CN113846049A - Application of cassia twig, poria cocos and bighead atractylodes rhizome decoction in relieving renal tubular epithelial cell hypoxia/reoxygenation injury - Google Patents
Application of cassia twig, poria cocos and bighead atractylodes rhizome decoction in relieving renal tubular epithelial cell hypoxia/reoxygenation injury Download PDFInfo
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Abstract
The invention provides application of cassia twig, poria cocos and bighead atractylodes rhizome decoction in relieving renal tubular epithelial cell hypoxia/reoxygenation injury, and belongs to the technical field of biological medicines and molecular biology. The invention proves that the cassia twig, poria cocos and bighead atractylodes rhizome decoction has a protective effect on H/R injury of renal proximal tubular HK-2 cells, and further researches on the mechanism of the decoction prove that GZQGFLBZS protects HK-2 cells from H/R injury by activating PI3K/Akt/eNOS pathways and inhibiting MAPKs pathways, so that the decoction can be used as an H/R-induced renal tubular epithelial cell repairing agent, can be further used for basic scientific research, and has good value in practical application.
Description
Technical Field
The invention belongs to the technical field of biological medicine and molecular biology, and particularly relates to application of cassia twig, poria cocos and bighead atractylodes rhizome decoction in relieving renal tubular epithelial cell hypoxia/reoxygenation injury.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Ischemia/reperfusion (I/R) is a common cause of acute kidney injury, and Acute Kidney Injury (AKI) in hospitalized non-ICU patients is associated with early and late mortality. Unfortunately, there is currently no specific therapy for ischemic AKI. Proximal tubular cells are reported to be the most sensitive renal cells to I/R injury, and are the major site of acute renal injury. Renal tubular epithelial cells have a strong repairing ability, and even if the damage is severe, renal function may be completely restored to normal as long as the damaging factor is removed. The search for drugs that promote tubular cell regeneration has become a hot topic.
Guizhi Gui Jia Fuling Baizhu Tang (GZQGFLBZS) is a Chinese medicinal formula with the functions of easing pain, relieving fever, perspiration and promoting urination. Recently, the protective effect of GZQGFLBZS on AKI patients has been reported. It was noted that after ischemia reperfusion injury, rapid recovery of renal function was achieved with the addition of GZQGFLBZS based on conventional combination therapy, and that renal injury markers (NGAL and kim-1) were significantly reduced. However, the mechanism of GZQGFLBZS to protect the postischemic kidney has not been elucidated.
The regulatory mechanisms of tubular cell remodeling are quite complex, and previous studies have shown that a variety of signaling pathways play important roles in this process, including the PI3K/AKT/eNOS signaling pathway. The PI3K/Akt pathway is involved in protecting the kidney from I/R injury by modulating oxidative stress and inflammation. eNOS is a key enzyme regulating the production of endothelial-derived NO, and can be regulated and controlled by a PI3K/Akt signal pathway, thereby relieving kidney injury caused by I/R.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the application of the cassia twig, poria cocos and bighead atractylodes rhizome decoction in relieving renal tubular epithelial cell hypoxia/reoxygenation injury. The present inventors have found that GZQGFLBZS has a protective effect on hypoxia/reoxygenation (H/R) damage of renal proximal tubular HK-2 cells, and further have demonstrated that GZQGFLBZS protects HK-2 cells from H/R damage by activating PI3K/Akt/eNOS pathway and inhibiting MAPKs pathway, thereby completing the present invention.
Specifically, the invention relates to the following technical scheme:
in a first aspect of the invention, there is provided the use of a product comprising cinnamon twig, poria cocos and atractylodes macrocephala decoction for attenuating hypoxic/reoxygenation damage to renal tubular epithelial cells.
The product containing the cassia twig, tuckahoe and bighead atractylodes rhizome decoction can be a concentrated solution of the cassia twig, tuckahoe and bighead atractylodes rhizome decoction or a medicated serum of the cassia twig, tuckahoe and bighead atractylodes rhizome decoction.
The attenuation of the hypoxia/reoxygenation injury of the renal tubular epithelial cells is characterized in that:
(a) reducing H/R-induced intracellular ROS production by renal tubular epithelial cells;
(b) increasing tubular epithelial cell eNOS activity and NO production;
(c) (ii) reducing H/R-induced apoptosis of renal tubular epithelial cells;
(d) activating PI3K/Akt/eNOS pathway of renal tubular epithelial cells;
(e) inhibiting H/R-mediated activation of NF- κ B and MAPKs in HK-2 cells;
(f) promoting miR-21 up-regulation expression of renal tubular epithelial cells;
(g) inhibiting the expression level of mRNA of ERS related genes of renal tubular epithelial cells;
(h) inhibit the expression of caspase-3 and caspase-8 proteins of renal tubular epithelial cells.
Wherein the renal tubular epithelial cells are HK-2 cells.
In the (g), ERS-associated genes include IRE-1 α, XBP-1 and GRP 78.
