CN108309987B - Pcn在制备用于治疗急性肾损伤相关病症的药物中的应用 - Google Patents
Pcn在制备用于治疗急性肾损伤相关病症的药物中的应用 Download PDFInfo
- Publication number
- CN108309987B CN108309987B CN201810121267.4A CN201810121267A CN108309987B CN 108309987 B CN108309987 B CN 108309987B CN 201810121267 A CN201810121267 A CN 201810121267A CN 108309987 B CN108309987 B CN 108309987B
- Authority
- CN
- China
- Prior art keywords
- pcn
- cisplatin
- induced
- cells
- apoptosis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 208000009304 Acute Kidney Injury Diseases 0.000 title claims description 29
- 208000033626 Renal failure acute Diseases 0.000 title claims description 29
- 201000011040 acute kidney failure Diseases 0.000 title claims description 29
- 239000003814 drug Substances 0.000 title claims description 13
- 238000002360 preparation method Methods 0.000 title claims description 7
- 229960004316 cisplatin Drugs 0.000 claims description 63
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 claims description 63
- 230000014509 gene expression Effects 0.000 claims description 29
- 230000006907 apoptotic process Effects 0.000 claims description 27
- 108010001511 Pregnane X Receptor Proteins 0.000 claims description 13
- 102000000804 Pregnane X Receptor Human genes 0.000 claims description 13
- 230000002757 inflammatory effect Effects 0.000 claims description 9
- 230000028327 secretion Effects 0.000 claims description 7
- 239000000556 agonist Substances 0.000 claims description 3
- 230000004898 mitochondrial function Effects 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 description 37
- 238000011282 treatment Methods 0.000 description 24
- 208000028867 ischemia Diseases 0.000 description 19
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 10
- 238000010186 staining Methods 0.000 description 10
- 230000006378 damage Effects 0.000 description 9
- 239000003642 reactive oxygen metabolite Substances 0.000 description 9
- 238000003753 real-time PCR Methods 0.000 description 9
- 108020005196 Mitochondrial DNA Proteins 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 210000004369 blood Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 102000055102 bcl-2-Associated X Human genes 0.000 description 6
- 229940109239 creatinine Drugs 0.000 description 6
- 229940079593 drug Drugs 0.000 description 6
- 230000003907 kidney function Effects 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 210000004926 tubular epithelial cell Anatomy 0.000 description 6
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 5
- 102100021569 Apoptosis regulator Bcl-2 Human genes 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 101000971171 Homo sapiens Apoptosis regulator Bcl-2 Proteins 0.000 description 5
- 102000004889 Interleukin-6 Human genes 0.000 description 5
- 108090001005 Interleukin-6 Proteins 0.000 description 5
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 5
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 5
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 5
