CN111110835A - New application of intestinal alkaline phosphatase and cell activity detection method of preparation of intestinal alkaline phosphatase - Google Patents
New application of intestinal alkaline phosphatase and cell activity detection method of preparation of intestinal alkaline phosphatase Download PDFInfo
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- CN111110835A CN111110835A CN202010073662.7A CN202010073662A CN111110835A CN 111110835 A CN111110835 A CN 111110835A CN 202010073662 A CN202010073662 A CN 202010073662A CN 111110835 A CN111110835 A CN 111110835A
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- alkaline phosphatase
- tnf
- intestinal alkaline
- human
- secretion
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention adopts a cell model of human leukocyte secretion inflammatory factors TNF- α and IL-6 which are extracted freshly, researches the inhibition effect of the intestinal alkaline phosphatase on the human leukocyte secretion inflammatory factors under the condition of existence and absence of LPS, finds that the intestinal alkaline phosphatase can inhibit the secretion of TNF- α and IL-6 of the human leukocyte under the condition of absence of LPS (without depending on endotoxin), has an action mechanism that a corresponding substrate is dephosphorylated to generate substances such as adenosine and the like so as to inhibit the secretion of TNF- α and IL-6, discloses the application of the intestinal alkaline phosphatase as an inhibitor of the secretion of TNF- α and/or IL-6 of the human leukocyte independent of endotoxin, discloses the application of the intestinal alkaline phosphatase in preparing an industrial medicament for treating inflammatory diseases related to the increase of the secretion of TNF- α and/or IL-6 of the human leukocyte independent of endotoxin, and discloses a cell activity widening detection method of a commercialized or commercialized intestinal alkaline phosphatase preparation, and greatly improves the application of the alkaline phosphatase.
Description
Technical Field
The invention relates to the field of alkaline phosphatase, in particular to a new application of intestinal alkaline phosphatase and a cell activity detection method of a preparation of the intestinal alkaline phosphatase.
Background
Alkaline phosphatase is classified into various subtypes (TNAP, IAP, PLAP), and is widely distributed in various tissues of the human body, including epithelial cells of heart, brain, placental blood vessels, intestinal mucosa, liver cells, and the like ( references 1, 2, 3, 4, 5). However, the physiological function of intestinal alkaline phosphatase has not been elucidated for half a century. It was first generally accepted that alkaline phosphatase functions to participate in bone calcification (reference 6). The inventors used gene transfer technology to introduce alkaline phosphatase gene into different cells in the early 90 s, and found that alkaline phosphatase is associated with pathological vascular calcification (references 7 and 8). What is the main physiological function of such widely distributed alkaline phosphatase? Poelstra and Meijer, scientists in the netherlands, revealed the mysteries (reference 9) and the activity of intestinal alkaline phosphatase to inactivate endotoxin was first reported. This study suggests that a wide distribution of alkaline phosphatase inhibits endotoxin-induced inflammation in and on the body. By knocking out the intestinal alkaline phosphatase gene, Bhan and Sonoko found that animals without intestinal alkaline phosphatase developed diabetes (10) and hyperlipidemia (11). Peters and Lukas, AM-Pharma, biopharmaceutical companies in the Netherlands, use recombinant human intestinal alkaline phosphatase to treat endotoxin-related sepsis kidney injury and colitis (1213).
A number of studies have shown that neutrophils in leukocytes are important cells for inducing inflammatory diseases in humans (references 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24), and that migration of neutrophils into inflamed tissues acts like a sword, which helps to remove invading microorganisms but induces inflammatory diseases, one of the mechanisms of action of which is the induction of inflammatory diseases through secretion of the major inflammatory factor TNF- α.
The corresponding references mentioned above are:
1.Schultz-Hector S,Balz K,
M,et al.Cellular localization ofendothelial alkaline phosphatase reaction product and enzyme protein in themyocardium[J].Journal of Histochemistry&Cytochemistry Official Journal of theHistochemistry Society,1993,41(12):1813.
2.Bell M A,Scarrow W G.Staining for microvascular alkalinephosphatase in thick celloidin sections of nervous tissue:Morphometric andpathological applications[J].Microvascular Research,1984,27(2):189-203
3.Birgit H,Sarah C,Nina M,et al.Relation of placental alkalinephosphatase expression in human term placenta with maternal and offspring fatmass[J].International Journal of Obesity,2018.
4.Bell L,Williams L.Histochemical demonstration of alkalinephosphatase in human intestine,normal and diseased[J].Histochemistry,1979,60(1):85-89.
5.HAGerstrand I,Lindholm K,Lindroth Y.Endothelial and BileCanalicular Alkaline Phosphatase in Human Liver and Serum[J].ScandinavianJournal of Clinical&Laboratory Investigation,1976,36(2):131-135.
6.Millan JL,Whyte MP(2016),Alkaline phosphatase and hypophosphatasia,Calcif Tissue Int 98(4):398-416.
7.Hui M,Li SQ,Holmyard D,Cheng PT(1997)Stable transfection of non-osteo-genic cell lines with tissue non specific alkaline phosphatase enhancesmineral deposition both in the presence and absence of beta glycerophosphate:Possible role for alkaline phosphatase in pathologi-cal mineralization.CalcifTissue Int 60:467-472.
8.Hui M,TenenbaumH C.New face of an old enzyme:Alkaline phosphatasemay contribute to human tissue aging by inducing tissue hardening andcalcification[J].Anatomical Record,1998,253(3):91-94.
9.Poelstra K,Bakker WW,Klok PA,Hardonk MJ,Meijer DK(1997),Aphysiologic function for alkaline phosphatase:endotoxin detoxification.LabInvest,76:319–327.
10.Bhan A,Alam S N,Raychowdhury A,et al.Intestinal alkalinephosphatase prevents metabolic syndrome in mice.[J].Proc Natl Acad Sci U S A,2013,110(17):7003-7008.
