 min and storing the sample in-20 ℃for later experiments.

3.3 Cell culture and groupingHK-2 cells were incubated at 37 ℃in an incubator with 5% carbon dioxide and air-saturated humidity，followed by observing the cell growth under an inverted phase contrast microscopy. The cells were incubated to 80%～90% confluence and subsequently passaged to a ratio of 1∶2. Cells from the same generation were randomly divided into seven groups：（1）control group：DMEM/F12 medium+5.6 mmol/L glucose；（2）mannitol group：DMEM/F12 medium+24.5 mmol/L mannitol；（3） high glucose group：DMEM/F12 medium+30 mmol/L glucose；（4）low-dose QSG group：DMEM/F12 medium+30 mmol/L glucose+5% QSG drug-containing serum+15% blank rabbit serum；（5）medium-dose QSG group：DMEM/F12 medium+30 mmol/L glucose+10% QSG drug-containing serum+10% blank rabbit serum；（6）high-dose QSG group：DMEM/F12 medium+30 mmol/L glucose+20% QSG drug-containing serum+20% blank rabbit serum；（7）PDTC group：DMEM/F12 medium+30 mmol/L glucose+5 μmol/L PDTC（PDTC concentration was referred to a previous study［9］）. The morphological changes of HK-2 cells were observed by phase-contrast microscopy，and the cell viability was measured using 3-［4，5-dimethylthiazol-2-yl］-2，5-diphenyl tetrazolium bromide（MTT）assay.

3.4 Detection of ROS and malondialdehyde（MDA）content，and superoxide dismutase（SOD）activity in HK-2 cellsThe content of intracellular ROS was measured by flow cytometry，with the excitation and emission at 485 nm and 530 nm wavelengths，respectively. ELISA assay was used to detect MDA content and SOD activity. Each group of cells was digested with 0.25% trypsin and collected by cell centrifugation，followed by three rounds of PBS-washing，addition of 100 μL cell lysis buffer to lyse the cells，8 000 ×gcentrifugation for 10～15 min，and collection of the supernatant to detect the total protein concentration using Bradford protein assay kit. MDA contents and SOD activity were detected in accordance with the specific instructions of the reagent kits.

3.5 Detection of DNA binding activity of NK-κB p65 in HK-2 cells using electrophoretic mobilityshift assay（EMSA）After three rounds of PBS-washing，each group of cells was added with buffer and protease inhibitors. The mixture was subjected to highspeed vortexing and centrifugation for nuclear protein extraction according to the instructions included with the nuclear protein extraction kit. Bradford protein assay was used to detect the nuclear protein concentrations strictly in accordance with the manufacturer’s instructions of the EMSA kit. Conventional T4 oligonucleotide enzymatic method was used to label NF-κB and oligonucleotide probes with γ-32P-ATP，followed by purifying the labeled probes. Ten micrograms extracted nuclear proteins were reacted with 0.5 ng γ-32P-labeled oligonucleotide containing NF-κB binding sites at room temperature for 30 min. The DNA-protein complexes were analyzed by 4% non-denaturing polyacrylamide gel electrophoresis. After the electrophoresis，the gels were placed on 3 mm filter paper（80 ℃，2 h）to dry the gel and subsequently perform X-ray autoradiograph at -70 ℃for 36 h. The absorbance scanning was performed on the developed film using a UVP gel image scanning system. The absorbance value of each band was analyzed using the Image Jsoftware. The experimental results are here presented as the absorbance value of the experimental group/the absorbance value of the control group，which represented the probe-binding activity of NF-κB in each group of extracted nuclear proteins.