- 230000003698 anagen phase Effects 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 238000000684 flow cytometry Methods 0.000 description 5
- 230000003834 intracellular effect Effects 0.000 description 5
- 206010061481 Renal injury Diseases 0.000 description 4
- 230000005754 cellular signaling Effects 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 239000007928 intraperitoneal injection Substances 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 210000005084 renal tissue Anatomy 0.000 description 4
- 108090000672 Annexin A5 Proteins 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 239000006180 TBST buffer Substances 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 208000035850 clinical syndrome Diseases 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 210000003470 mitochondria Anatomy 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- -1 20 mg/kg) Substances 0.000 description 2
- 208000003918 Acute Kidney Tubular Necrosis Diseases 0.000 description 2
- 102000004121 Annexin A5 Human genes 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 2
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 102000013519 Lipocalin-2 Human genes 0.000 description 2
- 108010051335 Lipocalin-2 Proteins 0.000 description 2
- 206010038540 Renal tubular necrosis Diseases 0.000 description 2
- 206010063837 Reperfusion injury Diseases 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 238000001378 electrochemiluminescence detection Methods 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 208000012947 ischemia reperfusion injury Diseases 0.000 description 2
- 208000037806 kidney injury Diseases 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 210000001700 mitochondrial membrane Anatomy 0.000 description 2
- 230000003589 nefrotoxic effect Effects 0.000 description 2
- 231100000381 nephrotoxic Toxicity 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- 210000005239 tubule Anatomy 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- 230000002407 ATP formation Effects 0.000 description 1
- 208000010444 Acidosis Diseases 0.000 description 1
- 102100034283 Annexin A5 Human genes 0.000 description 1
- 201000003126 Anuria Diseases 0.000 description 1
- 102000007272 Apoptosis Inducing Factor Human genes 0.000 description 1
- 108010033604 Apoptosis Inducing Factor Proteins 0.000 description 1
- 108010063104 Apoptosis Regulatory Proteins Proteins 0.000 description 1
- 102000010565 Apoptosis Regulatory Proteins Human genes 0.000 description 1
- 208000037157 Azotemia Diseases 0.000 description 1
- 102000051485 Bcl-2 family Human genes 0.000 description 1
- 108700038897 Bcl-2 family Proteins 0.000 description 1
- JUQPZRLQQYSMEQ-UHFFFAOYSA-N CI Basic red 9 Chemical compound [Cl-].C1=CC(N)=CC=C1C(C=1C=CC(N)=CC=1)=C1C=CC(=[NH2+])C=C1 JUQPZRLQQYSMEQ-UHFFFAOYSA-N 0.000 description 1
- 102000003952 Caspase 3 Human genes 0.000 description 1
- 108090000397 Caspase 3 Proteins 0.000 description 1
- 102000011727 Caspases Human genes 0.000 description 1
