Application of ellagic acid in preparation of medicine for preventing acute kidney injury induced by aristolochic acid I
阅读说明：本技术 鞣花酸在制备预防由马兜铃酸i诱导的急性肾损伤的药物中的应用 (Application of ellagic acid in preparation of medicine for preventing acute kidney injury induced by aristolochic acid I ) 是由 邓旭坤 付千 舒广文 于 2020-12-17 设计创作，主要内容包括：本发明涉及鞣花酸的医药新用途技术领域,具体公开了鞣花酸在制备预防马兜铃酸I诱导的急性肾损伤的药物中的应用。本发明通过建立体内外马兜铃酸I诱导的急性肾损伤模型研究鞣花酸的作用。本发明首次发现,马兜铃酸I诱导的HK-2细胞损伤中,NF-κB信号通路被异常激活,激活的NF-κB进一步活化NLRP3炎性小体,进而导致HK-2细胞的损伤；鞣花酸可通过抑制NF-κB/NLRP3炎性小体的激活来减缓马兜铃酸I诱导的肾细胞死亡,从而对马兜铃酸I诱导的体内体外急性肾损伤起到一定的保护作用。本发明拓展了鞣花酸的医药用途,为开发治疗马兜铃酸肾毒性的药物提供研究基础及理论依据。(The invention relates to the technical field of new medical application of ellagic acid, and particularly discloses application of ellagic acid in preparation of a medicine for preventing acute kidney injury induced by aristolochic acid I. The invention researches the action of ellagic acid by establishing an in vivo and in vitro aristolochic acid I induced acute kidney injury model. The invention discovers for the first time that in HK-2 cell damage induced by aristolochic acid I, the NF-kB signal path is abnormally activated, and the activated NF-kB further activates NLRP3 inflammasome, thereby causing the damage of HK-2 cells; the ellagic acid can slow down aristolochic acid I-induced renal cell death by inhibiting the activation of NF-kB/NLRP 3 inflammasome, thereby playing a certain role in protecting aristolochic acid I-induced acute kidney injury in vitro and in vivo. The invention expands the medical application of the ellagic acid and provides a research basis and a theoretical basis for developing the medicine for treating the aristolochic acid nephrotoxicity.)
1. Application of ellagic acid in preparing medicine for preventing acute kidney injury induced by aristolochic acid I is provided.
2. Application of ellagic acid in preparing medicine for preventing acute HK-2 cell injury induced by aristolochic acid I is provided.
3. Use of ellagic acid in the manufacture of a medicament for inhibiting activation of NF- κ B/NLRP3 inflammasome induced by aristolochic acid I.
Technical Field
The invention relates to the technical field of new medical application of ellagic acid, in particular to application of ellagic acid in preparing a medicine for preventing acute kidney injury induced by aristolochic acid I.
Background
Aristolochic Acids (AAs) are natural polyphenolic compounds obtained from aristolochia plants and various natural herbs and have anti-inflammatory, anti-tumor, anti-infective, anti-fertility, blood pressure regulating, etc. effects (wandexing et al, 2014). Therefore, traditional Chinese medicines containing AAs are commonly used for treating various diseases such as hepatitis, pneumonia, snake bite, stroke, arthritis, gout, and cardiovascular system diseases. It has been found that excessive intake of AA is a major cause of kidney damage, and is characterized by direct damage to tubular epithelial cells, a progressive tubulointerstitial nephritis that can cause apoptosis and necrosis of tubular epithelial cells, and clinical manifestations of irreversible renal function deterioration, such as uncontrolled, which can ultimately lead to end-stage renal disease (ESRD) and urothelial malignancy (XieX C, et., 2017). In recent years, although AA is controlled in many countries, several aristolochic acid-containing medicinal materials are prohibited in the "chinese pharmacopoeia" and the dosage thereof is strictly controlled, some AA-containing Chinese herbal medicines and preparations thereof which are not collected in the "chinese pharmacopoeia" in folk or in some areas are still used by doctors, such as wooly datchmanspipe, pinellia ternate cough syrup and the like, and acute kidney injury can be caused by taking a small dose of AA-containing Chinese patent medicine by renal patients (ZhangHM, oral, 2018). However, the clinical efficacy of AA is not negligible, with coverage for coronary heart disease as large as cough as small (ZengY, et., 2017). Although tubular epithelial cells are considered to be the main target of action of AA nephrotoxicity, their exact pathogenesis is not clear. Therefore, further studies on the molecular mechanisms underlying the onset of AA nephrotoxicity are still urgently needed in order to develop new therapeutic strategies.
The nuclear factor kB (NF- κ B), a transcription regulator, has a great role in regulating the expression of many pro-inflammatory factors and apoptosis (KimBW, et. NF- κ B may be activated by a variety of stimulants, such as Lipopolysaccharide (LPS), the inflammatory factor tumor necrosis factor α (TNF- α), free radicals, etc. (Royal et al, 2019). Normally, both subunits (dimers) I κ B α (NF- κ B inhibitor) and NF- κ Bp65, p50 are present in the cytoplasm in an inactive state (HuangB, et., 2010). When an organism is subjected to some stimulation, I κ B kinase is activated (IKK), the activated IKK causes phosphorylation and ubiquitination of I κ B α protein, and then I κ B α protein is degraded, so that a dimer formed by NF- κ Bp65 and p50 is released, and then two subunits of NF- κ B are activated from an inactivated state and transferred from cytoplasm into nucleus, and bind to corresponding inflammation-related genes to initiate transcription of inflammatory cytokines to induce inflammation (BrasierA R, 2006). Thus, it is thought that inhibition of NF-. kappa.B activation may contribute to the reduction of persistent inflammation.