According to the invention, researches show that the cassia twig cinnamon-removed and poria cocos-bighead atractylodes rhizome decoction containing the serum can effectively weaken the hypoxia/reoxygenation injury of the renal tubular epithelial cells, so that the cassia twig cinnamon-removed and poria cocos-bighead atractylodes rhizome decoction can be used as a hypoxia/reoxygenation injury repairing agent for the renal tubular epithelial cells, and further scientific experimental researches can be conveniently carried out.
In a second aspect of the invention, the invention provides a renal tubular epithelial cell hypoxia/reoxygenation injury repair agent, and the active ingredients of the repair agent comprise cassia twig-added poria cocos-bighead atractylodes rhizome decoction concentrated solution or cassia twig-added poria cocos-bighead atractylodes rhizome decoction medicated serum.
In a third aspect of the present invention, there is provided a use of the cassia twig, cinnamomum cassia, poria cocos and rhizoma atractylodis macrocephalae decoction concentrated solution or the cassia twig, cinnamomum cassia, poria cocos and rhizoma atractylodis macrocephalae decoction containing the drug serum in any one or more of the following:
(a) preparing a product that reduces H/R-induced intracellular ROS production by renal tubular epithelial cells;
(b) preparing a product for increasing eNOS activity and NO production of renal tubular epithelial cells;
(c) preparing a product that reduces H/R-induced apoptosis of tubular epithelial cells;
(d) preparing a product for activating PI3K/Akt/eNOS pathway of renal tubular epithelial cells;
(e) preparing a product that inhibits H/R-mediated activation of NF- κ B and MAPKs in HK-2 cells;
(f) preparing a product for promoting miR-21 of renal tubular epithelial cells to up-regulate expression;
(g) preparing a product for inhibiting the mRNA level of ERS related genes of renal tubular epithelial cells;
(h) prepare the products for inhibiting the protein expression of the caspase-3 and caspase-8 of the renal tubular epithelial cells.
Wherein the renal tubular epithelial cells are HK-2 cells.
In the (g), ERS-associated genes include IRE-1 α, XBP-1 and GRP 78.
In a fourth aspect of the invention, there is provided a method of attenuating hypoxic/reoxygenation injury to renal tubular epithelial cells, the method comprising: and adding concentrated decoction of cassia twig-plus-poria-bighead atractylodes rhizome or medicated serum of cassia twig-plus-poria-bighead atractylodes rhizome into H/R induced renal tubular epithelial cells, preferably the medicated serum of cassia twig-minus-poria-bighead atractylodes rhizome.
The beneficial technical effects of one or more technical schemes are as follows:
the research of the technical scheme proves that the cassia twig-cinnamomum cassia-poria cocos-bighead atractylodes rhizome decoction has a protective effect on H/R injury of renal proximal tubular HK-2 cells, and further researches on the mechanism of the cassia twig-cinnamomum cassia-poria cocos-bighead atractylodes rhizome decoction prove that GZQGFLBZS protects HK-2 cells from H/R injury by activating PI3K/Akt/eNOS pathways and inhibiting MAPKs pathways, so that the Guizhi-poria cocos-bighead atractylodes rhizome decoction can be used as an H/R-induced renal tubular epithelial cell repairing agent and further can be used for basic research, and has good practical application value.
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The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a graph relating to the reduction of H/R-induced tubular HK-2 cell damage by GZQGFLBZS in example 1 of the present invention, wherein A is a graph showing the cytotoxic effect of GZQGFLBZS against H/R damage at various concentrations; b is H/R-induced renal tubular HK-2 cell Lactate Dehydrogenase (LDH) release at various concentrations of GZQGFLBZS.
FIG. 2 is a graph showing the effect of varying concentrations of GZQGFLBZS on H/R-induced eNOS activity in tubular HK-2 cells in example 1 of the present invention.
FIG. 3 is a graph showing the effect of varying concentrations of GZQGFLBZS on H/R-induced NO production by tubular HK-2 cells in example 1 of the present invention.
FIG. 4 is a graph of the H/R-induced ROS production in tubular HK-2 cells by GZQGFLBZS at various concentrations in example 1 of the present invention.
FIG. 5 is a graph showing that GZQGFLBZS reduces H/R-induced apoptosis of HK-2 cells in example 1 of the present invention.
FIG. 6 is a graph showing the effect of GZQGFLBZS on the PI3K/Akt/eNOS signaling pathway in HK-2 cells in example 1 of the present invention.
FIG. 7 shows that GZQGFLBZS inhibits H/R-mediated activation of NF-. kappa.B and MAPKs in HK-2 cells in example 1 of the present invention.
FIG. 8 is a graph showing that GZQGFLBZS promotes H/R-mediated proliferation of HK-2 cells in example 2 of the present invention; note: p <0.05vs control group; # p <0.05vs H/R intervening cell groups.
FIG. 9 is a graph showing the expression of caspase-3 protein in each set of cells in example 2 of the present invention.