- 108010076667 Caspases Proteins 0.000 description 1
- 102000018832 Cytochromes Human genes 0.000 description 1
- 108010052832 Cytochromes Proteins 0.000 description 1
- 206010014418 Electrolyte imbalance Diseases 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 101000911513 Homo sapiens Uncharacterized protein FAM215A Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010023421 Kidney fibrosis Diseases 0.000 description 1
- 206010027417 Metabolic acidosis Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010052641 Mitochondrial DNA mutation Diseases 0.000 description 1
- 108010067028 Mitochondrial Permeability Transition Pore Proteins 0.000 description 1
- 208000034486 Multi-organ failure Diseases 0.000 description 1
- 208000010718 Multiple Organ Failure Diseases 0.000 description 1
- 208000004221 Multiple Trauma Diseases 0.000 description 1
- 208000023637 Multiple injury Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 206010030302 Oliguria Diseases 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 102100026728 Uncharacterized protein FAM215A Human genes 0.000 description 1
- 238000008083 Urea Assay Methods 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 208000012998 acute renal failure Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 229940052223 basic fuchsin Drugs 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000001964 calcium overload Effects 0.000 description 1
- 238000010523 cascade reaction Methods 0.000 description 1
- 230000006727 cell loss Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 208000020832 chronic kidney disease Diseases 0.000 description 1
- 229940105442 cisplatin injection Drugs 0.000 description 1
- 238000009096 combination chemotherapy Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000004163 cytometry Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000008260 defense mechanism Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000012137 double-staining Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 208000037888 epithelial cell injury Diseases 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000006676 mitochondrial damage Effects 0.000 description 1
- 230000004065 mitochondrial dysfunction Effects 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 208000029744 multiple organ dysfunction syndrome Diseases 0.000 description 1
- 210000003739 neck Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 231100000417 nephrotoxicity Toxicity 0.000 description 1
- 231100000637 nephrotoxin Toxicity 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000010627 oxidative phosphorylation Effects 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 230000000861 pro-apoptotic effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000012959 renal replacement therapy Methods 0.000 description 1
- 230000010410 reperfusion Effects 0.000 description 1