The NLRP3 inflammasome is a multiprotein complex that produces pro-inflammatory cytokines and consists of three parts: the sensor molecules NLRP3, apoptosis-related plaque spotting protein (ASC) and effector protease Caspase 1(Caspase-1) (ChenF, et., 2018). Upon activation of the NLRP3 inflammasome, the PYD domain of the NLRP3 receptor protein interacts with the CYD domain of the ASC adaptor protein, which in turn interacts with the CARD domain of the effector protein Caspase-1, ASC recruits Caspase-1 and induces its activation, and activated Caspase-1 can process IL-1 β and IL-18 into mature forms and induce its release, causing an inflammatory response and cell death (LiuS, et., 2018). There are studies reporting that activation of NF- κ B induces transcription of NLRP3, as well as other key proinflammatory factors, including the proinflammatory factor IL-1 β (mangann sj, et., 2018). NLRP3 inflammasome has been shown to play a key role in kidney disease, such as inhibition of renal inflammatory responses and ischemia reperfusion injury (Chang a, et al, 2014).
Ellagic Acid (EA) has various pharmacological effects, such as antioxidant, antitumor, antiinflammatory, antibacterial, antiallergic, etc. Ellagic acid is widely applied in the aspects of food, cosmetics, medicines and the like, and is a hot medicine for domestic and foreign research. Ellagic acid is known to have antioxidant, anti-apoptotic and anti-inflammatory effects on diabetic nephropathy (Zhoushhong et al, 2016) and cisplatin (Dengxahun et al, 2019) induced kidney damage. However, it is not clear whether ellagic acid has a protective effect on aristolochic acid nephrotoxicity.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide the application of ellagic acid in preparing a medicament for preventing acute kidney injury induced by aristolochic acid I.
In order to achieve the above purpose, the invention adopts the following technical scheme:
application of ellagic acid in preparing medicine for preventing acute kidney injury induced by aristolochic acid I is provided.
The HK-2 cells are taken as research objects, and the HK-2 cells are induced to be damaged by giving AAI so as to establish a model of kidney cell damage in vitro. Detecting the influence of AAI and EA with different concentrations on the activity of HK-2 cells and the protection effect of EA on HK-2 cell death induced by AAI by adopting an MTT method, and screening out the optimal administration dose; lactate Dehydrogenase (LDH) kit to evaluate the effect of EA on HK-2 cytotoxicity caused by AAI; microscopic photographing to observe the cell morphology, and observing the influence of EA on the apoptosis of HK-2 cells by Hoechst33258 fluorescent staining; detecting the expression of nuclear factor kappa B (NF-kappa B) signal channel and NLRP3 inflammasome-related protein under the action of AAI at different concentrations and different times and after EA pretreatment by a protein immunoblotting method (WesternBlot); the PCR technology detects the transcription expression level of NLRP3 inflammasome related gene, and clarifies the in vitro kidney protection function and mechanism of EA. The above results indicate that EA alleviates AAI-induced inflammation and apoptosis of HK-2 cells by inhibiting the activation of the NF-. kappa.B/NLRP 3 inflammasome.
In order to further determine the protection effect and the molecular mechanism of EA on AAI-induced kidney injury, the invention establishes an in-vivo kidney injury model by injecting 10mg/kg of AAI into the abdominal cavity. 40 male Kunming mice were randomly divided into a Control group (Control), a model group (AAI10mg/kg), an ellagic acid low dose group (AAI10mg/kg + EA10mg/kg) and an ellagic acid high dose group (AAI10mg/kg + EA30mg/kg), 10 mice per group were administered at a dose volume of 0.1mL/10 g. The control mice were gavaged daily with normal saline for 12 days. The mice of the ellagic acid administration group are continuously gavaged with ellagic acid with different doses for 12d every day, and after the mice are administered for 2h on the 7 th day, AAI is intraperitoneally injected for 5d every day by the model group and the ellagic acid administration group, so as to establish an acute kidney injury model. And (3) after the mice are fasted and not forbidden for 12 hours after the last administration, the mice are treated. Weighing kidney and calculating kidney index, detecting creatinine (Cr), urea nitrogen enzyme (BUN) and interleukin-1 beta (IL-beta) and tumor necrosis factor-alpha (TNF-alpha) level in serum; detecting the concentration of protein in urine by a Coomassie brilliant blue method; h & E staining and Masson staining for renal pathological changes and fibrosis; detecting the apoptosis condition of the kidney cells by Hoechst33258 and TUNEL fluorescent staining; the expression of proteins such as I kappa B alpha, P-I kappa B alpha, NF-kappa Bp65, P-NF-kappa Bp65, Nuclear-NF-kappa B, NLRP3, ASC, Caspase-1, IL-1 beta, Bax, Bcl-2, cleared-Caspase-3 and the like is detected by a WesternBlot method and an immunohistochemical method (IHC); the PCR technology is used for further verifying genes such as NLRP3, ASC, Caspase-1, IL-1 beta and the like. The results show that EA has good protection effect on mouse kidney injury caused by AAI, and the protection effect of EA is probably related to reduction of inflammatory response and inhibition of renal tubular epithelial cell apoptosis.
Compared with the prior art, the method has the advantages and beneficial effects as follows:
the action of ellagic acid is researched by establishing an in vivo and in vitro aristolochic acid I induced acute kidney injury model. The invention discovers for the first time that in HK-2 cell damage induced by aristolochic acid I, the NF-kB signal path is abnormally activated, and the activated NF-kB further activates NLRP3 inflammasome, thereby causing the damage of HK-2 cells; the ellagic acid can slow down aristolochic acid I-induced renal cell death by inhibiting the activation of NF-kB/NLRP 3 inflammasome, thereby playing a certain role in protecting aristolochic acid I-induced acute kidney injury in vitro and in vivo. The invention expands the medical application of the ellagic acid and provides a research basis and a theoretical basis for developing the medicine for treating the aristolochic acid nephrotoxicity.