11.Sonoko Narisawa,Lei Huang,Arata Iwasaki,Hideaki Hasegawa,DavidH.Alpers,and Jose Luis Millan(2003),Accelerated Fat Absorption in IntestinalAlkaline Phosphatase Knockout Mice.MOLECULAR AND CELLULAR B IOLOGY,23(21):7525–7530.
12.Peters E,Mehta R L,Murray P T,et al.Study protocol for amulticentre randomised controlled trial:Safety,Tolerability,efficacy andquality of life Of a human recombinant alkaline Phosphatase in patients withsepsis-associated Acute Kidney Injury(STOP-AKI)[J].BMJ Open,2016,6(9):e012371
13.Lukas M1,Drastich P,Konecny M,Gionchetti P,Urban O,Cantoni F,Bortlik M,Duricova D,Bulitta M.，Exogenous alkaline phosphatase for thetreatment of patients with moderate to severe ulcerative colitis[J].Inflammatory Bowel Diseases,2010,16(7):1180-1186.
14.Moritz Peiseler,Paul Kubes.More friend than foe:the emerging roleof neutrophils in tissue repair[J].The journal of clinical investigation,2019,129(7):2629-2639.
15.Suzuki K,Exhaustive Exercise-Induced Neutrophil-Associated TissueDamage and Possibility of its Prevention[J].Journal of Nanomedicine&Biotherapeutic Discovery,2017,07(02).
16.Davalyn R.Powell,Anna Huttenlocher.Neutrophils in the TumorMicroenvironment[J].Trends in Immunology,2016,37(1):41-52.
17.Alice E Jasper,William J McIver,Elizabeth Sapey,etc.Understandingthe role of neutrophils in chronic inflammatory airway disease[J].F1000Research,2019,8:557-574.
18.Kovtun A,Messerer D A C,Scharffetter-Kochanek K,et al.Neutrophilsin Tissue Trauma of the Skin,Bone,and Lung:Two Sides of the Same Coin[J].Journal of Immunology Research,2018,2018:1-12.
19.Williams A E,Chambers R C.Neutrophils and tissue damage:is hypoxiathe key to excessive degranulation？[J].Thorax,2016,71(11):977-978.
20.Wright H L,Moots R J,Bucknall R C,et al.Neutrophil function ininflammation and inflammatory diseases[J].Rheumatology,2010,49(9):1618-1631.
21.Katsuhiko Suzuki.Involvement of neutrophils in exercise-inducedmuscle damage and its modulation[J].General Internal Medicine and ClinicalInnovations,2018，3(3):1-8.
22.Can Yang Zhang,Xinyue Dong,Jin Gao,etc.Nanoparticle-inducedneutrophil apoptosis increases survival in sepsis and alleviates neurologicaldamage in stroke[J].Sci.Adv.,2019,5(11):2375-2548.
23.Esmaeil Mortaz,Shamila D.Alipoor,Ian M.Adcock,etc.Update onNeutrophil Function in Severe Inflammation[J].Front Immunol,2018,9:2171-2185.
24.Peters E,Heuberger J A A C,Tiessen R,et al.PharmacokineticModeling and Dose Selection in a Randomized,Double-Blind,Placebo-ControlledTrial of a Human Recombinant Alkaline Phosphatase in Healthy Volunteers[J].Clinical Pharmacokinetics,2016,55(10):1227-1237.
therefore, the above prior arts have limited research on the bioactivity, physiological function and clinical application of alkaline phosphatase, and have room for further development.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a new application of alkaline phosphatase.
The present invention uses the cytological method of freshly extracted human leucocyte to secrete inflammatory factors TNF- α and IL-6, and under the condition of existence and absence of LPS the inhibition action of intestinal alkaline phosphatase on human leucocyte inflammatory factor is studied。It was found by corresponding studies that intestinal alkaline phosphatase could inhibit TNF- α and/or IL-6 secretion from human leukocytes in the absence of LPS (endotoxin independent).
The novel application of the alkaline phosphatase of the invention is obtained based on the above research, and comprises the following technical scheme:
use of intestinal alkaline phosphatase as an inhibitor of TNF- α and/or IL-6 secretion from human leukocytes in the absence of endotoxin.
As a further development of the invention, the intestinal alkaline phosphatase inhibits the secretion of human leukocytes TNF- α and/or IL-6 by dephosphorylating the substrates ATP, ADP and/or AMP, which are produced.
Further, the intestinal alkaline phosphatase inhibits the secretion of human leukocyte TNF- α and/or IL-6 by dephosphorylating a substrate which is any one of GTP, CTP, TTP, UTP, and GDP, GMP, CDP, CMP, TDP, TMP, UDP, UMP in a cell degradation product, or a cell surface protein.
Further, the intestinal alkaline phosphatase acts at the disease site where human leukocytes migrate away from the accumulation.
Further, the intestinal alkaline phosphatase is in the form of an injection.
Further, the intestinal alkaline phosphatase is recombinant or extracted intestinal alkaline phosphatase.
In another aspect, based on the above studies, the present invention also provides the use of intestinal alkaline phosphatase for the manufacture of a medicament for the treatment of inflammatory disorders associated with increased secretion of TNF- α and/or IL-6 at sites of human leukocyte disorders in the absence of endotoxin and for the treatment of inflammatory disorders associated with increased secretion of TNF- α and/or IL-6 at sites of disorders at which human leukocyte removal and accumulation occurs in the absence of endotoxin.
In still another aspect, based on the above research, the present invention provides a method for detecting cell activity of an industrial or commercial intestinal alkaline phosphatase preparation, wherein the cell activity is detected by using a cell model of human or animal leukocyte secretion of TNF- α and IL-6, and the activity of the intestinal alkaline phosphatase preparation is determined by detecting the levels of TNF- α and IL-6 secreted by human or animal leukocyte in the absence of endotoxin.
Furthermore, the cell model for the human or animal leukocyte TNF- α and IL-6 secretion adopts an interaction model between leukocytes and a human umbilical vein endothelial cell line (HUVECs) or an interaction model between leukocytes, erythrocytes and the human umbilical vein endothelial cell line, and the animal is a dog.