- 230000027756 respiratory electron transport chain Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000005382 thermal cycling Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 210000005233 tubule cell Anatomy 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了一种pregnenolone‑16a‑carbonitrile(PCN)的新适应症。本发明提出了PCN可以减轻顺铂和缺血再灌注诱导的急性肾损伤相关病症，包括急性肾小管损伤、肾功能、肾小管细胞凋亡、炎症因子的表达及分泌等病症，主要是通过抑制肾小管细胞的凋亡来减轻急性肾损伤。
Description
技术领域
本发明涉及PCN的新应用，具体地涉及PCN在制备用于减轻缺血再灌注和顺铂诱导的急性肾损伤相关病症的药物中的应用。
背景技术
急性肾损伤(acute kidney injury，AKI)是指突然发生的肾功能下降，可伴或不伴有少尿或无尿，为由肾小球、肾小管、肾间质或血管等病变引起的肾功能在短期内急剧下降或丧失的临床综合征，表现为突然不能保持正常的水电解质平衡，体内代谢产物堆积，出现氮质血症、水及电解质紊乱和代谢性酸中毒等症状。急性肾损伤是一种可继发于多种疾病的临床综合征，在重症监护病房更为常见，具有发病急、进展快、病死率高的特点，是慢性肾脏病的重要病因之一。它是一组严重的临床综合征群，死亡率高，延误诊治或治疗不当可导致肾功能呈不可逆改变，使患者进入维持性透析，给社会经济带来很大负面影响。
AKI的患病率社区为1％，在医院为7.1％。发病率为每年医院获得的AKI的病死率为10％～80％，合并多脏器功能衰竭的病死率>50％，需要肾替代疗法的病死率是80％，因此AKI具有高发病率和致死率。它的主要病因是缺血，肾有毒物质所致或原发性肾脏病，肾小管上皮细胞(Renal tubular epithelial cells，RTEC)损伤是AKI的主要病理基础。在疾病状态下，功能活跃的RTEC更容易受到肾毒素等造成的损伤而发生急性肾小管坏死(AcuteTubular Necrosis，ATN)，引起AKI及肾功能衰竭。
缺血再灌注(ischemia reperfusion，IR)损伤是临床AKI的常见病因。缺血再灌注损伤涉及多种损伤机制，包括自由基的作用、细胞内钙超载和白细胞激活。而肾脏小管上皮细胞损伤又是各种损伤因子的靶细胞。虽然目前治疗方法有了改进，但是缺血再灌注的死亡率仍然居高不下，所以寻找新的治疗方法迫在眉睫。
顺铂(Cisplatin，CP)是临床上治疗实体肿瘤包括头部、颈部、卵巢和睾丸最常用及最有效的化疗药物之一，因其抗癌作用强，与其他肿瘤药物无交叉耐药，也是联合化疗中最常用的抗肿瘤药物之一。临床调查表明，由于其具有剂量依赖性急性肾脏毒性作用，极大地限制其临床应用，顺铂诱导的AKI发生率为25％～35％，其引起损伤的主要机制包括诱导肾小管上皮细胞的氧化应激引起细胞凋亡，并且能够触发炎症反应参与肾脏损伤。如何切实有效地预防和减轻顺铂引起的AKI，更好地发挥顺铂抗肿瘤作用已成为当前亟待解决的问题。
在病人和动物模型中肾小管细胞丢失——包括小管凋亡和坏死被认为是AKI发生发展中主要的参与细胞。另外，脱落的肾小管细胞与蛋白在管腔中形成管型阻断尿流，损伤的小管可能会进一步促进肾脏间质纤维化、间质炎症、毛细血管丢失。所以如何阻止肾小管细胞丢失是治疗AKI的关键。
线粒体是细胞的“能量工厂”，是合成ATP、电子传递和氧化磷酸化的主要场所。线粒体功能障碍在肾损伤中的作用已得到广泛关注。业已证明，肾毒性药物所致的急性肾损伤模型中，均存在线粒体形态异常、线粒体DNA突变、线粒体DNA拷贝数下降以及细胞凋亡。线粒体在细胞凋亡中具有核心地位和作用，是细胞凋亡发生的主要途径。顺铂进入细胞后能够激活Bcl-2家族中的促凋亡蛋白如Bax，促使线粒体通透性转换孔开放，细胞色素C、凋亡诱导因子等促凋亡因子从线粒体释放到胞浆，产生caspase级联反应，最终导致凋亡的发生。
PCN是孕烷X受体(pregnane X receptor，PXR)的激动剂。PXR作为内源及外源物激活受体在机体的防御机制中发挥重要的生物调节作用和＂解毒＂功能。此外，大量研究还表明PXR通过调节下游靶基因的表达而广泛参与机体的物质及能量代谢，并在某些疾病的发生发展中发挥重要作用。目前关于PXR的研究较多集中于肝脏脂质代谢、心血管、肥胖等方面。而在AKI方面尚未有研究报道。目前，也尚未有PCN可以对抗缺血再灌注和顺铂引起的AKI的报道。
发明内容
本发明的目的在于提供一种PCN在制备用于减轻缺血再灌注和顺铂诱导的AKI相关病症的药物中的应用。
本发明通过动物实验和体外细胞实验两个方面发现PCN对于减轻缺血再灌注或顺铂诱导的AKI相关病症的药物中的应用。
具体地，本发明提出了PCN在制备用于减轻缺血再灌注和顺铂诱导的AKI肾功能的药物中的应用。
本发明还提出了PCN在制备用于减轻顺铂诱导的肾脏炎症分子的表达及分泌的药物中的应用。
进一步地，本发明发现了PCN在制备用于减轻顺铂诱导的肾小管细胞凋亡的药物中的应用。
同时，本发明还发现PCN在制备用于顺铂诱导的肾脏小管细胞线粒体损伤药物中的应用。
附图说明
图1为用顺铂和缺血再灌注后造模以及用PCN治疗后的肾功能及糖原染色(PAS)结果；
图2为QPCR法及ELISA法研究PCN对顺铂诱导的肾脏炎症分子的表达及分泌的影响；
图3为QPCR法研究PCN对顺铂诱导的小管细胞凋亡及肾脏损伤相关分子的影响；
图4为流式细胞及QPCR法检测PCN在体外对顺铂诱导的小管细胞凋亡、活性氧及线粒体DNA拷贝数的影响；
图5为Western blot法研究PCN在体外对顺铂诱导的肾脏小管细胞凋亡的影响。
具体实施方式
本发明中Western blot、实时荧光定量PCR、PAS染色、流式细胞法的具体操作步骤如下：
Western blot：
组织裂解液提取肾脏组织总蛋白，采用BCA法测定蛋白质浓度，取30μg蛋白上样，10％或12％聚丙烯酰胺凝胶电泳(SDS.PAGE)，300mA×1.5h湿转转至PVDF膜上，封闭液室温封闭1h，TBST洗脱后加入一抗，Bax(Cell Signaling Technology)，Bcl-2(CellSignalingTechnology)，cleaved caspase3(Cell SignalingTechnology)，PXR(Abcam)，GAPDH(Cell SignalingTechnology)，4℃孵育过夜。TBST洗膜5次，每次5min，以相应二抗室温孵育1h，TBST洗膜5次。抗原.抗体复合物用增强化学发光法(ECL)显示，暗室X线胶片曝光并扫描，蛋白质定量采用对目的条带进行灰度值分析，以目的条带灰度值/GAPDH灰度值表示目的蛋白的相对表达量。
实时荧光定量PCR(QPCR)：
提取肾脏组织总RNA，用分光光度法测定RNA溶液浓度和纯度。利用逆转录试剂盒(Takara,DaLian)将1μgRNA逆转录成cDNA，按照下面的反应体系检测不同基因的变化情况。
a.反应体系
b.PCR热循环参数
55℃-95℃,snap every 0.5℃,repeat 81circles.