3.6 Western blot analysis of NF-κB p65，phosphorylated inhibitor of kappa B alpha（p-IκBα），inhibitor of kappa B kinase alpha（IKKα），monocyte chemoattractant protein-1（MCP-1）and intercellular adhesion molecule-1（ICAM-1）in HK-2 cellsThe cells in each group were seeded in three wells of the six-well plates and harvested at the density of 1×106cells/group，followed by three times of PBS-washing，adding cell lysis buffer to lyse the cells on ice for 30 min，centrifugation at 12 000×gand 4 ℃ for 20 min to collect supernatants. The total protein concentration in the supernatant was detected by bicinchoninic acid（BCA）assay according to the kit's instructions.Buffer was added to each supernatant sample to denature the protein，followed by separating the denatured protein in SDS-PAGE. The protein gels were transferred to polyvinylidene difluoride （PVDF） membranes，which were blocked by 5% skim milk at room temperature for 2 h. Each protein blot was washed in Tris-buffered saline（TBS）containing 0.05% Tween-20（TBST）for three times and incubated with the specific primary antibody overnight at 4 ℃. Dilution factors of primary antibodies used in this study were all 1∶1 000. After the TBST-washing，each protein blot was incubated with 1∶1 000 horseradish peroxidase（HRP）-labeled secondary antibodies at room temperature for 90 min. After the TBST-washing，enhanced chemiluminescence（ECL）reagent was used for protein blot exposure，and the signals were scanned using Bio-Rad Gel Imaging System. ImageJ software was used to analyze the integrated gray value of each positive band，and the corresponding value of β-actin in each lane was used as an internal reference to calculate the relative expression of each target protein.

3.7 NF-κB p65，α-smooth muscle actin（α-SMA）protein expression detected by immunofluorescenceThe cells in each group were seeded in three wells of the 24-well plates. After the cells grew into a monolayer，they were further cultured in DMEM/F12 medium containing 0.2% fetal bovine serum（FBS）for 24 h，followed by changing and incubating in the corresponding culture medium of each experimental group for 6 h. The cell slides were collected on schedule，washed three times in PBS，and subsequently fixed in 4% paraformaldehyde solution. Endogenous peroxidase activity of each cell sample was blocked using 30 mL/L hydrogen peroxide. Each cell slide was independently incubated with 1∶1 000 NF-κB p65 and 1∶1 000 α -SMA primary antibodies overnight at 4 ℃. After three times of PBS-washing，each cell slide was incubated with 1∶250 universal anti-IgG antibody-HRP polymer.After three rounds of washing on PBS，3，3′-diaminobenzidine（DAB），an HRP substrate，was used for color development of the cell slides，and hematoxylin dye was used for nuclear counterstaining. PBS was used to replace primary antibody as a blank control in each experiment. The immunofluorescence results were observed under a light microscope to randomly select five fields of vision under×100 magnification and calculate the percentage of positive cells（positive rate）using Image-Pro Plus software.

4 Statistical analysis

SPSS 17.0 software was used for statistical analysis. The data were presented as mean± standard deviation（mean±SD）. Comparison between groups was performed using one-way ANOVA. Data not normally distributed or with inequality of variance were analyzed using non-parametric tests.P＜0.05 was considered statistically significant.

RESULTS

1 Morphological changes of HK-2 cells observed under light microscope

The HK-2 cells in control group were pebble- or paving stone-shaped with blunt cell edges，strongly refractive，adherent，and dense，with neat arrangement.With the extension of high glucose stimulation，the HK-2 cells gradually elongated toward both ends，eventually taking on a long spindle or irregular shape，with radial edge，enlarged cell gaps，decreased refractive index and irregular cell arrangement（Figure 2）.

Figure 2. Morphological changes of HK-2 cells（scale bar=50 μm）. The HK-2 cells in control group were pebble- or paving stone-shaped with blunt cell edges，strongly refractive，adherent，and dense，with neat arrangement. The HK-2 cells stimulated by high glucose for 48 h gradually elongated toward both ends，eventually taking on a long spindle or irregular shape，with radial edge，enlarged cell gaps，decreased refractive index and irregular cell arrangement.

2 MTT assay for cell viability analysis

No change in cell viability was found between control group and mannitol group over time. However，the cell viability in high glucose group was continuously decreased with time and dropped to approximately 50% at 48 h. Therefore，we selected 48 h as the appropriate drug reaction time in the subsequent experiments. The cell viability in 10 and 15 μmol/L PDTC groups after 24 h of treatment was significantly lower than that in high glucose group. Therefore，we selected 5 μmol/L as the appropriate concentration of PDTC and studied the morphological changes of the cells after 48 h of treatment.