By adopting the technical scheme, the invention at least has the following advantages:
1. the invention discovers for the first time that the intestinal alkaline phosphatase can inhibit the secretion of human leukocyte TNF- α and IL-6 without depending on LPS, and further can be used for treating inflammatory diseases related to TNF- α and/or IL-6 increase secreted at the disease part where the leukocyte is removed and gathered, belongs to a broad-spectrum anti-inflammatory drug, greatly widens the application of the existing intestinal alkaline phosphatase, and ensures that the intestinal alkaline phosphatase not only has the treatment effect on TNF- α and IL-6 inflammatory factors induced by endotoxin.
2. The intestinal alkaline phosphatase of the invention mainly acts in blood, ATP in the blood exists as energy but not as an inflammatory factor (ATP exists as an inflammatory factor in intestinal tracts), the intestinal alkaline phosphatase obtains intermediate product AMP and final product adenosine by dephosphorylating ATP in the blood, wherein the final product adenosine can inhibit the secretion of TNF- α and IL-6 by freshly extracted human leucocytes, and the intermediate product AMP can partially inhibit the secretion of TNF- α and IL-6 by the human leucocytes.
3. Aiming at the newly discovered action mechanism of the intestinal alkaline phosphatase, the invention provides a corresponding cell activity detection method of an industrialized or commercialized preparation, which is used for quality control of whether the product is qualified or not.
Drawings
The foregoing is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and the detailed description.
FIG. 1 is a graph showing the results of SDS-PAGE electrophoresis of a solution containing high concentrations of recIAP after depyrogenation;
FIG. 2 is a graph showing the results of a high pressure liquid phase HPLC molecular sieve column detection of a solution containing a high concentration of recIAP after depyrogenation;
FIG. 3 is a graph of the dose-dependent decrease in LPS viability and recIAP after 3 hours of incubation of recIAP with LPS;
FIG. 4 is a graph showing the results of examination of the effect of LPS on TNF- α secretion using human leukocytes and HUVECs and a model of interaction between leukocytes, erythrocytes and HUVECs;
FIG. 5(A, B, C, D, E, F) is a graph showing the results of measurements of the inflammatory factors TNF- α and IL-6 secreted by human leukocytes (mainly human neutrophils) by intestinal alkaline phosphatase in the presence or absence of LPS;
FIG. 6(A, B, C, D) is a graph showing the results of measurements of extracted bovine intestinal alkaline phosphatase (bIAP) and recIAP in the presence or absence of LPS to inhibit TNF- α and IL-6 secretion from human leukocytes;
FIG. 7(A, B, C) is a graph showing the results of testing recIAP at various pH conditions for dephosphorylation of ATP, ADP, AMP and the production of products ADP, AMP and adenosine;
FIG. 8(A, B, C, D) is a graph showing the results of measurement of inhibitory effects of ATP and its dephosphorylated products, ADP, AMP, and adenosine, on TNF- α secreted from leukocytes.
FIG. 9(A, B, C, D) is the dephosphorizing effect of recIAP (0.043U/mL) on 0.5mM GTP, CTP, UTP and TTP under different pH conditions.
FIG. 10(A, B, C, D) is a graph showing the inhibitory effect of dephosphorylated products of GTP, CTP, UTP and TTP, guanosine, cytidine, uridine and thymidine, on TNF- α secreted from leukocytes.
Detailed Description
The present invention is illustrated by the following specific examples, which are presented herein for the purpose of illustration and explanation and are not intended to be limiting.
Example 1
The purpose is as follows: recombinant human intestinal alkaline phosphatase expression and production
The method comprises the following steps: the present invention used a chimeric recombinant human intestinal alkaline phosphatase (recIAP) cDNA (references 24, 25) to construct an expression vector pMH3-recIAP (reference 26), and then transferred pMH3-recIAP into CHO-S cells (CVCL _7183, Life Technologies). A DMEM/F12 culture solution containing 10% Fetal Bovine Serum (FBS) is subjected to G418 pressure screening to obtain a stable and high-expression cell clone as a seed cell line for harvesting protein, the cell clone is subjected to expanded culture by an animal cell bioreactor to obtain a harvest solution, the harvest solution is subjected to purification treatment, the harvest solution is centrifugally concentrated and added into an anion column (QXK50, GEHealthcare), and an eluent containing a high-concentration recoP part is collected and subjected to pyrogen removal by a hollow fiber column (Bogelong biotechnology limited) for later use. The solution containing high-concentration recIAP after depyrogenation is subjected to purity detection by SDS-PAGE electrophoresis and a high-pressure liquid-phase HPLC molecular sieve column, and the obtained recIAP protein solution with the purity of 92.45 percent is used for the following research.
Discussion: the concentration of each injection of recIAP from AM-Pharma was 7036U (11.26mg)/ml (reference 24), the specific activity was 625U/mg, and the purity of the injection was 99%. The specific activity of recIAP used in the present invention is 578U/mg, the purity is 92.45%, and the specific activity is close to that of the recIAP of AM-Pharma.
And (4) conclusion: from the results shown in FIGS. 1 and 2, it can be seen that the recIAP protein solution of 92.45% purity was obtained for the following example studies.
Reference documents:
25.Tina K M,Sheen C R,Silva G K C D,et al.Catalytic Signature of aHeat-Stable,Chimeric Human Alkaline Phosphatase with Therapeutic Potential[J].PLoS ONE,2014,9(2):e89374-.