FIG. 10 is a graph of the upregulation of miR-21in H/R-treated HK-2 cells by GZQGFLBZS in example 2 of the present invention.
FIG. 11 is a graph showing the expression of mRNA levels of various groups of ERS-associated genes in example 2 of the present invention.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. The experimental procedures, if specific conditions are not indicated in the following detailed description, are generally in accordance with conventional procedures and conditions of molecular biology within the skill of the art, which are fully explained in the literature. See, e.g., Sambrook et al, "molecular cloning: the techniques and conditions described in the laboratory Manual, or according to the manufacturer's recommendations.
The present invention is further illustrated by reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions not specified in the examples are specified, they are generally according to the conventional conditions, or according to the conditions recommended by the sales companies; materials, reagents and the like used in examples were commercially available unless otherwise specified.
In a typical embodiment of the present invention, there is provided a use of a cinnamon twig, poria cocos, white atractylodes rhizome decoction for attenuating hypoxic/reoxygenation damage of renal tubular epithelial cells.
In another embodiment of the present invention, the product containing Guizhi Xiaguaiao Fuling Baizhu decoction may be a Guizhi Xiaguaiao Fuzhu decoction concentrated solution or a Guizhi Xiaguaiao Fuzhu decoction containing serum.
In another embodiment of the present invention, the cassia twig, poria cocos and bighead atractylodes rhizome decoction containing the blood serum is obtained by performing intragastric gavage on rats with the cassia twig, poria cocos and bighead atractylodes rhizome decoction and then collecting the blood.
In another specific embodiment of the present invention, the cassia twig, poria cocos and bighead atractylodes rhizome decoction comprises the following raw materials, by mass: 10-16 parts of white peony root, 8-12 parts of honey-fried licorice root, 12-16 parts of ginger, 12-16 parts of bighead atractylodes rhizome, 10-16 parts of poria cocos and 18-25 parts of Chinese date.
In another specific embodiment of the present invention, the cassia twig, poria cocos and bighead atractylodes rhizome decoction comprises the following raw materials, by mass: 15 parts of white peony root, 10 parts of honey-fried licorice root, 15 parts of ginger, 15 parts of bighead atractylodes rhizome, 15 parts of poria cocos and 20 parts of Chinese date.
In yet another embodiment of the present invention, the attenuating of hypoxic/reoxygenation injury of renal tubular epithelial cells is embodied by:
(a) reducing H/R-induced intracellular ROS production by renal tubular epithelial cells;
(b) increasing tubular epithelial cell eNOS activity and NO production;
(c) (ii) reducing H/R-induced apoptosis of renal tubular epithelial cells;
(d) activating PI3K/Akt/eNOS pathway of renal tubular epithelial cells;
(e) inhibiting H/R-mediated activation of NF- κ B and MAPKs in HK-2 cells;
(f) promoting miR-21 up-regulation expression of renal tubular epithelial cells;
(g) inhibiting the expression level of mRNA of ERS related genes of renal tubular epithelial cells;
(h) inhibit the expression of caspase-3 and caspase-8 proteins of renal tubular epithelial cells.
In yet another embodiment of the present invention, the renal tubular epithelial cells are HK-2 cells.
In still another embodiment of the present invention, in the (g), the ERS-associated genes include IRE-1 α, XBP-1 and GRP 78.
According to the invention, researches show that the cassia twig cinnamon-removed and poria cocos-bighead atractylodes rhizome decoction containing the serum can effectively weaken the hypoxia/reoxygenation injury of the renal tubular epithelial cells, so that the cassia twig cinnamon-removed and poria cocos-bighead atractylodes rhizome decoction can be used as a hypoxia/reoxygenation injury repairing agent for the renal tubular epithelial cells, and further scientific experimental researches can be conveniently carried out.
In another embodiment of the present invention, the present invention provides a renal tubular epithelial cell hypoxia/reoxygenation injury repair agent, wherein the active ingredients of the repair agent comprise cassia twig and poria cocos and rhizoma atractylodis macrocephalae decoction concentrated solution or cassia twig and poria cocos and rhizoma atractylodis macrocephalae decoction drug-containing serum.
In another embodiment of the present invention, the application of the cassia twig, poria cocos and white atractylodes rhizome decoction concentrated solution or the cassia twig, poria cocos and white atractylodes rhizome decoction containing the serum is provided in any one or more of the following:
(a) preparing a product that reduces H/R-induced intracellular ROS production by renal tubular epithelial cells;
(b) preparing a product for increasing eNOS activity and NO production of renal tubular epithelial cells;
(c) preparing a product that reduces H/R-induced apoptosis of tubular epithelial cells;
(d) preparing a product for activating PI3K/Akt/eNOS pathway of renal tubular epithelial cells;
(e) preparing a product that inhibits H/R-mediated activation of NF- κ B and MAPKs in HK-2 cells;
(f) preparing a product for promoting miR-21 of renal tubular epithelial cells to up-regulate expression;
(g) preparing a product for inhibiting the mRNA level of ERS related genes of renal tubular epithelial cells;
(h) prepare the products for inhibiting the protein expression of the caspase-3 and caspase-8 of the renal tubular epithelial cells.