PAS染色：
4％多聚甲醛固定组织48h，石蜡包埋切片，脱蜡至水，蒸馏水冲洗，70％酒精冲洗3次。浸入高碘酸酒精溶液10min(此溶液温度以17－20度为好)，70％酒精洗后，入还原液中1min(此溶液温度以17－20度为好)，70％酒精洗后，入无色盐基性品红溶液1-1.5h，冬天室温较低时，可放入37度温箱。流水冲洗10min，用Mayer\'s苏木素复染液复染细胞核3-5min，再用1％盐酸酒精分化，流水冲洗后，脱水透明，最后封片。
Annexin V/PI凋亡双染实验:
取对数生长期肾小管上皮细胞(RTEC)接种于6孔板中，分别给予顺铂及PCN，药物作用24h后，用无EDTA胰酶消化细胞，以1000rpm转速离心5min，收集细胞，弃掉培养基。用预冷过的PBS溶液洗涤细胞两次，在其中加入400μL1×Binding Buffer悬浮细胞，细胞密度大约为1×106cells/mL。每组细胞悬液分别加入5μLAnnexin V和5μL PI，轻轻混匀后室温避光条件下孵育15min。1h内用流式细胞仪检测。
线粒体膜电位(JC-1)检测：
线粒体膜电位的下降是细胞凋亡早期的一个标志性事件。通过JC-1从红色荧光到绿色荧光的转变可以很容易地检测到细胞膜电位的下降，同时也可以用JC-1从红色荧光到绿色荧光的转变作为细胞凋亡早期的一个检测指标。选择对数生长期的细胞接种于6孔板中，分别给予顺铂及PCN，药物作用24h后，用无EDTA胰酶消化细胞，以1000rpm转速离心5min，收集细胞，弃掉培养基。用预冷过的PBS溶液洗涤细胞两次，加入1mL JC-1染色工作液，充分混匀。细胞培养箱中37℃孵育20min。在孵育期间，按照每1mL JC-1染色缓冲液(5×)加入4mL超纯水的比例，配制适量的JC-1染色缓冲液(1X)，并放置于冰浴。37℃孵育结束后，吸除上清，用JC-1染色缓冲液(1X)洗涤2次。加入2mL细胞培养液，在1h内用流式细胞仪检测。
活性氧(ROS)检测：
选择对数生长期的细胞接种于6孔板中，分别给予顺铂及PCN，药物作用24h后，用无EDTA胰酶消化细胞，以1000rpm转速离心5min，收集细胞，弃掉培养基。用预冷过的PBS溶液洗涤细胞一次，加入500μLmitoSOX染色工作液(1X)，充分混匀。细胞培养箱中37℃孵育20min。37℃孵育结束后，离心吸除上清，PBS溶液洗涤细胞两次，加入500μL细胞培养液，在1小时内用流式细胞仪检测。
下面通过具体的实施例详细说明本发明。
实施例1PCN对顺铂和缺血再灌注诱导的急性肾损伤肾功能的影响。
取体重18～22g的雄性C57BL/6小鼠，分为3组，即对照组，顺铂模型组以及PCN治疗组(n＝8)。
对照组：每日1次腹腔注射等体积介质，共5天；
顺铂模型组：腹腔注射，20mg/kg，单次给药；
PCN治疗组：PCN提前给药2天(腹腔注射，100mg/kgPCN，200μL/次)，每日1次，随后单次给药顺铂(腹腔注射，20mg/kg)，PCN再治疗3天，顺铂注射72h后取血，留取肾组织。
将血标本离心(20min，3000r/min),用肌酐试剂盒(Creatinine Assay Kit(cat:K625-100,biomars))、尿素氮试剂盒(QuantiChrom Urea Assay kit(cat:DIUR-500,Hayward,CA))测定血肌酐、尿素氮，实验结果见图1A。
同时我们用缺血再灌注模型，PCN对缺血再灌注损伤是否有治疗作用。取体重18～22g的雄性C57BL/6小鼠，分为3组，即对照组，缺血再灌注模型组以及PCN治疗组(n＝8)。
对照组：每日1次腹腔注射等体积介质，共3天；
缺血再灌注组：给予单纯介质每日1次，共3天；
PCN治疗组：PCN提前给药2天(腹腔注射，100mg/kgPCN，200μL/次)，每日1次，随后对小鼠肾脏缺血再灌注，PCN再治疗1天，缺血再灌注24h后取血，留取肾组织。利用上文方法分别测取各组血肌酐、尿素氮，实验结果见图1A。
图1B为用顺铂和缺血再灌注造模以及用PCN治疗后的PAS染色结果，从结果中可以看出，使用顺铂和缺血再灌注以后造模成功，血肌酐、尿素氮显著升高，提示肾脏受到损伤。而使用PCN治疗后，可以明显改善肾功能，肌酐、尿素氮水平与模型组相比模型降低，p<0.05。根据PAS染色结果，顺铂组和缺血再灌注组肾小管结构破坏，蛋白管型形成，PCN能够显著改善顺铂和缺血再灌注诱导的肾脏损伤。
实施例2PCN对顺铂诱导的肾脏炎症分子的表达及分泌的影响。
利用QPCR法及ELISA法研究PCN对顺铂诱导的肾脏炎症分子的表达及分泌的影响。
如图2A所示，利用ELISA法研究PCN对顺铂诱导的肾脏炎症分子的表达及分泌的影响。在顺铂诱导的AKI模型中，顺铂模型组的炎症因子IL-6、TNF-α表达水平与对照组相比明显升高，p<0.001。而PCN治疗组能够显著降低IL-6、TNF-α表达水平，p<0.05。
如图2B所示，利用QPCR法研究PCN对顺铂诱导的肾脏炎症分子的表达及分泌的影响。在顺铂诱导的AKI模型中，顺铂模型组的炎症因子IL-6、TNF-α表达水平与对照组相比明显升高，p<0.05。而PCN治疗组能够显著降低IL-6、TNF-α表达水平，p<0.01。