3 α-SMA protein expression

Percentage of the α-SMA-positive cells was significantly higher in high glucose group than that in control group（P＜0.05）. The percentages of the α-SMA-positive cells in different doses of QSG groups and PDTC group were significantly lower than that in high glucose group （P＜0.05）. Among different doses of QSG groups，low-dose QSG group had the lowest percentage of α-SMA-positive cells（Figure 3）.

Figure 3. Immunofluorescence staining of α-SMA in HK-2 cells of each group（scale bar=10 μm）. Mean±SD. n=3.*P＜0.05 vs control group；#P＜0.05 vs high glucose group；&P＜0.05 vs low-dose QSG group.

4 ROS and MDA content and SOD activity

The levels of ROS and MDA were significantly higher in high glucose group than those in control group（P＜0.05）. Compared with high glucose group，the ROS and MDA levels in different doses of QSG groups and PDTC group were significantly decreased （P＜0.05），and low-dose QSG group had the minimal ROS and MDA levels（P＜0.05）. The SOD activity in high glucose group was significantly lower than that in control group（P＜0.05）. The SOD activity in different doses of QSG groups and PDTC group was higher than that in high glucose group（P＜0.05），and low-dose QSG group showed the strongest SOD activity（P＜0.05）. See Figure 4.

5 NF-κB p65 DNA binding activity

The NF-κB p65-DNA binding activity in high glucose group and mannitol group was significantly higher than that in control group（P＜0.05）. The NF-κB p65-DNA binding activity in different doses of QSG groups and PDTC group was lower than that in high glucose group（P＜0.05）. The NF-κB p65-DNA binding activity in medium-dose QSG group was the lowest. See Figure 5.

6 NF-κB p65，p-IκBα，IKKα，MCP-1 and ICAM-1 protein levels

The protein levels of NF-κB p65，p-IκBα，IKKα，MCP-1，and ICAM-1 in high glucose group were significantly higher than those in control group（P＜0.05）.The protein levels of NF-κB p65，p-IκBα，IKKα，MCP-1 and ICAM-1 in different doses of QSG groups and PDTC group were significantly lower than those in high glucose group（P＜0.05）. Among the groups receiving different doses of QSG，low-dose QSG group had the lowest protein levels of p-IκBα，IKKα and ICAM-1（P＜0.05）. See Figure 6.

Figure 4. Comparisons of ROS and MDA content and SOD activity in HK-2 cells of different groups. A：quantification of ROS levels；B：detection of MDA by ELISA；C：detection of SOD by ELISA；D：flow cytometric detection of ROS. Mean±SD. n=3.*P＜0.05 vs control group；#P＜0.05 vs high glucose group；$P＜0.05 vs low-dose QSG group.

Figure 5. Changes of NF-κB p65 DNA binding activity in HK-2 cells of different groups. A：original strip of EMSA（1：negatice control reaction；2：control group；3：probe cold competitive reaction；4：cold competitive reaction of a mutant probe；5：super-shift reaction；6：mannitol group；7：high glucose group；8：low-dose QSG group；9：medium-dose QSG group；10：high-dose QSG group；11：PDTC group）；B：the absorbance value of each band analyzed by the ImageJ software.Mean±SD. n=3.*P＜0.05 vs control group；#P＜0.05 vs high glucose group；&P＜0.05 vs medium-dose QSG group.

7 NF-κB p65 expression

The percentage of the NF-κB p65-positive cells was significantly higher in high glucose group than that in control group（P＜0.05）. The percentages of the NF-κB p65-positive cells in different doses of QSG groups and PDTC group were significantly lower than that in high glucose group（P＜0.05）. Among different doses of QSG groups，low-dose QSG group had the lowest percentage of NF-κB p65-positive cells（Figure 7）.