In yet another embodiment of the present invention, the renal tubular epithelial cells are HK-2 cells.
In still another embodiment of the present invention, in the (g), the ERS-associated genes include IRE-1 α, XBP-1 and GRP 78.
In yet another embodiment of the present invention, there is provided a method of attenuating hypoxic/reoxygenation injury to renal tubular epithelial cells, the method comprising: and adding concentrated decoction of cassia twig-plus-poria-bighead atractylodes rhizome or medicated serum of cassia twig-plus-poria-bighead atractylodes rhizome into H/R induced renal tubular epithelial cells, preferably the medicated serum of cassia twig-minus-poria-bighead atractylodes rhizome.
In another embodiment of the present invention, the cassia twig, poria cocos and bighead atractylodes rhizome decoction containing the blood serum is obtained by performing intragastric gavage on rats with the cassia twig, poria cocos and bighead atractylodes rhizome decoction and then collecting the blood.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1
1 materials and methods
1.1 drugs and reagents
The product consists of 6 traditional Chinese medicines: 15g of white peony root, 10g of honey-fried licorice root, 15g of ginger, 15g of bighead atractylodes rhizome, 15g of poria cocos and 20g of Chinese date. The drugs in the above prescriptions were purchased from the third hospital of Shandong province. After the decoction, the drug was sterilized by filtration and stored in a refrigerator at 4 ℃.
The following primary antibodies were purchased from cell signaling technology (Danvers, MA): rabbit anti-phosphorylated ERK1/(2 phosphorylated p 44/42; Thr202/Tyr204), rabbit anti-ERK 1/2(p44/42), rabbit anti-phosphorylated Akt (Ser473), and rabbit anti-Akt. Rabbit anti-phosphorylation eNOS (serine1177), rabbit anti-eNOS (EPR19296), rabbit anti-IkB alpha, rabbit phosphorylated IkB alpha (Ser36), rabbit anti-p 38MAPK (Thr180/Tyr182), rabbit anti-p 38MAPK, rabbit anti-p-1/2/3 (Thr183/185/221), rabbit anti-JNK 1/2/3, rabbit anti-caspase-3, rabbit anti-PARP, rabbit anti-GAPDH monoclonal antibodies were purchased from Cambridge Abcam, UK.
1.2 preparation of serum containing tested drug and drug therapy
Male SD rats of 8 weeks old were purchased from jinanxingkang laboratory animals ltd, and placed in a room at a temperature of 2125 ℃, a relative humidity of 50-60%, and a light/dark cycle of 12 hours. All animal experiments were performed in hospitals according to the third animal experiment guideline in Shandong province. That intervention treatment: rats were randomized into low dose (5g/kg/d, n-10), medium dose (10g/kg/d, n-10) and high dose (20g/kg/d GZQGFLBZS, n-10) and normal controls (saline, n-11). All rats were individually gavaged for 7 days. Blood was aseptically obtained from the abdominal aorta of rats 1 hour after the last administration, and then serum was obtained by centrifugation at 720Xg for 20 minutes. After filtration twice with a 0.22 μm filter, the serum was bottled, heated in 56 ℃ water for 30 minutes, and stored for use at 20 ℃. According to experimental groups, the prepared drug-containing serum was added to the HK-2 cell culture medium sequentially 2h before h/R induction, and incubated at 37 ℃.
1.3 cell culture and H/R Induction
Human proximal tubular cell lines (HK-2) were purchased from ATCC American type culture specimen Bank, Masassas, Va. HK-2 cells were cultured in 10% FBS-containing MEM medium (KeyGenBiotech, Nanjing, China), 37 ℃, 5% CO2Culturing under the condition. Intervention was performed when cells were grown to 80% confluence. An H/R model of simulating I/R kidney injury of HK-2 cells in vivo is established: HK2 cells were cultured in serum-free medium for 24 hours, then the medium was discarded, different media containing (or no drug) was added 2 hours in advance according to the group, and then the cells were placed in an anoxic incubator (37 ℃ C., 95% N)2，5％CO2) Placed for 24 hours, and then placed in an normoxic incubator (5% CO2, 21% O)2，74％N2) For 2 hours. LY294002 was dissolved in dimethyl sulfoxide. Cells were pretreated with LY294002(20 μ M) to inhibit Akt prior to H/R.
1.4 cell viability and lactate dehydrogenase Release assay
Cell viability was quantified using MTT colorimetry (Solarbio, beijing, china), which measures mitochondrial activity in living cells. Briefly, cells were seeded at a density of 5000 cells/well in 96-well plates. Treating with serum containing drugs with different concentrations under normoxic or anoxic conditions for 24h, adding MTT solution, incubating at 37 deg.C for 4h, taking out culture medium, and adding 110 μ l methoxypyrimidine solution into each well. The absorbance at 490nm was determined by a microplate reader (TECAN, Switzerland). Cytotoxicity was quantified by measuring LDH release in the medium during different reagent exposures. The lactate dehydrogenase detection kit (beotimer biotech, beijing, china) was used according to the manufacturer's instructions.