26.Qian Jia,HongTao Wu,XingJun Zhou,Jian Gao,Wei Zhao,JouDi Aziz,JingShuang Wei,Lihua Hou,Shuyin Wu,Ying Zhang,XiangFeng Dong,YanMin Huang,WeiYuan Jin,HongJie Zhu,XinHui Zhao,ChunHua Huang,LiPingXing,Liwen Li,JunMa,Xiyan Liu,Ran Tao,ShuaiDong Ye,YiGao Song,LingLing Song,GuanPing Chen,ChunLing Du,XueTing Zhang,Bo Li,YanTao Wang,Wei Yang,Gilbert Rishton,YuYangTeng,GouQing Leng,LuanFeng Li,WenXi an Liu,Li Jun Cheng,QiuBo Liang,ZhengWuLi,XiuQin Zhang,Yajun Zuo,We i Chen,Huicheng Li,Matthew(Mizhou)Hui.A“GC-rich”method for mammalian gene expression:a dominant role of non-coding DNA GCcontent inregulation of mammalian gene expression.Science China Life Science,2010,53(1):94–100.
example 2
The purpose is as follows: inactivation of LPS-dependent pH and dosage by recombinant human intestinal alkaline phosphatase
The method comprises the following steps:
1. intestinal alkaline phosphatase Activity detection
The alkaline phosphatase activity was measured by using disodium phenylphosphate colorimetry (TE0007, Beijing Rayleigh Biotech Co., Ltd.). Disodium phenylphosphate is hydrolyzed by alkaline phosphatase under alkaline conditions to form free phenol and phosphate. Phenol is combined with aminoantipyrine under alkaline conditions, and the red quinoid structure with different depths is generated by oxidation. Absorbance was measured at 510nm and the level of alkaline phosphatase activity was calculated by colorimetric analysis. One activity unit (U/L) is defined as the reaction of 100ml of the test sample with disodium phenyl phosphate at 37 ℃ for 15min to yield 1mg of phenol.
2. Determination of inactivation Effect of LPS by recombinant human intestinal alkaline phosphatase
The recIAP was diluted to 50U/mL using 50mmol/L Tris-HCl buffers at different pH (6.0-8.0). 990. mu.l of recIAP and 10. mu.l of LPS solution (E.coli 0111: B4, Sigma) were mixed well (final concentration of LPS 1ug/mL) and incubated at 37 ℃ for 3 hours. The absorbance at 545nm was measured by TAL method (Tachypleus tridentatus kit, EC32545S, Xiamen Tachypleus tridentatus reagent Biotech Co., Ltd.) and LPS activity units were determined by comparison with a standard curve. Under the condition of physiological pH7.4, recIAP (enzyme activity is 1, 5 and 10U/ml) and LPS (5ng/ml) are incubated for 3 hours at 37 ℃, and the content of LPS is measured again. Blanks were made after treatment with Tris-HCl buffer solutions containing recIAP alone at various pH values (6.0-8.0) for 60 minutes at 65 ℃.
As a result:
the significant LPS inactivation effect of repap at pH7.0-8.0 and at pH6.5 (table 1) suggests that LPS inactivation by intestinal alkaline phosphatase is effective over the general range of pH variation in the extracellular fluid and blood of humans. recIAP (10U/ml) reduced the LPS (5ng/ml) viability by more than 50% after incubation for 3 hours at 37 ℃ (FIG. 3).
TABLE 1 pH range for efficient endotoxin inactivation (EU/ml) by recombinant human intestinal alkaline phosphatase (50U/ml)
Note: the LPS activity is reduced by about 60% when the pH value is 7.0 and 7.5; the LPS activity was reduced by 76.85% at pH 8.0. The data are expressed as mean ± standard deviation (n ═ 3), P <0.05 and P <0.0001 in the recIAP + LPS group compared to the LPS group.
As shown in FIG. 3, the recIAP was incubated at concentrations of 1, 5, 10U/ml and 5ng/ml LPS at 37 ℃ for 3 hours, and the results showed that the decrease in LPS activity was positively correlated with the dose of recIAP. Data are expressed as mean ± standard deviation (n ═ 3), P <0.05 in the recIAP + LPS group compared to the LPS group.
Discussion numerous studies have shown that neutrophils are important cells in the initiation of inflammatory diseases in humans (references 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24), that migration of neutrophils into inflamed tissues behaves like a sword-like sword, helping to eliminate invading microorganisms, but initiating inflammatory diseases.A method for the cytological activity of intestinal alkaline phosphatase on the influence of LPS-dependent or independent induction of TNF- α and IL-6 secretion from human leukocytes (FIG. 4, FIG. 5-CDEF). LPS stimulates human leukocytes (mainly human neutrophils) to secrete inflammatory factors TNF- α and IL-6. intestinal alkaline phosphatase is effective in inactivating LPS at pH7.0-8.0 (Table 1).
Example 3
Purpose, LPS stimulation human blood leukocyte TNF- α and IL-6 secretion model
The method comprises the following steps:
1. human venous blood leukocyte isolation
The healthy volunteers were 6 persons, aged 26 ± 5 years, and blood collection was approved by vinpocetine and ethics committee of medical care in surgical hospitals and with consent of the same. The invention adopts a sugar density gradient centrifugation method, namely a human venous blood leukocyte separation kit (endotoxin)<0.1EU, tianjin tertiary science biotechnology limited), venous blood was isolated. Collecting human venous blood at normal temperature, centrifuging at 1800rpm for 25min, sucking a mononuclear cell layer (lymphocyte and a small part of mononuclear cells) and a multinucleated cell layer (mainly neutrophilic granulocyte), mixing, fully cracking erythrocytes, repeatedly washing twice, and suspending with 3ml of 1640 medium containing 10% FBS for later use. Staining with leukocyte classification staining solution to observe cell morphology, and adjusting density to 1 × 106One/ml for later use. Blood from different volunteers was collected each time to rule out individual differences and to ensure that the experiment was reproducible.
2. Human leukocyte secretion inflammatory factor TNF- α/IL-6 model
Human umbilical vein endothelial cell lines (HUVECs) were cultured in DMEM/F12 medium containing 10% FBS adherent at 37 deg.C, suspended by trypsinization, and cell density adjusted to 1X 105One per ml. 100ul of cells were added to each well, inoculated in a 96-well plate, and cultured at 37 ℃ for 24 hours. On the day of the experiment, freshly extracted human venous blood leukocytes were cultured alone or added to 96-well plates of monolayer-cultured HUVECs, and a model of interaction between leukocytes and HUVECs and leukocytes, erythrocytes (leukocytes: erythrocytes ═ 1: 5) and HUVECs was established. LPS(0.00, 0.05, 0.10 and 0.50ng/ml) and two cell models are cultured for 24 hours, and then the secretion levels of TNF- α and IL-6 are detected.
Human ELISA kit was used for the detection of TNF- α and IL-6 levels in cell culture media, as specified by the manufacturer (TNF- α Elisa kit, R & D system, USA; IL-6 Elisa kit, Wash. CSB-E04638 h).
As a result:
HUVECs extracted from fresh human blood and human vascular endothelial cells containing a small number of monocytes and a large number of lymphocytes also have a small amount of TNF- α secretion, red blood cells are substantially free of TNF- α and IL-6 secretion.
TABLE 2 conditions of TNF- α and IL-6 secretion from human leukocytes, human vascular endothelial cells and human erythrocytes, which contain neutrophils and lymphocytes.
Note: the human leucocyte is separated into three kinds of cells of human neutrophilic granulocyte, mononuclear cell and erythrocyte by density gradient centrifugation.
The invention establishes a model (figure 4) of interaction among human leucocytes, HUVECs and leucocytes, erythrocytes and HUVECs, and is used for researching the new biological activity, new physiological function and potential new clinical effect of the intestinal alkaline phosphatase on human.
As shown in FIG. 4, the present inventors established a model of the interaction between human leukocytes and HUVECs and leukocytes, erythrocytes and HUVECs to study the novel anti-inflammatory biological activity of intestinal alkaline phosphatase in humans, and also showed that LPS dose-dependently promoted secretion of TNF- α from leukocytes in the presence of erythrocytes and HUVECs at concentrations ranging from 0.0 to 0.5ng/ml, note that the data are expressed as mean. + -. standard deviation (n. 4.) in the model of human leukocytes and HUVECs, P <0.05 and P <0.0001 in the model of LPS versus control, P <0.05 and P <0.0001 #.in the model of human leukocytes, erythrocytes and HUVECs.
Discussion: numerous studies have shown that neutrophils are important cells in the initiation of inflammatory diseases in humans (references 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24), and that migration of neutrophils into inflamed tissue acts like a double sword, although helping to eliminate invading microorganisms, but initiating inflammatory diseases. The invention establishes a model (figure 4) of interaction among human leucocytes, HUVECs and leucocytes, erythrocytes and HUVECs, and is used for researching the new biological activity, new physiological function and potential new clinical effect of the intestinal alkaline phosphatase on human.
The present invention shows that LPS in the concentration of 0-0.5ng/ml promotes TNF- α secretion in the presence of leucocyte, erythrocyte and HUVECs in dosage dependent mode.
Example 4
The aim is that the recombinant human intestinal alkaline phosphatase inhibits the secretion of TNF- α and IL-6 from leucocytes independent of LPS
The method comprises the following steps:
1. human venous blood leukocyte isolation: the same as in example 3.
The HUVECs culture method comprises the following steps: as in example 3.
3. On the day of the experiment, recIAP (5, 10, 20U/ml) was incubated with 0.5ng/ml LPS for 3 hours in advance or with addition of stimulating leukocytes simultaneously, cultured at 37 ℃ for 24 hours, and then culture supernatant was collected to detect TNF- α. recIAP (0.5, 1.0, 2.0, 4.0U/ml) and 0.5ng/ml LPS were simultaneously added to stimulate leukocytes, and TNF- α and IL-6 levels were detected by ELISA after 24 hours of culture.
As a result:
first, the recIAP was incubated with LPS for 3 hours in advance or added with the LPS to stimulate TNF- α secretion by leukocytes, which stimulated TNF- α secretion to a similar extent (FIG. 5AB), suggesting that recombinant human intestinal alkaline phosphatase inhibits TNF- α secretion by leukocytes independently of LPS, and that recIAP also inhibits TNF- α secretion by leukocytes in the absence of LPS (FIG. 5CD), indicating that recIAP significantly reduces TNF- α secretion in the presence or absence of LPS, and further that recIAP also inhibits IL-6 secretion by leukocytes in the absence of LPS (FIG. 5EF), indicating that recIAP significantly reduces IL-6 secretion in the presence or absence of LPS.
As shown in fig. 5, the inhibitory effect of recipp on inflammatory factors TNF- α and IL-6 was independent of LPS-recipp (5, 10, 20U/ml) incubated with LPS (0.5ng/ml) for 3 hours in advance or with LPS to stimulate the secretion of TNF- α - α to a similar extent (A B). recIAP (5U/ml) also inhibited the secretion of TNF- α (CD) by leukocytes in the absence of LPS, indicating that recipp (5U/ml) significantly reduced the secretion of TNF- α in the presence or absence of LPS, further, iapcec (5U/ml) also inhibited the secretion of IL-6(E F) in the absence or presence of LPS, indicating that recIAP (5U/ml) significantly reduced the secretion of IL-6 in the presence or absence of LPS, wherein the interplay of leukocytes, erythrocytes and HUVECs (ACE), the interplay of leukocytes and HUVECs (5U/ml) are poor compared to control # 0.0001 #. # 0.0001 < LPS-0001 #. # 0001.
The present invention relates to a method for inhibiting inflammatory factors TNF- α and IL-6 secreted by human leucocyte (mainly human neutrophil) under the condition of that the intestinal alkaline phosphatase can inhibit LPS induction, and it is not closely related to extent of inhibiting LPS by using intestinal alkaline phosphatase, and it is indicated that the inhibition action of intestinal alkaline phosphatase on TNF- α secreted by human leucocyte is not related to extent of inhibiting LPS by using intestinal alkaline phosphatase.
250, 500, 1000U/kg (12) of recIAP from AM-Pharma, up to an average weight of 50kg in humans requiring 12500, 25000, 50000U per treatment, the effective dose of the inflammatory factors TNF- α and IL-6 of the present invention using recIAP to inhibit secretion of human leukocytes is 2.5-5U/ml, up to an average weight of 50kg in humans (5L blood, 3L plasma), the concentration of each injection of recIAP from 7500 and 15000 U.AM-Pharma is 7036U (11.26mg)/ml per injection (ref 24), the specific activity is 625U/mg, the purity of the injection is 99% per treatment, the specific activity of the recIAP used herein is 625U/mg, the purity is 92.45%, the specific activity of the recIAP close to AM-Pharma is near to that of TNF- α more specific activity associated with secretion of TNF-35578 in humans.
Conclusion the present invention demonstrates that intestinal alkaline phosphatase inhibits the inflammatory factors TNF- α and IL-6 secreted by LPS-induced human leukocytes (mainly human neutrophils). The present invention found that intestinal alkaline phosphatase still inhibits the inflammatory factors TNF- α and IL-6 secreted by human leukocytes (mainly human neutrophils) in the absence of LPS (FIGS. 5-C and D, E and F). The results of the above studies indicate that intestinal alkaline phosphatase inhibits the secretion of TNF- α and IL-6 by human leukocytes, not by inactivation of LPS, but directly inhibits the secretion of inflammatory factors TNF- α and IL-6 by human neutrophils.
Example 5
Aims at extracting the bovine intestinal alkaline phosphatase, which inhibits the secretion of TNF- α and IL-6 by human leucocyte like the recombinant human intestinal alkaline phosphatase.
The method comprises the following steps:
1. human venous blood leukocyte isolation: the same as in example 3.
The HUVECs culture method comprises the following steps: as in example 3.
3. On the day of the experiment, 5U/ml of recIAP and bIAP (P6774-2KU, Sigma) were added together with 0.5ng/ml of LPS to stimulate leukocytes, and the culture broth after 24 hours of culture was collected to detect TNF- α and IL-6 by ELISA.
As a result:
extraction of both bovine intestinal alkaline phosphatase (bIAP) and recIAP inhibited TNF- α and IL-6 secretion from human leukocytes in the presence and absence of LPS (FIG. 6ABCD), suggesting that intestinal alkaline phosphatase has inflammatory factor-inhibiting and anti-inflammatory effects on the intestinal mucosa.
As shown in fig. 6, both the balp and the recap inhibited the secretion of TNF- α and IL-6 by human leukocytes in the presence and absence of LPS (fig. 6ABCD), wherein the model of interaction between leukocytes, erythrocytes and HUVECs (AC) and the model of interaction between leukocytes and HUVECs (BD), the data of the present invention are expressed as mean ± standard deviation (n ═ 4), P <0.0001# #### # compared to the control group and P <0.0001^ compared to the LPS group.
Discussion: the bIAP is also used to treat LPS-related diseases in humans in the same manner as recIAP, and the effect is substantially the same as that of recIAP.
As a result, both bIAP and recIAP inhibit the secretion of TNF- α and IL-6 by human leukocytes in the presence or absence of LPS.
Example 6
Aims at inhibiting the action mechanism of TNF- α secreted by leucocyte by recombinant human intestinal alkaline phosphatase
The method comprises the following steps:
1. phosphomolybdic acid method for determining free phosphorus release amount of intestinal alkaline phosphatase substrate
Preparing 10mM ATP, ADP, AMP solutions (ATP, Biotopped; ADP and AMP, Zostera Marinae) at different pH (6.0-8.0) using 50mmol/L Tris-HCl buffers at different pH (6.0-8.0); taking recIAP (1000U/ml) for gradient dilution to be 0.5, 5.0 and 50.0U/ml; 950. mu.l of recIAP at different concentrations and 50. mu.l of ATP, ADP and AMP solutions (each at a final concentration of 0.5mM) at different pH values (6.0-8.0) were mixed well and incubated at 37 ℃ for 60 minutes. Blanks were made after treatment with Tris-HCl buffer solutions containing recIAP alone at various pH values (6.0-8.0) for 60 minutes at 65 ℃. The content of free phosphorus Pi released from the recIAP-treated ATP, ADP and AMP solutions (final concentration 0.5mM) at different pH values was measured using an inorganic phosphorus assay kit (C006-1-1, Nanjing institute of bioengineering).
2. Human venous blood leukocyte isolation: the same as in example 3.
The HUVECs culture method comprises the following steps: as in example 3.
4. On the day of the experiment, low concentrations (0.10, 0.25, 0.50, 1.00uM) of ATP, ADP, AMP, and adenosine were added to the model of interaction between leukocytes and vascular endothelial cells, and culture supernatants were collected after 24 hours of culture to determine the level of TNF- α secretion.
As a result:
the Lukas of AM-Pharma uses recombinant human intestinal alkaline phosphatase for endotoxin-related colitis (reference 13), suggesting that recIAP inhibits ATP-induced inflammation by intestinal microorganisms by dephosphorylating ATP, but ATP is in the intestinal tract, unlike blood, where it acts as an inflammatory factor, and is used to provide energy in the blood without inflammation, by reducing the concentration of inflammatory factor, thereby reducing the amount of inflammatory factor induced by it, whereas in the present invention, adenosine, which is a product of dephosphorylation, is used to inhibit inflammatory factors that have already been produced by dephosphorylation of non-inflammatory factors, and additionally, the present results indicate that recIAP dephosphorylates ATP, ADP, and AMP in a dose-dependent manner, and produces the products ADP, AMP and adenosine (FIG. 7 ABC). The present invention also found that AMP and adenosine, which are products of dephosphorylation, have a partial effect of inhibiting TNF- α secretion by human leukocytes (FIG. 8 CD).
As shown in fig. 7, the present results indicate that recap dephosphorizes ATP, ADP, AMP in dose and pH dependent manner and produces the final product adenosine the present invention also found that ATP dephosphorized product adenosine and AMP inhibited TNF- α partially secreted from human leukocytes the present data are expressed as mean ± standard deviation (n ═ 3) with P <0.0001 ×, compared to LPS group.