Drawings
FIG. 1 is a graph showing the effect of different concentrations of AAI on the survival rate of HK-2 cells;
FIG. 2 is a schematic representation of the effect of different concentrations of EA on the viability of HK-2 cells;
FIG. 3 is a graphical representation of the effect of EA on AAI-induced survival of HK-2 cells;
FIG. 4 is a graph showing the improvement of EA in cytotoxicity induced by AAI (A: the effect of EA on the levels of LDH in AAI-induced HK-2 cells; B: the effect of EA on the morphology of AAI-induced HK-2 cells);
FIG. 5 is a graph showing the effect of EA on AAI-induced apoptosis of HK-2 cells (A: Hoechst33258, B: Hoechst33258 fluorescence intensity);
FIG. 6-1 is a schematic representation of the effect of AAI on the NF-. kappa.B/NLRP 3 signaling pathway (A: the effect of different concentrations of AAI on NF-. kappa.Bp 65 and IkappaB α and their phosphorylated protein expression levels after acting on HK-2 cells; B: the effect of different concentrations of AAI on NLRP3 inflammasome-related mRNA expression after acting on HK-2 cells; C: the effect of different times of AAI on NF-. kappa.Bp 65 and IkappaB α and their phosphorylated protein expression levels after acting on HK-2 cells; D: the effect of different times of AAI on NLRP3 inflammasome-related mRNA expression after acting on HK-2 cells);
FIG. 6-2 is a schematic illustration of the effect of AAI on the NF-. kappa.B/NLRP 3 signal pathway;
FIG. 7 is a schematic representation of the effect of EA on the NF-. kappa.B/NLRP 3 signaling pathway in AAI-induced HK-2 cells (A: the effect of EA on NF-. kappa.Bp 65 and IkappaB α and their phosphorylated protein expression levels in AAI-induced HK-2 cells; B: the effect of EA on NLRP3 inflammatory-corpuscle-associated protein expression in AAI-induced HK-2 cells; C: the effect of EA on NLRP3 inflammatory-corpuscle-associated mRNA expression in AAI-induced HK-2 cells);
FIG. 8 is a graph showing the effect of ellagic acid on kidney function in AAI-induced kidney injury mice (A: kidney index; B: urine protein concentration; C: urea nitrogen (BUN) level; D: creatinine (Cr) level);
FIG. 9 is a schematic illustration of the effect of ellagic acid on AAI-induced kidney histopathology in kidney injured mice;
FIG. 10 is a graph showing the effect of ellagic acid on AAI-induced inflammatory factors in kidney-injured mice (A: Tumor Necrosis Factor (TNF) - α level; B: Interleukin (IL) -1 β level; C: mRNA expression levels of TNF- α and IL-1 β; D: mRNA expression levels of TNF- α and IL-1 β are quantified);
FIG. 11 is a schematic representation of the effect of ellagic acid on AAI-induced apoptosis in kidney injured mice (A: Hoechst 33258; B: TUNEL fluorescent staining);
FIG. 12 is a graph showing the effect of ellagic acid on AAI-induced apoptosis-related proteins in kidney-injured mice (A: western blot of Bax and Bcl-2; B: protein quantification of Bax and Bcl-2; FIG. C: immunohistochemistry of Bax and Bcl-2);
FIG. 13 is a schematic representation of the effect of ellagic acid on the AAI-induced NF-. kappa.B/NLRP 3 signaling pathway (A: immunoblotting and protein quantitation of NF-. kappa.B/IkB α; B: immunoblotting and protein quantitation of NLRP3 inflammasome and downstream proteins. C: mRNA expression levels and quantitation of NLRP3 inflammasome and IL-1 β).
Detailed Description
In order to illustrate the invention more clearly, the applicant now further describes the invention in relation to preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
In the following examples:
1. experimental Material
Ellagic acid (EA, purity more than or equal to 98%, Chenguan optical technology of Baoji city); aristolochic acid I (AAI, purity not less than 99%, Jiangsu Yongjian technology pharmaceutical Co., Ltd.); hoechst33258 staining solution (bio-organism), Tunel staining solution, BCA protein concentration determination kit, and hypersensitivity ECL luminescence kit (Biyuntian biotechnology research institute); tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta) kit (Wuhanbei Rhine biotech GmbH); trizol lysate (Nanjing Nozam Biotech, Inc.); reverse transcription/amplification kit (Tiangen Biochemical technology Co., Ltd.); LDH, urea nitrogen (BUN) and creatinine (Cr) detection kit (Nanjing institute of biological research); MEM medium (Wuhan Punuo Seiki Life technologies, Ltd.).
2. Test cells and animals
Human renal tubule epithelium (HK-2) cell line was purchased from the university of Wuhan's book reservation center and stored by the laboratory for self-passage. 40 SPF-grade KM mice with the weight of 18-22g are provided by Hubei disease prevention and control center, and the production license number of experimental animals is as follows: SCXK (jaw) 2016-. Mice were given sufficient water and feed daily and the experiment was started 1 week later.
3. Preparation of drugs and reagents
(1) Preparation of ellagic acid solution
Precisely weighing appropriate amount of ellagic acid powder, adding a small amount of DMSO, ultrasonic dissolving to make its concentration be 10mM, filtering the solution with sterile microporous membrane on a super clean bench, storing the filtered solution at 4 deg.C in refrigerator, and diluting with MEM medium to desired concentration before experiment.
(2) Preparation of aristolochic acid I
Precisely weighing appropriate amount of aristolochic acid I powder, addingAdding a proper amount of 1 wt% NaHCO3The solution was dissolved in warm water under heating to a concentration of 1mg/mL, filtered through a sterile microporous membrane on a clean bench, and the filtered solution was stored in a refrigerator at 4 ℃ and diluted to the desired concentration with MEM medium before the experiment.