1.5 determination of NO level and eNOS Activity
Each set of cell culture media was collected and the NO levels and NOS activity released by HK-2 cells were calculated using NO and NOS detection kits according to manufacturer's instructions (institute of bioengineering, Tokyo, Nanjing, China).
1.6 intracellular reactive oxygen species detection
Reactive oxygen species generation was assessed using ROS sensitive fluorescent indicator 2 ', 7' -dichlorodihydrofluorescein diacetate (DCFH-DA, beijing, china, Solarbio). After the different treatments, 10 μ MDFH-DA was incubated at 37 ℃ for 30 minutes, the ROS levels were determined by observing DCFH-DA staining with a fluorescence microscope, and the expression levels were quantified as the sum of pixel values using ImageProPlus image analysis software.
1.7 flow cytometry
Apoptotic cells were analyzed by flow cytometry using the annexin V-PE/7-AAD double staining apoptosis kit (Meilong, China). After 24 hours of treatment with different concentrations of drug-containing serum (low, medium, high dose GZQGFLBZS) under normoxic or hypoxic conditions, cells were harvested and resuspended in 500. mu.l of binding solution. Subsequently, 5. mu.l of annexin V-PE and 10. mu.l of 7-AAD were incubated for 15 minutes in the dark. Cells were immediately analyzed using a flow cytometer (BDBiosciences, USA) and analyzed using flowjo10.0 software.
1.8 immunoblotting
Total protein was solubilized in a RIPA cracker (Beijing Beoterm, China) and quantified using the BCA kit. The proteins of each sample were separated on a 10% gel by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then electroblotted onto a polyvinylidene fluoride membrane. Incubation with blocking buffer (5% skim milk) for 60 min at room temperature followed by primary antibody (rabbit anti-eNOS, 1: 1000; rabbit anti-phosphorylated eNOS, 1: 1000; rabbit anti-Akt, 1: 1000; rabbit anti-phosphorylated Akt, 1: 1000; rabbit anti-ERK, 1: 1000; rabbit anti-phosphorylated ERK, 1: 5000; rabbit anti-IkB α,1: 1000; rabbit phosphorylated-IkB α,1: 1000; rabbit anti-phospho p38MAPK, 1: 1000; rabbit anti-phospho JNK1/2/3,1: 1000; rabbit anti-JNK 1/2/3,1: 1000; rabbit anti-lytic caspase-3,1: 500; rabbit anti-caspase-3, 1: 1000; rabbit anti-lytic PARP, 1: 1000). After three washes with TBST, incubation with anti-rabbit secondary antibody for 60 minutes at room temperature, 3 washes with TBST. Blots were then developed using the ECLPlus detection kit (milbebo, massachusetts, usa) to generate chemiluminescent signals and captured on X-ray film. Computer-assisted density measurement (ImageJ) was used to quantify bands captured on radiographic film.
1.9 statistical analysis
All experimental measurements are expressed as means ± standard deviation (mean ± SD) and statistically analyzed using the sps 22.0 software. Differences between groups were evaluated using one-way analysis of variance (ANOVA) and then the Least Significant Differences (LSD) were performed on multiple mean comparisons where appropriate. A value of P <0.05 was considered as statistically significant difference between the mean values.
2 results
2.1GZQGFLBZS reduces H/R-induced tubular HK-2 cell injury
The effect of GZQGFLBZS on HK-2 cell viability at different concentrations of H/R injury was studied. H/R injury reduced cell viability to 40%. GZQGFLBZS significantly prevented the cytotoxic effects of H/R injury in a concentration-dependent manner up to the mid-dose concentration (P <0.01) (fig. 1A). The high concentration GZQGFLBZS has the effect of inhibiting the proliferation of HK-2 cells. As shown in figure 1B, H/R damage resulted in a significant increase in LDH release, and GZQGFLBZS significantly attenuated the increase in LDH in the medium (P < 0.01). These results indicate that GZQGFLBZS protects HK-2 cells from H/R-induced cellular damage.
2.2GZQGFLBZS reduces H/R-induced ROS production in HK-2 cells
It was further investigated whether GZQGFLBZS is involved in ROS regulation. As shown in FIG. 4, H/R increased ROS production (P <0.001), while GZQGFLBZS significantly inhibited H/R (P <0.01) by modulating ROS production.
2.3 increase of eNOS Activity and NO production in HK-cells by GZQGFLBZS the effect of varying concentrations of GZQGFLBZS on eNOS activity and NO production in HK-2 cells was observed
As shown in FIGS. 2 and 3, H/R treatment increased eNOS activity and NO production by HK-2 cells compared to normoxic cells (control). GZQGFLBZS further increased eNOS activity and NO production, and within the mid-dose concentration range, GZQGFLBZS increased eNOS levels dose-dependently (P < 0.05). The above experiments show that medium concentration drug serum containing GZQGFLBZS has better cell survival promoting effect than other concentrations, and therefore the concentration is selected as the optimal concentration for subsequent studies.