As shown in fig. 8, the present inventors also found that adenosine, ADP and AMP, final products of ATP dephosphorylation, had a partial inhibitory effect on TNF- α secreted from human neutrophils (ABC). the present data are expressed as mean ± standard deviation (n ═ 3), and P <0.05 x and P <0.0001 x in the treated group compared to the control group.
Discussion:
in addition to inhibiting LPS activity (fig. 5-a and B), another potential mechanism of intestinal alkaline phosphatase for inhibition of the cellular inflammatory factor TNF- α is dephosphorylation of ATP, which dephosphorylates intestinal alkaline phosphatase matrix ATP and gradually produces ADP, AMP, and adenosine, the present inventors found that alkaline phosphatase partially inhibits the inflammatory factor TNF- α secreted by human leukocytes (mainly human neutrophils) by dephosphorylating substrates ATP, ADP, AMP, etc. to form adenosine and AMP in the absence of LPS, indicating that adenosine has an anti-inflammatory effect that partially reduces TNF- α production by human neutrophils (fig. 8-D), and thus intestinal alkaline phosphatase also inhibits the secretion of inflammatory factor TNF- α by dephosphorylation of human leukocytes by other substrates, including the degradation products adenine, guanosine, cytosine, thymidine, adenosine and RNA degradation products adenine, guanosine triphosphate, guanine and uridine, guanine triphosphate, adenosine triphosphate, and RNA degradation products, which are also dephosphorylated by physiological substrates ATP-6, alkaline phosphatase is also a highly effective substance for degradation of intracellular TNF-366, and alkaline phosphatase to inhibit the intracellular degradation of inflammatory factors.
And (4) conclusion:
the results of the invention show that alkaline phosphatase partially inhibits inflammatory factor TNF- α secreted by human leucocytes (mainly human neutrophils) by dephosphorylating substrates such as ATP, ADP, AMP and the like to form adenosine and AMP in the absence of LPS.
Example 7
Aims at inhibiting the action mechanism of TNF- α secretion of leucocyte by the degradation product of recombinant human intestinal alkaline phosphatase
The method comprises the following steps:
1. phosphomolybdic acid method for determining free phosphorus release amount of intestinal alkaline phosphatase substrate
Preparing 10mM CTP, GTP, TTP and UTP (Shanghai-derived leafy organisms) solutions with different pH values (6.0-8.0) by using 50mmol/L Tris-HCl buffer solutions with different pH values (6.0-8.0); taking recIAP (1000U/ml) and diluting the recIAP to 0.05U/ml in a gradient way; 950. mu.l of recIAP (0.05U/ml) and 50. mu.l of CTP, GTP, TTP and UTP solutions (final concentration 0.5mM each) with different pH values (6.0-8.0) were mixed well and incubated at 37 ℃ for 60 minutes. Blanks were made after treatment with Tris-HCl buffer solutions containing recIAP alone at various pH values (6.0-8.0) for 60 minutes at 65 ℃. The content of free phosphorus Pi released by recIAP-treated CTP, GTP, TTP and UTP solutions (final concentration 0.5mM) with different pH values was measured using an inorganic phosphorus test kit (C006-1-1, Nanjing institute of bioengineering).
2. Human venous blood leukocyte isolation: the same as in example 3.
The HUVECs culture method comprises the following steps: as in example 3.
4. On the day of the experiment, low concentrations (0.10, 0.25, 0.50, 1.00uM) of guanosine, cytidine, uridine, thymidine were added to the model of interaction between leukocytes and vascular endothelial cells, and culture supernatants were collected after 24 hours of culture to determine the level of TNF- α secretion.
As a result:
the Lukas of AM-Pharma uses recombinant human intestinal alkaline phosphatase for endotoxin-related colitis (reference 13), suggesting that recIAP inhibits ATP-induced inflammation by intestinal microorganisms by dephosphorylating ATP, but ATP is in the intestinal tract, unlike blood, where it acts as an inflammatory factor, and is used to provide energy in the blood without inflammation, by reducing the concentration of inflammatory factor, thereby reducing the amount of inflammatory factor induced by it, whereas in the present invention, adenosine, which is a product of dephosphorylation, is used to inhibit inflammatory factors that have already been produced by dephosphorylation of non-inflammatory factors, and additionally, the present results indicate that recIAP dephosphorylates ATP, ADP, and AMP in a dose-dependent manner, and produces the products ADP, AMP and adenosine (FIG. 7 ABC). The present invention also found that AMP and adenosine, which are products of dephosphorylation, have a partial effect of inhibiting TNF- α secretion by human leukocytes (FIG. 8 CD).
As shown in fig. 7, the present results indicate that recap dephosphorizes ATP, ADP, AMP in dose and pH dependent manner and produces the final product adenosine the present invention also found that ATP dephosphorized product adenosine and AMP inhibited TNF- α partially secreted from human leukocytes the present data are expressed as mean ± standard deviation (n ═ 3) with P <0.0001 ×, compared to LPS group.
As shown in fig. 8, the present inventors also found that the ATP dephosphorylated end products adenosine ADP and AMP had partial inhibition of TNF- α secretion from human neutrophils (ABC). the present data are expressed as mean ± standard deviation (n ═ 3), P <0.05 and P <0.0001 in the treated groups compared to the control group.
Discussion:
in addition to inhibiting LPS activity (fig. 5-a and B), another potential mechanism of intestinal alkaline phosphatase for inhibition of the cellular inflammatory factor TNF- α is dephosphorylation of ATP, intestinal alkaline phosphatase dephosphorylates intestinal alkaline phosphatase matrix ATP and gradually produces ADP, AMP, and adenosine, it was found in the present invention that alkaline phosphatase partially inhibits the inflammatory factor TNF- α secreted by human leukocytes (mainly human neutrophils) by dephosphorylating substrates ATP, ADP, AMP, etc. in the absence of LPS (fig. 8-D), suggesting that adenosine has an anti-inflammatory effect that partially reduces the production of TNF- α by human neutrophils (fig. 8-D), and thus intestinal alkaline phosphatase also inhibits the secretion of the inflammatory factor TNF- α by dephosphorylation of human neutrophils via dephosphorylation of other substrates, including the degradation products purine nucleoside triphosphate, guanosine triphosphate, cytosine nucleoside triphosphate, thymidine nucleoside and RNA degradation products adenine nucleoside triphosphate, guanine nucleoside triphosphate, and RNA degradation products, etc. as well as a result of alkaline phosphatase degradation of intracellular alkaline phosphatase substrate ATP, alkaline phosphatase, ATP, AMP, and AMP, which are also a biological substrate, a biological substance capable of degrading, and other substrate, and a biological substance capable of rapidly degrading human leukocyte, and a biological substance capable of inhibiting the inflammatory factor, and a biological substance capable of degrading inflammatory factor, and a biological substance, or a biological substance capable of degrading human neutrophil, such as shown in the result of a biological substance.