(3) Preparation of complete Medium
According to the MEM medium: fetal bovine serum: diabesin (penicillin, streptomycin) ═ 9: 1: the mixture was prepared at a ratio (volume ratio) of 0.1 and stored in a refrigerator at 4 ℃.
(4) Preparation of frozen stock solution
According to the MEM medium: fetal bovine serum: DMSO ═ 5.5: 4: 0.5 (volume ratio), and is used when the cells are frozen.
(5) Preparation of 5mg/mLMTT
0.5g of MTT was weighed out and dissolved in 100ml of PBS solution, filtered through a 0.22 μm filter membrane, and stored in a refrigerator at-20 ℃ in the dark.
(6) Preparation of TBST solution
24.2g of Tris and 80g of NaCl were taken and the volume was adjusted to 1L, and the pH was adjusted to 7.6 with HCl. When in use, 900mL of double distilled water and 1mL of Tween 20 are added into 100mL of the mixture.
(7) 5% confining liquid (5% skimmed milk powder)
1g of skim milk powder was weighed and dissolved in 20ml of LTBST solution, and stirred to dissolve it sufficiently.
(8)0.01M phosphate buffer (PBS solution)
Weighing 8g of NaCl, 0.2g of KCl and 1.44g of Na2HPO4And 0.24gKH2PO4Dissolving in 800mL of distilled water, adjusting the pH value of the solution to 7.4 by using HCl, and finally adding distilled water to a constant volume of 1L.
(9) Preparation of 1 Xelectrophoresis buffer
Weighing 1.5g of Tris, 7.2g of glycine and 0.5g of SDS into a beaker, adding 300mL of distilled water, performing ultrasonic treatment to fully dissolve the Tris, the glycine and the SDS, then diluting to 500mL, and storing the obtained solution at room temperature.
(10) Preparation of 1 Xtransmembrane buffer
Weighing 5.8g of Tris and 2.9g of glycine into a beaker, adding 200mL of methanol and 500mL of distilled water, carrying out ultrasonic treatment to fully dissolve the Tris and the glycine, then carrying out constant volume to 1000mL, and storing the obtained solution at room temperature.
The experimental materials not described are also common commercial products.
Example 1 protective Effect of ellagic acid on Aristolochic acid I-induced HK-2 cell injury
1. Cell culture
HK-2 cells were cultured in MEM complete medium containing 10% fetal bovine serum, penicillin 100U/mL and streptomycin 100U/mL, and placed at 37 ℃ in a 5% CO atmosphere2Culturing in a constant temperature incubator.
Detection of cell viability by MTT method
Taking cells in logarithmic growth phase, re-suspending and inoculating the cells in a 96-well plate, wherein the cell density of each well is 1 multiplied by 1045% CO2Incubate at 37 ℃ for 24 h. Then adding AAI with different concentrations of 0, 5, 10, 20 and 40 mu g/mL respectively, and incubating for 24h or 48 h; similarly, EA with different concentrations of 0, 2.5, 5, 10, 20 and 40 μ M is added respectively for 24 h. Screening out AAI and EA with optimal concentration according to the cell viability; the cells were treated according to the above procedure and divided into blank Control group (Control), AAI group (adding 20. mu.g/mLAAI for 24h), and AAI + EA low, medium and high three dose groups (adding 10, 20 and 40. mu.M EA for 2h, adding AAI and continuing the culture for 24 h). After the culture, 10 mu of LMTT solution is added into each hole, and the culture is continued for 4 hours. The culture medium was discarded, 150. mu.L of DMSO was added to each well, and the mixture was shaken on a shaker at low speed for 10 min. The absorbance value (OD) of each well at 490nm was measured on a microplate reader, 5 parallel wells were set for each group, and the repetition was repeated 3 times, and the cell viability was calculated as (measurement well OD value-blank well OD value)/(control well OD value-blank well OD value) × 100%.
3. Cellular morphological analysis
Taking cells in logarithmic growth phase, digesting and centrifuging by pancreatin, and then re-suspending and inoculating the cells in a 96-well plate, wherein the cell density of each well is 1 multiplied by 1055% CO2After incubation at 37 ℃ for 24h, different concentrations of EA (20. mu.M, 40. mu.M) were added for 2h, followed by addition of AAI at a final concentration of 20. mu.g/mL for 24h, the cells were observed for morphological changes under an inverted microscope and photographed.
LDH detection
HK-2 cells in a logarithmic growth phase are inoculated in a 12-well plate and incubated for 24h, then the cells are divided into a blank Control group (Control), an AAI group (AAI20 mu g/mL acts for 24h), and two dosage groups of low AAI + EA (EA20 mu M and 40 mu M act for 2h respectively, then AAI with the final concentration of 20 mu g/mL is added for continuous culture for 24h), three parallel groups are arranged in each group, cell culture solution supernatant is collected after the culture is finished, and the LDH activity is detected according to the kit instructions.
Detection of apoptosis by Hoechst33258 staining method
Cells were seeded in 12-well plates (2.5X 10)5seed/mL) for 24 hours, adding EA (20 mu M, 40 mu M) with different concentrations for treatment for 2 hours, and adding AAI with the final concentration of 20 mu g/mL for stimulation for 24 hours in each group; 0.5mL of Hoechst33258 stain was added to each well and incubated for 30min, and the Hoechst33258 stain solution was aspirated, then washed 3 times with PBS for 5min each, observed under a fluorescent microscope and photographed.