2.4GZQGFLBZS can alleviate H/R-induced apoptosis of HK-2 cells
H/R increased the expression of cleaved caspase-3 and cleaved PARP in HK-2 cells, indicating that H/R induced apoptosis. However, GZQGFLBZS significantly blocked H/R-induced caspase-3(P <0.001) and PARP cleavage (P <0.01) (fig. 5). The number of apoptotic cells after H/R treatment was further measured by flow cytometry with double staining of Annexin-PE and 7-AAD. The number of apoptotic cells after H/R exposure was significantly increased compared to normal control cells (P < 0.01). In turn, GZQGFLBZS pretreatment significantly reduced H/R-induced apoptosis (fig. 5) (P < 0.01). Therefore, GZQGFLBZS has an anti-apoptotic effect on H/R damage of HK-2 cells.
2.5 Effect of GZQGFLBZS on the PI3K/Akt/eNOS signaling pathway in HK-2 cells
Western blot analysis showed a significant increase in the ratio of p-Akt/total-Akt and p-eNOS/totaleNOS in HK-2 cells compared to the control (p <0.001) (FIG. 6). The GZQGFLBZS pretreatment further significantly improves the expression level of p-Akt (p < 0.01). HK-2 cells compared to H/R, p-eNOS (p <0.001) group. To further confirm the role of PI3K/Akt/eNOS pathway in the protection of GZQGFLBZS-induced H/R injury, LY294002 was detected in HK-2 cells. LY294002 is a synthetic compound designed as an inhibitor of PI3K and has a reversible inhibitory effect on the PI3K/AKT pathway. LY294002 can effectively inhibit the protective action of GZQGFLBZS. These changes suggest that GZQGFLBZS has an anti-H/R injury effect by activating the PI3K/Akt/eNOS pathway.
2.6GZQGFLBZS inhibits H/R-mediated activation of NF-. kappa.B and MAPKs in HK-2 cells
To further investigate the molecular mechanisms associated with H/R renal injury, the effects of NF-. kappa.B and MAPKs (p38MAPK, JNK1/2/3 and ERK1/2) signaling pathways were examined. H/R activated NF-kB (P <0.001) by western blot analysis of increased phosphorylation of IkB α/total IkB α proteins. Compared to the control group, H/R increased phosphorylation of p38MAPK, ERK1/2, and JNK1/2/3 proteins without affecting the corresponding total protein levels (FIG. 7). These effects were completely reversed after administration of GZQGFLBZS. These data indicate that intervention in NF-kB and MAPKs transduction is involved in GZQGFLBZS against H/R injury.
In conclusion, GZQGFLBZS has an improved effect on H/R-induced HK-2 cell damage. The protective effect is that the PI3K/Akt/eNOS signal path is activated by eliminating ROS, and the NF-kB and MAPKs path mediation is inhibited. Therefore, GZQGFLBZS is expected to be a potential repairing agent for promoting tubular cell recovery after H/R injury.
Example 2
1 materials and methods
1.1 cell culture and cell IRI model establishment
Human proximal tubule cell line HK-2 was purchased from American TypeCultureCo, ATCC (Manassas, VA, USA). HK-2 cells were cultured (37 ℃, 5% CO) in MEM medium (KeyGenBiotech, Nanjing, China) containing 10% FBS (Gibco technologies)2) When cells were grown to 80% confluence, 0.25% pancreatin digestion, 1: 3-1: passage 4, and when the cells grow to 80%, performing intervention treatment. An H/R model of the HK-2 cells is established: inoculating cells in 6-well plate, culturing in serum-free culture medium for 24 hr after cell adherence, removing original serum-free culture medium, adding different medicated (or non-medicated) culture media according to groups 2 hr in advance, placing cells in culture conditions of 37 deg.C and 95% N2+5％CO2(V/V) in an incubator, performing anaerobic culture for 24h, taking out cells, placing in an incubator adjusted to 37 ℃, 95% air + 5% CO2The incubator of (2) is used for reoxygenation culture for 12 h.
1.2 preparing the cassia twig, poria cocos and bighead atractylodes rhizome decoction: 15g of white peony root, 10g of honey-fried licorice root, 15g of ginger, 15g of bighead atractylodes rhizome, 15g of poria cocos and 20g of Chinese date. Weighing the mass of each medicine in the above formula according to a proportion, adding 500ml of water according to the calculated amount, soaking for 30min, putting into a decocting machine, adjusting the pressure to 1-1.25 kg, decocting for 45min at 110 ℃, filtering to remove residues, filtering the filtered decoction, putting into a constant temperature drying oven at 100 ℃ after filtering through a filter flask, evaporating and concentrating to 100ml, containing the concentration of the crude medicine of 90mg/ml, bottling, sealing, and storing in a refrigerator at 4 ℃ for later use.