And (4) conclusion:
the results of the invention show that alkaline phosphatase partially inhibits inflammatory factor TNF- α secreted by human leucocytes (mainly human neutrophils) by dephosphorylating substrates such as ATP, ADP, AMP and the like to form adenosine and AMP in the absence of LPS.
Example 8
The purpose is as follows: the statistical analysis method applied to examples 1-6 above.
The method comprises the following steps: all data are expressed as mean ± standard deviation or standard deviation, both data are compared using two t-tests in the batch, P <0.05 and P <0.01 are considered statistically significant and very statistically significant, respectively. Cell culture results were taken in triplicate and repeated at least 2 times. The data analysis was performed using Graph prism 6.0, and the results are shown in the table or Graph.
As a result: see examples 1-7.
And (4) conclusion: the statistical analysis method is simple and can be effectively applied to the statistical analysis of the results of examples 1-6.
In summary, the present invention relates to a new human leukocyte production model for TNF- α and IL-6 secretion, which is found to increase the secretion of TNF- α and IL-6 in a dose-dependent manner by using freshly extracted human leukocytes, and to a new human leukocyte production model for the reduction of endotoxin-induced TNF- α and IL-6 secretion, which is found to inhibit the secretion of freshly extracted human leukocyte TNF- α and IL-6 in the absence of endotoxin, which indicates that the intestinal alkaline phosphatase inhibits the secretion of human leukocyte TNF- α and IL-6 by an endotoxin-independent mechanism, and is an inhibitor of the secretion of human leukocyte TNF-5639 and IL-6, which is a non-endotoxin-dependent human leukocyte secretion inhibitor of TNF- α and IL-6, which suggests that the secretion of human leukocyte production of TNF- α and IL-356 by using a new human leukocyte production model for the reduction of TNF-6 secretion, which is found to inhibit the accumulation of human leukocyte accumulation of TNF-5636 and IL-356 in a healthy human leukocyte production model for the production of human inflammatory diseases.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the present invention in any way, and it will be apparent to those skilled in the art that the above description of the present invention can be applied to various modifications, equivalent variations or modifications without departing from the spirit and scope of the present invention.
Claims (10)
1. Use of intestinal alkaline phosphatase as an inhibitor of TNF- α and/or IL-6 secretion from human leukocytes in the absence of endotoxin.
2. Use according to claim 1, wherein the intestinal alkaline phosphatase inhibits the secretion of human leukocytes TNF- α and/or IL-6 by dephosphorylating the substrates ATP, ADP and/or AMP, which produce adenosine or AMP.
3. The use according to claim 1, wherein the intestinal alkaline phosphatase inhibits the secretion of human leukocytes TNF- α and/or IL-6 by dephosphorylation of the substrate;
the substrate is any one of GTP, CTP, TTP, UTP and GDP, GMP, CDP, CMP, TDP, TMP, UDP and UMP in cell degradation products;
or the substrate is a cell surface protein.
4. The use of claim 1, wherein the intestinal alkaline phosphatase is active at the disease site where human leukocytes migrate away from the accumulation.
5. The use according to claim 1, wherein the intestinal alkaline phosphatase is in the form of an injection.
6. Use according to claim 1, wherein the intestinal alkaline phosphatase is recombinant or extracted intestinal alkaline phosphatase.
7. Use of intestinal alkaline phosphatase in the preparation of a medicament for the treatment of inflammatory diseases associated with increased secretion of TNF- α and/or IL-6 from human leukocyte disease sites in the absence of endotoxin.
8. Use of intestinal alkaline phosphatase for the treatment of inflammatory disorders associated with increased secretion of TNF- α and/or IL-6 from a diseased part of human leukocytes in the absence of endotoxin.
9. An industrialized or commercialized method for detecting cell activity of intestinal alkaline phosphatase preparation, characterized in that cell activity detection is carried out by using cell model secreted by human or animal leukocyte TNF- α and IL-6;
the activity of the intestinal alkaline phosphatase preparation is judged by measuring the levels of TNF- α and IL-6 secretion by human or animal leukocytes in the absence of endotoxin.
10. The method for detecting the activity of the cells according to claim 9, wherein the cell model for the secretion of human or animal leukocytes TNF- α and IL-6 uses:
model of interaction between leukocyte and endothelial cell line of umbilical vein;
or a model of interaction between leukocytes, erythrocytes and human umbilical vein endothelial cell lines;
the animal is a dog.
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| CN1917899A (en) * | 2004-02-04 | 2007-02-21 | 塞浦西斯阿瓦药物集团 | Use of alkaline phosphatase for the detoxification of lps |
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| CN1917899A (en) * | 2004-02-04 | 2007-02-21 | 塞浦西斯阿瓦药物集团 | Use of alkaline phosphatase for the detoxification of lps |
| WO2011100543A2 (en) * | 2010-02-12 | 2011-08-18 | The General Hospital Corporation | Methods of reducing or inhibiting toxic effects associated with a bacterial infection using alkaline phosphatase |
| WO2012054057A1 (en) * | 2010-10-22 | 2012-04-26 | Zoltan Kiss Consulting | Alkaline phosphatase to correct insulin resistance, hyperinsulinemia, and dyslipidemia |
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