结果表明，PCN能够明显降低顺铂诱导的AKI模型中IL-6、TNF-α表达水平。
实施例3PCN对顺铂诱导的小管细胞凋亡及肾脏损伤相关分子的影响。
利用QPCR法检测肾脏凋亡及损失相关分子的表达水平来测定PCN对顺铂诱导的AKI的改善情况，结果如图3所示，顺铂模型组的凋亡相关分子Bax表达明显升高，Bcl-2表达明显降低；损伤相关分子Kim-1、NGAL表达均明显升高，p<0.05。而PCN治疗组能够显著降低Bax、Kim-1、NGAL的表达水平，升高Bcl-2的表达，p<0.05。
结果表明，PCN能够明显降低顺铂诱导的肾脏凋亡及损失相关分子的表达水平。
实施例4PCN在体外对顺铂诱导的小管细胞凋亡、活性氧及线粒体DNA拷贝数的影响。
体外应用RTEC细胞(小鼠肾小管上皮细胞系)，选择对数生长期的细胞接种于6孔板中，分别给予顺铂(5μg/mL)及PCN(5μM)，药物作用24h后，利用流式细胞仪检测小管细胞的凋亡发生率和活性氧水平，QPCR法检测小管细胞线粒体DNA拷贝数的变化。结果如图4A所示，顺铂模型组细胞发生凋亡的比率明显升高，可以观察到约30％的凋亡率，PCN治疗组与顺铂模型组相比，发生凋亡的比率明显降低，观察到约20％的凋亡率。如图4B所示，顺铂模型组细胞内的活性氧水平与对照组相比明显升高，P<0.001。PCN治疗组与顺铂模型组相比能够明显降低细胞内的活性氧水平，P<0.05。如图4C所示，顺铂模型组细胞线粒体DNA拷贝数与对照组相比明显降低，P<0.05。PCN治疗组与顺铂模型组相比能够明显升高细胞线粒体DNA拷贝数，P<0.01。
结果表明，顺铂刺激RTEC细胞24h，可以明显诱导细胞凋亡的发生，并能导致细胞内ROS水平升高、线粒体DNA拷贝数的减少。给予PCN治疗后，能够明显减少顺铂诱导的细胞凋亡的发生，并能降低细胞内ROS水平、增加线粒体DNA拷贝数，提示PCN有可能改善顺铂诱导的线粒体功能损伤。
实施例5PCN在体外对顺铂诱导的小管细胞凋亡的影响。
体外应用RTEC细胞(小鼠肾小管上皮细胞系)，选择对数生长期的细胞接种于6孔板中，分别给予顺铂(5μg/mL)及PCN(5μM)，药物作用24h后，利用western blot检测Bax、Bcl-2、cleaved-caspase 3的表达，同时检测PXR的蛋白表达情况。结果如图5所示，顺铂模型组，PXR的表达下调，同时凋亡相关蛋白Bax及cleaved-caspase 3表达水平上调，Bcl-2表达下调。PCN治疗组与顺铂模型组相比，能够明显上调PXR、Bcl-2的表达，下调Bax及cleaved-caspase3表达水平。
结果表明，顺铂刺激RTEC细胞24h，可以下调细胞内PXR的表达。给予PXR激动剂PCN处理能上调PXR的表达，同时抑制由顺铂所诱导的小管细胞凋亡。
Claims (1)
1.PCN在制备用于减轻顺铂诱导的急性肾损伤相关病症的药物中的应用，所述PCN为pregnenolone-16a-carbonitrile，是孕烷X受体的激动剂，所述减轻顺铂诱导的急性肾损伤相关病症为减轻顺铂诱导的肾小管细胞凋亡、减轻顺铂诱导的肾脏炎症因子的表达及分泌以及改善顺铂诱导的肾脏小管细胞线粒体功能。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810121267.4A CN108309987B (zh) | 2018-02-07 | 2018-02-07 | Pcn在制备用于治疗急性肾损伤相关病症的药物中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810121267.4A CN108309987B (zh) | 2018-02-07 | 2018-02-07 | Pcn在制备用于治疗急性肾损伤相关病症的药物中的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108309987A CN108309987A (zh) | 2018-07-24 |
| CN108309987B true CN108309987B (zh) | 2020-09-29 |
Family
ID=62903098
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810121267.4A Expired - Fee Related CN108309987B (zh) | 2018-02-07 | 2018-02-07 | Pcn在制备用于治疗急性肾损伤相关病症的药物中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108309987B (zh) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109908139B (zh) * | 2018-12-28 | 2022-02-22 | 南京市儿童医院 | 西洛司特在制备用于治疗急性肾损伤相关病症的药物中的用途 |