1.3 detection of cell proliferation Rate
The cassia twig, poria cocos and bighead atractylodes rhizome decoction is prepared into solutions with final concentrations of 50, 100 and 150mg/L respectively by using 10% complete culture media. MTT assay experimental grouping: (1) normal oxygen content group: HK-2; ② HK-2+50 mg/L; ③ HK-2+100 mg/L; HK-2+150 mg/L; (2) hypoxia/reoxygenation group: HK-2; ② HK-2+50 mg/L; ③ HK-2+100 mg/L; and HK-2+150 mg/L.
4x10 in terms of cell division4Laying 96-well plates per mL, culturing in serum-free culture solution for 24h after the cells are attached to the wall, synchronously treating, discarding the original serum-free culture solution, respectively adding 200uL of different drug-containing (or drug-free) culture media into each well, setting 3 parallel wells in each group, and setting a zero-adjusting group. After the normoxic culture or the anoxic/reoxygenation treatment, 20. mu.l of MTT (5mg/ml) was added to each well, and the culture was continued for 4 hours. The supernatant was aspirated, 200. mu.l DMSO was added to each well, the crystals were sufficiently dissolved by shaking for 10min, and the absorbance (OD value) of each well was measured with a microplate reader (490nm wavelength). And the cell proliferation rate was calculated by the following formula (OD value of experimental group-OD value of zero adjustment group)/(OD value of control group-OD value of zero adjustment group) × 100%. Determining the optimal intervention concentration.
1.4 Experimental groups
(1) Control group (control): normal HK2 cells were not treated with any special treatment;
(2) control + drug group (100 mg/L): control plus Guizhi Xigui-removed Fuling Baizhu Tang;
(3) group I/R: IRI processing;
(4) I/R + drug group (100mg/L), cassia twig, poria cocos and bighead atractylodes rhizome decoction are pretreated for 2 hours and then subjected to IRI treatment.
1.5 Western Blot assay
Extracting total cell protein with protein lysate (RIPA: PMSF ═ 100: 1), quantifying protein by BCA method, adding 5 Xprotein loading buffer solution, mixing, and denaturing at 100 deg.C in water bath for 10 min; protein samples were run on SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Chicago, IL, USA) and blocked with 5% skim milk for 1h at room temperature. The membranes were washed 3 times with TBST and then placed in the corresponding primary antibody and incubated overnight at 4 ℃. Taking out the protein strips the next day, washing the protein strips with TBST for 3 times, then placing the protein strips into HRP-coupled secondary antibody, incubating the protein strips for 1h at normal temperature, performing ECL luminescence imaging, scanning by using a gel imaging analyzer system, and performing density analysis on the protein strips by using ImageJ software.
1.6 silencing of miR-21 using miR-21inhibitor (available from Genephrma). First, HK-2 cells were seeded in 6-well plates prior to transfection. miR-21inhibitor, miR-21inhibitor negative control and lipofectamine2000 transfection reagent (Invitrogen, USA) were added to the medium for 6 hours of incubation, followed by 2 hours of incubation with media containing different drug-containing (or drug-free) sera instead of the previous media. Finally, the cells are subjected to hypoxia/reoxygenation interventions.
2 results of the experiment
2.1 cell proliferation Rate: MTT detection shows that the medium concentration (100mg/L) medicament has the best effect, can obviously promote the proliferation of the hypoxic reoxygenation cells and reduce the apoptosis (figure 8).
2.2 caspase-3 protein expression
The results of the WesternBlot test show that the protein expression of the hypoxia reoxygenation cell caspase-3 is significantly higher than that of the Guizhi Jia Fuling Baizhu Tang prepared group (figure 9).
2.3 Guizhi Xigui Jia Fuling Baizhu Tang upregulates miR-21in HK-2 cells treated with H/R
As shown in FIG. 10, after the Guizhi Jia Fuling Baizhu decoction treatment, the miR-21 of the HK-2 cells treated by the H/R is obviously higher than that of the control group (P is less than 0.01).
2.4 Cassia twig, cassia twig, poria cocos, and bighead atractylodes rhizome decoction down-regulates mRNA level of ERS related gene and mRNA level of TNF-alpha and NF-kappa B through miR-21
mRNA levels of ERS-associated genes, including IRE-1 α, XBP-1 and GRP78, were detected using RT-qPCR (FIG. 11). The results showed that the H/R group had significantly higher levels of IRE-1 α, XBP-1 and GRP78mRNA than the control group (P < 0.01). The ERS related gene is obviously reduced (P is less than 0.01) after the drug treatment, and the reduction effect is reversed (P is less than 0.01) after the miR-21inhibitor is applied. The expression of inflammation-related genes including TNF-alpha and NF-kappa B is detected, and the levels of TNF-alpha and NF-kappa BmRNA of the H/R group are obviously higher than those of a control group (P is less than 0.01). The ERS related gene is obviously reduced (P is less than 0.01) after the drug treatment, and the reduction effect is reversed (P is less than 0.01) after the miR-21inhibitor is applied.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. Application of a product containing GUIZHIQUGUIJIAOFUBAOAtractylodis rhizoma decoction in reducing anoxia/reoxygenation injury of renal tubular epithelial cell is provided.
2. The use of claim 1, wherein the product containing Guizhi Xiaguaiao Fuling Baizhu Tang is a Guizhi Xiaguaiao Fuling Baizhu Tang concentrated solution or a Guizhi xiujiafuliao Baizhu Tang containing serum.
3. The use of claim 2, wherein the serum containing cinnamon twig, cassia twig, poria cocos and bighead atractylodes rhizome decoction is obtained by performing intragastric gavage on a rat with the cinnamon twig, cassia twig, poria cocos and bighead atractylodes rhizome decoction and then collecting blood.
4. The application of claim 1, wherein the cassia twig, cinnamomum cassia, poria cocos and bighead atractylodes rhizome decoction comprises the following raw materials in parts by weight: 10-16 parts of white paeony root, 8-12 parts of honey-fried licorice root, 12-16 parts of ginger, 12-16 parts of bighead atractylodes rhizome, 10-16 parts of poria cocos and 18-25 parts of Chinese date;
preferably, the cassia twig, poria cocos and bighead atractylodes rhizome decoction comprises the following raw materials in parts by weight: 15 parts of white peony root, 10 parts of honey-fried licorice root, 15 parts of ginger, 15 parts of bighead atractylodes rhizome, 15 parts of poria cocos and 20 parts of Chinese date.
5. The use of claim 1, wherein the attenuation of hypoxic/reoxygenation injury to renal tubular epithelial cells is characterized by:
(a) reducing H/R-induced intracellular ROS production by renal tubular epithelial cells;
(b) increasing tubular epithelial cell eNOS activity and NO production;
(c) (ii) reducing H/R-induced apoptosis of renal tubular epithelial cells;
(d) activating PI3K/Akt/eNOS pathway of renal tubular epithelial cells;
(e) inhibiting H/R-mediated activation of NF- κ B and MAPKs in HK-2 cells;
(f) promoting miR-21 up-regulation expression of renal tubular epithelial cells;
(g) inhibiting the expression level of mRNA of ERS related genes of renal tubular epithelial cells;
(h) inhibit the expression of caspase-3 and caspase-8 proteins of renal tubular epithelial cells.
6. The use of claim 5, wherein the renal tubular epithelial cells are HK-2 cells;
in the (g), ERS-associated genes include IRE-1 α, XBP-1 and GRP 78.
7. A renal tubular epithelial cell hypoxia/reoxygenation injury repairing agent is characterized in that active ingredients of the repairing agent comprise cassia twig-poria cocos-bighead atractylodes rhizome decoction concentrated solution or cassia twig-poria cocos-bighead atractylodes rhizome decoction drug-containing serum.
8. The cassia twig, poria cocos and bighead atractylodes rhizome decoction concentrated solution or the cassia twig, poria cocos and bighead atractylodes rhizome decoction containing the medicine serum is applied to any one or more of the following components:
(a) preparing a product that reduces H/R-induced intracellular ROS production by renal tubular epithelial cells;
(b) preparing a product for increasing eNOS activity and NO production of renal tubular epithelial cells;
(c) preparing a product that reduces H/R-induced apoptosis of tubular epithelial cells;
(d) preparing a product for activating PI3K/Akt/eNOS pathway of renal tubular epithelial cells;
(e) preparing a product that inhibits H/R-mediated activation of NF- κ B and MAPKs in HK-2 cells;
(f) preparing a product for promoting miR-21 of renal tubular epithelial cells to up-regulate expression;
(g) preparing a product for inhibiting the mRNA level of ERS related genes of renal tubular epithelial cells;
(h) preparing products for inhibiting the expression of the renal tubular epithelial cell caspase-3 and caspase-8 proteins;
preferably, the renal tubular epithelial cells are HK-2 cells;
in the (g), ERS-associated genes include IRE-1 α, XBP-1 and GRP 78.
9. A method of attenuating hypoxic/reoxygenation injury to renal tubular epithelial cells, the method comprising: and adding concentrated decoction of cassia twig-plus-poria-bighead atractylodes rhizome or medicated serum of cassia twig-plus-poria-bighead atractylodes rhizome into H/R induced renal tubular epithelial cells, preferably the medicated serum of cassia twig-minus-poria-bighead atractylodes rhizome.
10. The method of claim 9, wherein the serum containing cinnamon twig, cassia twig, poria cocos and white atractylodes rhizome decoction is obtained by performing intragastric gavage on a rat with the cinnamon twig, cassia twig, poria cocos and white atractylodes rhizome decoction and then collecting blood.
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| CN111494407A (en) * | 2020-01-08 | 2020-08-07 | 南京市儿童医院 | Use of trehalose in the preparation of a medicament for alleviating conditions associated with acute renal injury induced by ischemia reperfusion |
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