WesternBolt assay for intracellular protein expression
Preparing two 6-well plates of cells, one of which is subjected to the grouping treatment of the cells according to the method in item "4"; after 24 hours of incubation, the cells in the other 6-well plate are divided into a blank Control group (Control), an NF-kB inhibitor group (Bay10 mu M) AAI group (AAI20 mu g/mL acts for 24 hours) and an AAI + Bay group (10 mu M Bay acts for 4 hours, then AAI with the final concentration of 20 mu g/mL is added for continuous culture for 24 hours), three parallel groups are arranged in each group, cell supernatant is collected after the culture is finished, and the supernatant is discarded after 12000g centrifugation for 5 minutes at low temperature; washing with PBS for three times, adding protein lysate (RIPA/PMSF 100:1), scraping the bottom of a six-well plate with a cell scraper lightly, performing lysis on ice for 15min, transferring the lysate to 1.5mLEP, centrifuging at 4 ℃ and 12000g for 5min, carefully sucking the supernatant into a clean EP tube, adding 1/4 volumes of protein loading buffer, mixing uniformly, and boiling the protein for 5min to denature the protein. Proteins were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to PVDF membranes. Non-specific binding was blocked by incubating the transferred PVDF membrane with 5% skim milk powder at room temperature. Washed three times with TBST and incubated with p-I κ B α (1:2000), I κ B α (1:1500), p-NF- κ Bp65(1:1000), NF- κ Bp65(1:1000), NLRP3(1:500), ASC (1:1000), Caspase-1(1:1000), GSDMD (1:1000), L-1 β (1:1000) and GAPDH (1:1000) for 2h at room temperature. After four washes with TBST, the PVDF membrane was incubated with goat anti-rabbit or goat anti-mouse secondary antibody (1:1000) for 1h at room temperature. After TBST membrane washing, the membranes were washed with BeyoECL PlusA and B in a 1:1 mix well for color development and ImageJ analysis software quantitates and analyzes the density of protein bands.
RNA extraction
HK-2 cells in logarithmic growth phase were seeded in 6-well plates at 5X 10 per well5Individual cells, the cells were grouped according to the method in item "4". Collecting cell supernatant, centrifuging at 12000g at low temperature for 5min, discarding the supernatant, collecting precipitated cells and cells in a six-hole plate, washing the cells and the cells in the six-hole plate with PBS for three times, adding 1mL of Trizol lysate into each hole to blow the cells, transferring the lysate into a 1.5mL centrifuge tube, repeatedly blowing the lysate into the centrifuge tube by a pipette gun until no obvious particle sample exists, and standing the iceberg for 5 min. Adding 200 μ L chloroform, shaking into emulsion, standing at 4 deg.C for 5min, and centrifuging at 12000g4 deg.C for 15 min. Taking out, carefully absorbing the upper aqueous phase into a new EP tube, adding equal volume of precooled isopropanol, turning upside down, mixing uniformly, standing at 4 ℃ for 10min, and centrifuging at 12000g4 ℃ for 10 min. Carefully discard the supernatant and add 1ml of pre-cooled 75% ethanol, wash the tube lid and tube wall thoroughly, flick the tube bottom, allow the precipitate to suspend, and stand for 5 min. Centrifuging at 12000g4 deg.C for 5min, discarding supernatant, drying for 3min, adding 40 μ L EPC water to dissolve precipitate, collecting 1 μ L RNA, detecting OD values at 260nm and 280nm on detector, and calculating OD260/OD280And the rest is stored at-80 ℃, and the operations are carried out under the condition of no enzyme. The RNA extracted above was immediately reverse transcribed and amplified with reference to the kit instructions, and the resulting cDNA was stored in a-80 ℃ freezer for subsequent detection.
8. Data statistics and analysis
The experimental results are all as followsAll data are plotted using graphpadprism5.0(GraphPad Software, LaJolla, CA, USA) Software. Differences between groups were statistically analyzed by one-way analysis of variance (ANOVA), P<0.05 the difference was considered statistically significant. And (4) carrying out quantitative treatment and Western-felt strip gray scale analysis on the fluorescence result graph by using Image J software.
9. Results of the experiment
1) Effect of different concentrations of AAI on HK-2 cell survival
The MTT method is adopted to detect the cell survival rates of AAI with different concentrations at 24h and 48h respectively. As shown in FIG. 1, the cell survival rate showed a downward trend with the increase of AAI concentration and time, at 24h, the cell survival rate was 70% at AAI concentration of 20. mu.g/mL, and at a concentration higher than 20. mu.g/mL, the cell survival rate was about 65%; at 48h, when the AAI concentration is 5 mug/mL, the cell survival rate is about 70%, when the AAI concentration is higher than 5 mug/mL, the cell viability is reduced to below 50%, and the optimal concentration and time of the AAI are determined to be 20 mug/mL for 24h through integrating the time and the dosage.
2) Effect of different concentrations of EA on HK-2 cell viability
To determine whether EA had an effect on HK-2 cells in vitro, the effect of EA on cell viability was examined using the MTT method and was found (see fig. 2): after 24h of EA treatment at concentrations of 0-40. mu.M, the viability of HK-2 cells was essentially unaffected. Concentrations of EA of 10, 20 and 40 μ M were therefore selected for subsequent experiments.
3) Effect of EA on AAI-induced survival of HK-2 cells
Based on the results in FIGS. 1 and 2, applicants chose a concentration of AAI of 20 μ g/mL for 24h and EA concentrations of 10, 20 and 40 μ M to examine the effect of EA on the survival of AAI-induced HK-2 cells. As shown in fig. 3: applicants found that EA had some relief from cell damage induced by AAI compared to the AAI group, where the relief from damaged cells was significant at EA concentrations of 20 and 40 μ M, and therefore 20 and 40 μ M of EA was selected for later cell experiments.
4) EA amelioration of AAI-induced cytotoxicity
LDH is an important indicator of cell death caused by damaged cell membranes. As shown in fig. 4, applicants found from the cell morphology and LDH detection results: compared with the blank group, the LDH level of the AAI group is obviously increased, the cells die in a large amount, and the LDH level and the cell morphology are obviously reduced and improved after EA pretreatment, which indicates that EA has a protective effect on HK-2 cytotoxicity induced by AAI.