| CN113730416A (zh) * | 2021-09-18 | 2021-12-03 | 南京市儿童医院 | Bix-01294在制备治疗急性肾损伤相关病症药物中的应用 |
| CN117122603B (zh) * | 2023-09-07 | 2024-03-19 | 南方医科大学顺德医院(佛山市顺德区第一人民医院) | 一种药物制剂及其应用 |
-
2018
- 2018-02-07 CN CN201810121267.4A patent/CN108309987B/zh not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| Aberrant DNA methylation of pregnane X receptor underlies metabolic gene alterations in the diabetic kidney;Atsushi Watanabe, et al.;《Am J Physiol Renal Physiol》;20171206;第314卷;F551–F560 * |
| 孕烷X 受体研究进展;蒲丹等;《生理科学进展》;20071231;第38 卷(第4 期);318-322 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108309987A (zh) | 2018-07-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | Maggot extracts alleviate inflammation and oxidative stress in acute experimental colitis via the activation of Nrf2 | |
| Yeh et al. | Propofol inhibits lipopolysaccharide-induced lung epithelial cell injury by reducing hypoxia-inducible factor-1α expression | |
| Shao et al. | Paeoniflorin ameliorates macrophage infiltration and activation by inhibiting the TLR4 signaling pathway in diabetic nephropathy | |
| Chen et al. | Effects of long non-coding RNA LINC00667 on renal tubular epithelial cell proliferation, apoptosis and renal fibrosis via the miR-19b-3p/LINC00667/CTGF signaling pathway in chronic renal failure | |
| Ni et al. | Gas6 attenuates sepsis-induced tight junction injury and vascular endothelial hyperpermeability via the Axl/NF-κB signaling pathway | |
| Cai et al. | The effect of HMGA1 in LPS-induced Myocardial Inflammation | |
| Xue et al. | Ginsenoside Rc alleviates myocardial ischemia-reperfusion injury by reducing mitochondrial oxidative stress and apoptosis: role of SIRT1 activation | |
| Guo et al. | Emodin attenuates acute lung injury in Cecal-ligation and puncture rats | |
| Wang et al. | Rutaecarpine alleviates renal ischemia reperfusion injury in rats by suppressing the JNK/p38 MAPK signaling pathway and interfering with the oxidative stress response | |
| CN108309987B (zh) | Pcn在制备用于治疗急性肾损伤相关病症的药物中的应用 | |
| Zhang et al. | Shikonin improve sepsis‐induced lung injury via regulation of miRNA‐140‐5p/TLR4—a vitro and vivo study | |