Figure 6. The protein levels NF-κB p65，p-IκBα，IKKα，MCP-1，and ICAM-1 in HK-2 cells of different group. Mean±SD. n=3.*P＜0.05 vs control group；#P＜0.05 vs high glucose group；&P＜0.05 vs low-dose QSG group.

Figure 7. Immunofluorescence staining of NF-κB p65 in HK-2 cells of each group（scale bar=10 μm）. Mean±SD. n=3.*P＜0.05 vs control group；#P＜0.05 vs high glucose group；&P＜0.05 vs low-dose QSG group.

DISCUSSION

RIF is the main pathological change of CKD caused by various pathogenic factors and is the final stage of the advanced renal disease［10］. Its main features include an increase of myofibroblasts in renal interstitium and ECM deposition. Approximately 36% of newly formed myofibroblasts in RIF are derived from EMT［11］. During EMT，the renal tubular epithelial cells first manifest as loss of epithelial cell phenotypes and achieving mesenchymal features（e. g.，by reducing E-cadherin expression and increasing α-SMA expression to eventually convert to myofibroblasts）. At that time，the cell morphology changes from the original paving stone-shape to spindle-shape. This change in cell morphology is accompanied by the destruction of basement membrane structures，enhancement of viability，migration of the cells to the renal interstitium，and the secretion of a large amount of ECM，which led directly to the development and progression of tubulointerstitial fibrosis［12-13］. E-cadherin is an adhesion receptor found between cells of the same type and extensively presents in various epithelial cells to act as cytoskeleton. Reduced expression of E-cadherin can cause cells to lose polarity and become isolated from the surrounding cells. Currently，reduced E-cadherin expression has been considered as an early event of EMT. A study has shown that the degree of E-cadherin expression is negatively correlated with the severity of renal interstitial damage［14］.α-SMA is one of the hallmark proteins of myofibroblasts［15］. A previous study confirmed that α -SMA-expressing myofibroblasts are absent from normal renal interstitium in humans［16］. Despite the weak expression in other parts of blood vessels，α-SMA is only expressed in smooth-muscle-derived cells in normal kidneys［17-18］.In this way，E-cadherin and α-SMA are two of the most important markers that confirm the transition of HK-2 cells.

This study also showed significant morphological changes of HK-2 cells induced by high glucose. HK-2 cells in the normal group showed pebble- or paving stone-shape with blunt cell edges，strong refractive，adherent，dense，and neat arrangement. As high-glucose stimulation continues，the morphology of HK-2 cells gradually changes. The cells become elongated toward both ends，eventually becoming a long spindle or irregular shape，with radial edge and enlarged cell gaps；the refractive index decreased and irregular cell arrangement，which had the morphological features of fibroblasts. Results of immunofluorescence showed the protein expression of α -SMA was significantly increased in HK-2 cells in the high glucose group，which further confirmed that EMT in HK-2 cells was induced by high glucose levels and not associated with osmotic pressure.

Oxidative stress is an increased production of oxygen radicals or decreased abilities in their elimination in tissues or cells，resulting in oxidative damage caused by the accumulation of ROS in tissues or cells. ROS is the key to initiation of oxidative stress and also a direct indicator to reflect the level of oxidative stress level in the cells. Under normal physiological conditions，certain concentrations of ROS are essential to maintaining normal viability and activity of cells，allowing them to participate in cell proliferation，apoptosis，and other physiological progresses. However，under pathological conditions，elevation of ROS concentration can cause protein，lipid，and DNA peroxidation damages in the body［19］.High glucose can induce oxidative stress and massive production of ROS，causing oxidative damage in cells. Intracellular NADH/NAD+ratio increases in high-glucose environment to increase the electron leakage of respiratory chain electron transport via NADH or FADH2，thereby enhancing the ROS production. In addition，high glucose causes a decline in the expression and activity of antioxidative enzymes to lower the scavenge ability of free radicals to further increase ROS concentrations and accumulation，which causes further damage of the intracellular oxidative stress［20］. HK-2 cell line cultured in high glucose environments for 24～48 h showed a significant increase in ROS contents. In addition，the mitochondrial membrane potential and mtDNA contents of these HK-2 cells are reduced，which increases the rate of apoptosis. These results indicate that excessive formation of ROS activated by high glucose-induced and mitochondrial oxidative damage are involve in the pathological processes of HK-2 cells［21］.

MDA is a product after lipid peroxidation that can cause the aggregation of macromolecules（e. g.，proteins and nucleic acids）and has cytotoxicity. MDA content directly represents the rate or intensity of intracellular lipid peroxidation and indirectly reflects the degree of cell damage. In this way，MDA can serve as a sensitive indicator of the state of oxidative stress［22-23］.SOD，a major antioxidant enzyme，is the first line of defense to eliminate ROS during oxidative stress［24］.SOD activity directly reflects the scavenging ability of oxygen free radicals in the body. Therefore，MDA and SOD are often used as the indicators of oxidation and anti-oxidation.In this study，high glucose was used to induce oxidative stress in cells and establish a RIF-model. The experimental results showed that the intracellular ROS and MDA contents of the high glucose group to be significantly increased and SOD contents to be reduced. The viability of the high glucose group was significantly reduced，and the cells in the high glucose group changed until their morphology matched that of fibroblasts.These results indicated that high glucose induced oxidative stress in HK-2 cells and enhanced the development of EMT.

NF-κB，a transcription factor widely found in tissues and cells，has multi-directional effects and plays an important role in cell signal transmission，immunity and inflammatory responses. Under physiological conditions，NF-κB presents in dimer form in cytoplasm. In addition，the dimeric structure of NF-κB binds to IκB，which is inactive and cannot bind with DNA. However，under the stimulation of a variety of external factors（e. g.，oxidative stress，UV，and inflammatory cytokines）in living organism，NF-κB dissociates and activates IκB to transfer into nuclei and bind to a variety of gene-promoter-specific sequences，thereby regulating the expression of numerous cytokines and affecting in cell proliferation，apoptosis，and immune responses［25］. NF-κB is an intracellular target of oxidative and high-glucose stress［26］. Studies have shown that NF-κB regulates apoptosis bidirectionally.ROS levels determine the activation and transcription of NF-κB. Mild increases in ROS activate NF-κB to inhibit apoptosis，and pronounced increases of ROS prevent the activation of NF-κB and promote apoptosis［27-28］. In this study，high glucose levels induced oxidative stress in HK-2 cells. In addition，the NF-κB p65-DNA binding activity of the nuclear protein extract measured by EMSA showed that the NF-κB signaling was activated.Results of Western blot and immunofluorescence showed increased expression of the related proteins in NF-κB signaling，suggesting that intracellular oxidative stress may lead to inflammatory cytokine production through the activation of NF-κB signaling，causing further apoptosis. Activation of NF-κB upregulated the expression of the inflammatory molecule MCP-1，which may serve as a target to promote renal tubular inflammation and EMT progression of the renal tubular epithelial cells，leading to renal fibrosis.

This study showed that high glucose conditions induced oxidative stress in HK-2 cells to increase intracellular ROS levels，attacking macromolecular component， accumulating MDA content， and consuming SOD，which aggravated oxidative stress and cytotoxicity and caused cell damage. ROS，acting as a second signal molecule，also activated NF-κB signaling to increase the expression of MCP-1 and ICAM-1 inflammatory molecules to further induce NF- κB activation，down-regulate E-cadherin levels，up-regulate α-SMA，and induce changes in cell morphology，resulting in the formation of interstitial fibrosis. QSG drug containing serum enhanced the antioxidant capacity of cells by increasing SOD contents to reduce MDA production，eliminate oxygen free radicals，and inhibit the development of oxidative stress. In addition，it directly inhibit-ed the activation of NF-κB pathway，reduced the production of MCP-1 and ICAM-1 inflammatory cytokines，down-regulated α -SMA levels，prevented changes in cell phenotype，and gradually restored cell morphology to normal. It stopped the EMT process，lowered the incidence of RIF，and played a role in kidney protection.