5) Effect of EA on AAI-induced apoptosis of HK-2 cells
Hoechst33258 fluorescent staining is a classical apoptosis detection method. As shown in FIG. 5, applicants found that the nuclei of the control group were normally blue, without blue fluorescent nuclei, indicating that the control group was not apoptotic. Compared with the control group, the cells in the AAI group are densely stained in a broken block shape, and a large amount of blue fluorescence indicates apoptosis. While in the EA pretreatment group, blue fluorescence gradually decreased, and apoptosis was significantly improved.
6) Effect of AAI on NF- κ B/NLRP3 Signal Path
There are studies reporting activation of the NLRP3 inflammatory bodies in AAI-induced HK-2 cells. A number of studies have shown that NF- κ B can activate NLRP3 inflammasome and its downstream inflammatory factors. However, whether AAI can activate the NF-. kappa.B signaling pathway has yet to be investigated. Thus, this experiment investigated the state of the NF-. kappa.B/NLRP 3 signaling pathway in AAI-induced HK-2 cells. As shown in fig. 6-1: compared with a blank control group, after AAI treatment of HK-2 cells at different concentrations and different action time, the phosphorylation levels of NF-kappa Bp65 and Ikappa B alpha protein are increased in a time-dependent and dose-dependent manner, and NLRP3 inflammatory corpuscle is activated in a time-dependent and dose-dependent manner, which is shown in that the mRNA expression levels of NLRP3, ASC, Caspase-1 and IL-1 beta which is a downstream inflammation-related factor are obviously increased. These data indicate that NF-. kappa.B signaling pathway and NLRP3 inflammasome are abnormally activated in AAI-induced HK-2 cells.
To further determine whether aberrant activation of the NLRP3 inflammasome is associated with NF-. kappa.B signaling pathway, this experiment examined protein expression of NF-. kappa.B and NLRP3 inflammasome in AAI-induced HK-2 cells by blocking the NF-. kappa.B signaling pathway using NF-. kappa.B inhibitors. As shown in fig. 6-2: compared with a blank control group, after 20 mu g/mL of AAI acts on HK-2 cells for 24 hours, the protein expression levels of p-NF-kappa B, NLRP3, Caspase-1 and IL-1 beta are consistent with the results detected before; compared with the AAI group, the phosphorylation expression of NF-kB is obviously blocked after the NF-kB inhibitor (10 mu MBay) is added, and meanwhile, the protein expression levels of NLRP3, Caspase-1 and IL-1 beta are also obviously reduced by inhibition. These results indicate that aberrant activation of the NF-. kappa.B signaling pathway in AAI-induced HK-2 cells further activates downstream NLRP3 inflammasome.
7) Effect of EA on AAI-induced NF-. kappa.B/NLRP 3 Signaling pathway in HK-2 cells
The experimental research finds that EA has a certain protective effect on the injury of HK-2 cells induced by AAI, and the NF-kB/NLRP 3 signal channel is activated in the HK-2 cell injury induced by AAI. Based on the above findings, this experiment further analyzed the molecular mechanisms of EA-induced HK-2 cell damage by AAI. Expression of NF-kB/IkB alpha related protein and NLRP3 inflammasome as well as protein expression and mRNA expression levels of downstream GSDMD and IL-1 beta are analyzed by Western-blot and PCR. As shown in fig. 7: after AAI administration, the phosphorylation levels of NF-kappa Bp65 and I kappa B alpha protein are obviously increased, the protein levels of NLRP3 inflammasome components (NLRP3, ASC and caspase-1) and GSDMD and IL-1 beta downstream of the inflammatory corpuscle components are also obviously increased, and the mRNA expression levels of NLRP3 inflammasome and IL-1 beta are also increased. EA pretreatment inhibited NF- κ Bp65 and Iκ B α protein phosphorylation and NLRP3 inflammasome-associated protein high expression to different extents compared to the AAI group. These results indicate that the protective effects of EA on AAI-induced renal inflammation are associated with inhibiting activation of the NF-. kappa.B/NLRP 3 signaling pathway.
Example 2 protective Effect of ellagic acid on Aristolochic acid I induced acute Kidney injury in mice
1. Establishment of animal model
40 male Kunming mice were randomly divided into a Control group (Control), a model group (AAI10mg/kg), an ellagic acid low dose group (AAI10mg/kg + EA10mg/kg) and an ellagic acid high dose group (AAI10mg/kg + EA30mg/kg), and 10 mice were administered at a dose volume of 0.1mL/10g per group. The control mice were gavaged daily with normal saline for 12 days. The mice of the ellagic acid administration group are continuously gavaged with ellagic acid with different doses for 12d every day, and after the mice are administered for 2h on the 7 th day, AAI is intraperitoneally injected for 5d every day by the model group and the ellagic acid administration group, so as to establish an acute kidney injury model. Feeding mice for the last time, fasting for 12h without water supply, collecting urine of the mice, taking eyeballs, taking blood, centrifuging at 3000rpm to obtain serum, and preserving at-80 ℃ for biochemical detection; the kidneys on both sides of the mouse were removed, washed with pre-cooled physiological saline, wiped dry, weighed and recorded (all procedures were performed on ice), the left kidney was placed in a tissue fixative for histopathology and the like, and the right kidney was stored at-80 ℃ for subsequent research of various indexes. Measurement of Kidney index and urine protein
After 12h of the last administration, the weight of the mouse is weighed and recorded, the urine of the mouse is collected, the kidney of the mouse is dissected and quickly cleaned in ice physiological saline, the weight of the kidney of the mouse is recorded, and the visceral organ index of the mouse is calculated as follows: organ index is organ weight/final body weight × 100. Urine protein concentration was measured by the Bradford method.
2. Determination of renal function index
The mouse was bled from the eyeball, the blood was left at room temperature for 1h and then centrifuged at 3000rpm for 10min, and the serum was separated in a clean EP tube. The content of creatinine (Cr) and urea nitrogen (BUN) in the serum was determined according to the kit instructions, and the remaining serum was stored in a-80 ℃ freezer for subsequent studies.
3. Determination of content of inflammatory factors TNF-alpha and IL-1 beta in serum
Serum obtained by centrifugation in the steps is used for detecting the levels of tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta). And (3) balancing the kit at room temperature for 20min according to the ELISA kit specification, preparing the required solution in advance for later use, and diluting the standard substance according to the experimental operation. Gradually adding sample, washing, sealing, adding enzyme labeled antibody, developing, etc. to the sample to be tested and the standard substance, determining OD value on an enzyme labeling instrument within 30min after reaction termination, drawing labeled yeast according to the kit instructions and calculating the content.
4. Determination of GSH, T-SOD and MDA content in kidney tissue
Repeatedly washing the right kidney of the mouse in ice physiological saline, and weighing the kidney tissue (g): preparing 10% homogenate with the volume of physiological saline (mL) being 1: 9, centrifuging at 3000 r/min for 10min, and collecting supernatant to determine the contents of T-SOD, MDA and GSH in kidney tissue according to the operation method of each test box.
5. Histopathological morphological examination of the kidney
Kidneys were fixed in 10% neutral formalin, gradually dehydrated with low to high concentration alcohol, and embedded in paraffin. The embedded wax blocks are fixed on a slicer and cut into slices with a thickness of 4-5 μm. Staining was performed according to standard hematoxylin-eosin (HE) and Masson kit instructions followed by addition of neutral gum seals, observation under a 200 x microscope, and photography.
Hoechst33258 staining
The wax block was processed as described in step 5 above, the paraffin embedded sections were dewaxed and hydrated conventionally, the water on the section surface was wiped off, a small amount of Hoechst33258 staining solution was added dropwise to cover the sample, and the stained nuclei were observed under a fluorescent microscope and photographed.
Detection of apoptosis by TUNEL method
Taking a paraffin section of the kidney tissue, drying the section at 65 ℃ for 1min, then carrying out conventional dewaxing hydration, gently wiping off distilled water on the surface of the section, digesting with proteinase K at 37 ℃ for 30min, washing with PBS for 3 times, washing for 5min each time, adding Tunel reaction liquid after cell permeation, and carrying out operation according to the kit instructions. After washing with PBS, staining was performed with reference to DAPI kit, and observation was performed under a fluorescent microscope.
8. Immunohistochemical analysis
Paraffin blocks of kidney tissue were cut into 4-5 μm thick sections, subjected to conventional deparaffinization hydration, antigen retrieval, washed, blocked and then added dropwise with NLRP3(1: 200), ASC (1: 200), Caspase-1(1: 100), IL-1 β (1: 200), Bax (1: 200) and Bcl-2 (1: 200) antibodies, incubated overnight at 4 ℃, rewarming for 10min, washed three times with PBS and then incubated for 30min with secondary antibody. Staining the slices with DAB and staining with hematoxylin, returning water to blue for 10min, dehydrating conventionally, sealing with neutral gum, observing under microscope, and taking pictures.
9. Extraction of renal tissue protein and WesternBolt detection
Weighing a proper amount of kidney tissues, putting the kidney tissues into a prepared tissue lysate (RIPA/PMSF is 100:1), homogenizing by a homogenizer to extract total proteins of the kidney tissues (the whole experiment operation is carried out in icebergs), carrying out ice-bath lysis for 30min, carrying out centrifugation for 5min at 4 ℃, 12000g, carefully sucking a supernatant into a clean EP tube, adding 5 Xloading buffer solution (the lysate: buffer is 4:1), and boiling the proteins for 5min to denature the proteins after uniform mixing. Proteins were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to PVDF membranes. Non-specific binding was blocked by incubating the transferred PVDF membrane with 5% skim milk powder at room temperature. Washed three times with TBST and incubated for 2h at room temperature with primary antibodies p-I κ B α (1:2000), I κ B α (1:1500), p-NF- κ Bp65(1:1000), NF- κ Bp65(1:1000), NLRP3(1:500), ASC (1:1000), Caspase-1(1:1000), GSDMD (1:1000), L-1 β (1:1000), cl-Caspase-3(1:1000), Bax (1:1000), Bcl-2 (1:500), GAPDH (1:1000), and the like. After four washes with TBST, the membranes were incubated with goat anti-rabbit or goat anti-mouse secondary antibodies (1:1000) for 1h at room temperature. After washing the membrane with TBST, the membranes were washed with BeyoECLPlusA and solution B at a ratio of 1:1 mix well for color development and ImageJ analysis software quantitates and analyzes the density of protein bands.
10. Extraction of RNA from renal tissue
Weighing 50mg of the fresh mouse kidney tissue treated in the step 1 into a 1.5mL lep tube, adding 1mL of precooled Trizol lysate, homogenizing by a homogenizer placed on ice in advance, and performing ice bath lysis on the obtained homogenate for 15 min. Adding 200 μ L chloroform, shaking into emulsion, standing at 4 deg.C for 5min, and centrifuging at 12000g4 deg.C for 15 min. Taking out, carefully absorbing the upper aqueous phase into a new EP tube, adding equal volume of precooled isopropanol, turning upside down, mixing uniformly, standing at 4 ℃ for 10min, and centrifuging at 12000g4 ℃ for 10 min. Carefully discard the supernatant and add 1ml of pre-cooled 75% ethanol, wash the tube lid and tube wall thoroughly, flick the tube bottom, allow the precipitate to suspend, and stand for 5 min. Centrifuging at 12000g4 deg.C for 5min, discarding supernatant, drying for 3min, adding 40 μ L EPC water to dissolve precipitate, collecting 1 μ L RNA, detecting 260nm and 280nm OD values on detector, and calculating OD260/OD280The remainder were stored at-80 ℃ (the entire RNA extraction process was performed in the absence of enzymes). The RNA extracted above was reverse transcribed and amplified with reference to the kit instructions, and the resulting cDNA was stored in a-80 ℃ freezer for subsequent detection.
11. Results of the experiment
1) Effect of ellagic acid on AAI-induced Kidney injury mouse Kidney function
The kidney index is a basic index reflecting kidney damage, and BUN, Cr and urine protein are clinically commonly used related indexes of kidney function. As shown in fig. 8, the kidney index, urine protein and BUN and Cr levels in serum of the mice were significantly increased after intraperitoneal injection of AAI, compared to the control group, indicating that AAI can induce kidney enlargement and cause renal dysfunction in the mice. Compared with AAI group, after pretreatment of EA, the kidney index of mice is obviously reduced, and the levels of urine protein and BUN and Cr in serum are reduced in a dose-dependent manner. The above results indicate that EA can effectively alleviate AAI-induced kidney enlargement and renal and hepatic dysfunction in kidney-injured mice.
2) Effect of ellagic acid on AAI-induced Kidney injury mouse Kidney histopathology
To more visually observe the condition of AAI-induced kidney injury in mice, the kidney tissues of the mice were H & E and Masson stained. Staining results show (fig. 9): the kidney of the control group of mice has no inflammatory cell infiltration, renal tubular epithelial edema, interstitial blood vessels have no congestion and expansion, and no pathological changes such as tissue fibrosis and the like are seen; the kidney of the AAI group mouse has obvious renal tubular dilatation, interstitial hyperemia and edema, partial renal tubular epithelial cell degeneration and necrosis, inflammatory cell exudation and mild fibrosis. After EA pretreatment with different concentrations of 10 and 30mg/kg, the renal tubular injury degree is obviously reduced, and pathological changes such as interstitial congestion, inflammatory cell infiltration, renal tubular epithelial cell degeneration necrosis, fibrosis and the like are obviously improved. Indicating that ellagic acid can dose-dependently alleviate AAI-induced renal pathology.
3) Effect of ellagic acid on AAI-induced Kidney injury mouse inflammatory factor
Numerous studies have shown that inflammatory factors are closely related to AAI-induced kidney injury. To investigate whether EA could protect kidney cells from AAI-induced nephritis response, applicants examined the expression of inflammation-related factors. As shown in FIG. 10, the levels of TNF-. alpha.and IL-1. beta. in the serum of the mice in the AAI group were significantly increased, while the levels of TNF-. alpha.and IL-1. beta. in the serum of the mice in the EA-pretreated group were significantly decreased as compared with the AAI group. In addition, applicants examined the transcriptional levels of TNF- α and IL-1 β in renal tissue. In contrast to the AAI group, which showed significantly higher mRNA expression levels of TNF- α and IL-1 β than the control group, EA dose-dependently reduced the transcript levels of TNF- α and IL-1 β in kidney tissue. The above experimental results show that the protective effect of ellagic acid on acute kidney injury of mice caused by AAI is related to inhibition of inflammatory reaction in kidney tissues.
4) Effect of ellagic acid on AAI-induced apoptosis in Kidney injured mice
As previously described, the apoptotic mechanism is associated with AAI-induced kidney injury. To determine whether EA pretreatment reduced AAI-induced tubular cell apoptosis, applicants performed Hoechst33258 and TUNEL fluorescent staining of renal tissue, and also examined the expression of the apoptosis-related markers cl-caspase-3, Bax, and Bcl-2. Hoechst33258 fluorescent staining is a classical apoptosis detection method. According to the staining result, the applicant finds that the renal cell nuclei of the control group are in normal blue and have no blue fluorescence cell nuclei, which indicates that the control group has no apoptosis. Compared with the control group, the kidney cells in the AAI group are densely stained in a broken block shape, and a large amount of blue fluorescence indicates apoptosis. While in the EA pre-treatment group the blue fluorescence gradually decreased and the cell morphology gradually became normal (see fig. 11). Furthermore, the TUNEL fluorescent staining further confirmed the Hoechst33258 staining. The control group has almost no positive expression, the positive expression of the AAI group is obviously increased compared with the control group, and the positive expression can be obviously reduced after EA pretreatment.
The pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2 are important factors in apoptosis. From the results of western applicants found that: the expression of cl-caspase-3 and Bax proteins was significantly increased, while the expression of Bcl-2 protein was significantly decreased after AAI treatment compared to the control group (see FIG. 12). EA pretreatment has a great inhibitory effect on these changes. Positive expression of apoptosis-immunohistochemical detection of related proteins Bax and Bcl-2 was consistent with the results of Westernblot analysis. These results above show that: EA can prevent AAI-induced apoptosis of renal tissue cells.
5) Effect of ellagic acid on AAI-induced NF- κ B/NLRP3 Signal pathway
To further determine the anti-inflammatory molecular mechanisms of EA on AAI-induced renal inflammatory responses, applicants analyzed the NF- κ B/I κ B α signaling pathway and the expression of NLRP3 inflammasome using Western-blot and immunohistochemistry. As shown in fig. 13: after AAI administration, the phosphorylation levels of NF-kappa Bp65 and I kappa B alpha protein are obviously increased, the protein levels of NLRP3 inflammasome components (NLRP3, ASC and caspase-1) and GSDMD and IL-1 beta downstream of the inflammatory corpuscle components are also obviously increased, and the mRNA expression levels of NLRP3 inflammasome and IL-1 beta are also increased. Compared with the AAI group, EA pretreatment has different degrees of inhibition effect on the high expression of NF-kB/NLRP 3 signal channel related proteins. In addition, the immunohistochemical results of NLRP3, ASC, Caspase-1 and IL-1. beta. were consistent with those of Western-blot. These results indicate that the protective effect of EA on AAI-induced kidney inflammation is associated with inhibition of activation of the NF-kb/NLRP 3 inflammasome signaling pathway.