| Chen et al. | Hydrogen sulfide reduces kidney injury due to urinary-derived sepsis by inhibiting NF-κB expression, decreasing TNF-α levels and increasing IL-10 levels | |
| Xue et al. | Protective effect of pterostilbene on sepsis-induced acute lung injury in a rat model via the JAK2/STAT3 pathway | |
| Yang et al. | SIRT1/IGFBPrP1/TGF β1 axis involved in cucurbitacin B ameliorating concanavalin A‐induced mice liver fibrosis | |
| Li et al. | Exosomal OIP5-AS1 attenuates cerebral ischemia-reperfusion injury by negatively regulating TXNIP protein stability and inhibiting neuronal pyroptosis | |
| Zhu et al. | Senolytic combination of dasatinib and quercetin protects against diabetic kidney disease by activating autophagy to alleviate podocyte dedifferentiation via the Notch pathway | |
| Zhao et al. | Berberine hydrochloride ameliorates PM2. 5-induced pulmonary fibrosis in mice through inhibiting oxidative stress and inflammatory | |
| Wu et al. | Inhibition of fatty acid synthase protects obese mice from acute lung injury via ameliorating lung endothelial dysfunction | |
| Zhang et al. | Integrated analysis of ATAC-seq and RNA-seq unveils the role of ferroptosis in PM2. 5-induced asthma exacerbation | |
| Chen et al. | Qinzhu Liangxue inhibits IL-6-induced hyperproliferation and inflammation in HaCaT cells by regulating METTL14/SOCS3/STAT3 axis | |
| Shu et al. | Protective effect of andrographolide against ulcerative colitis by activating Nrf2/HO-1 mediated antioxidant response | |
| Yan et al. | The protective effects of exogenous spermine on renal ischemia-reperfusion injury in rats | |
| Teng et al. | Peritoneal microvascular endothelial function and the microinflammatory state are associated with baseline peritoneal transport characteristics in uremic patients | |
| Li et al. | Growth differentiation factor 15 protects the airway by inhibiting cell pyroptosis in obese asthmatic mice through the phosphoinositide 3-kinase/AKT pathway | |
| Wu et al. | [Retracted] Exploring the Effect of Dapagliflozin on Alcoholic Kidney Injury and Renal Interstitial Fibrosis in Rats Based on TIMP‐1/MMP‐24 Pathway |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200929 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |