AIM: To delineate the mechanisms of renal vasocon- striction in hepatorenal syndrome (HRS), we investigated the expression of typeⅠinositol 1, 4, 5-triphosphate receptors (IP3RⅠ) of kidney in mice with fulminant hep...AIM: To delineate the mechanisms of renal vasocon- striction in hepatorenal syndrome (HRS), we investigated the expression of typeⅠinositol 1, 4, 5-triphosphate receptors (IP3RⅠ) of kidney in mice with fulminant hepatic failure (FHF). METHODS: FHF was induced by lipopolysaccharide (LPS) in D-galactosamine (GalN) sensitized BALB/c mice. There were 20 mice in normal saline (NS)-treated group, 20 mice in LPS-treated group, 20 mice in GalN- treated group, and 60 mice in GalN/LPS-treated group (FHF group). Liver and kidney tissues were obtained at 2, 6, and 9 h after administration. The liver and kidney specimens were stained with hematoxylin-eosin for studying morphological changes under light microscope. The expression of IP3RⅠin kidney tissue was tested by immunohistochemistry, Western blot and reverse transcription (RT)-PCR. RESULTS: Kidney tissues were morphologically normal at all time points in all groups. IP3RⅠproteins were found localized in the plasma region of glomerular mesangial cells (GMC) and vascular smooth muscle cells (VSMC) in kidney by immunohistochemical staining. In kidney of mice with FHF at 6 h and 9 h IP3RⅠstaining was up- regulated. Results from Western blot demonstrated consistent and significant increment of IP3RⅠexpression in mice with FHF at 6 h and 9 h (t = 3.16, P < 0.05; t = 5.43, P < 0.01). Furthermore, we evaluated IP3RⅠ mRNA expression by RT-PCR and observed marked up- regulation of IP3RⅠmRNA in FHF samples at 2 h, 6 h and 9 h compared to controls (t = 2.97, P < 0.05; t = 4.42, P < 0.01; t = 3.81, P < 0.01). CONCLUSION: The expression of IP3RⅠprotein increased in GMC and renal VSMC of mice with FHF, possibly caused by up-regulation of IP3RⅠmRNA.展开更多
OBJECTIVE Previous studies have demonstrated acetylcholine muscarinic 4(M4) receptor regulates DARPP-32 phosphorylation at Thr75 in isolated medium spiny neurons(MSNs),indicating antagonistic mechanism with D1 depende...OBJECTIVE Previous studies have demonstrated acetylcholine muscarinic 4(M4) receptor regulates DARPP-32 phosphorylation at Thr75 in isolated medium spiny neurons(MSNs),indicating antagonistic mechanism with D1 dependent signal cascade,but the exact molecular mechanisms remain unclearly.In this study,we investigated the roles of M4 receptor in modulation D1 dependent signal to integrate striatal DA inputs in isolated MSNs.METHODS(1)Lentivirus technology was employed to genetically knock down the M4 receptor of MSNs;(2) Apomorphine(APO),acts as a dopamine receptor agonist,while SCH23390,acts as a selective antagonist for D1,were used to study the pharmacologically profiles with D1 receptor stimulation or blockade,respectively.Then the no subtype-selective muscarinic agonist oxotremorine M(OX) were used to show that mAchRs activation,in order to dissect the particular function of M4,a selective M4 antagonist,MT3 was used;(3) Intracellular cAMP production of MSNs was measured by using time resolved fluorescence resonance energy transfer detection method;(4) Laser confocal was used to explore the expression of M4 and D1 in MSNs;(5) Immunofluorescence cytochemistry and Western blotting were used to confirm the alteration of signaling molecular including P-CREB,DARPP-32 P-Thr34,DARPP-32 P-Thr75,cyclin-dependent kinase 5(CDK5) as wel as p25/35,which are involved in DA-dependent signaling modulations.RESULTS Firstly,TR-FRET assay revealed APO(10-2 mol·L^(-1))significantly increased the level of intracellular cAMP(vs control,n=3,P<0.01),also Western blotting results showed that APO(10-6 mol · L^(-1))increased DARPP-32 Thr34 phosphorylation(vs control,n=3,P<0.01),and these effect were reversed by D1 receptor antagonist SCH23390(vs APO,n=3,P<0.01).Interestingly,we confirmed that OX(10-6 mol · L^(-1)) down-regulated APO-induced DARPP-32 Thr34 phosphorylation(vs APO,n=3,P<0.01),due to its effects on DARPP-32 phosphorylation at Thr75.The results presented the antagonistic mechanism of mAchRs stimulation with D1 dependent signal cascade in MSNs.Meanwhile,OX(10-7,10-6 and10^(-5) mol·L^(-1)) stimulated DARPP-32 phosphorylation at Thr75,and simultaneously up regulated P25/35 and CDK5 activity(vs control,n=3,P<0.01) by using Western blotting assay.Furthermore,roscovitine(10^(-5) mol · L^(-1)),acts as a CDK5 inhibitor,suppressed CDK5 activity(vs control,n=10,P<0.01),and fully inhibited OX-induced DARPP-32 Thr75 phosphorylation(vs OX,n=10,P<0.01).More important,pretreated with roscovitine(10^(-5) mol·L^(-1)),the effect of APO on DARPP-32 Thr34 phosphorylation was potentiated(vs APO,n=3,P<0.05).The result presented CDK5 is required in suppression of APO on DARPP-32 Thr34 phosphorylation mediated through mAchRs stimulation.In addition,laser confocal results showed that the CDK5 up-regulation was mostly confined to MSNs co-expressing M4,which means that M4 participated in CDK5-mediated phosphorylation of DARPP-32 at Thr75.Consistently,immunofluorescence and Western blotting results confirmed that both genetic knockdown and pharmacologic inhibition of M4 receptors with MT3(10-7 mol · L^(-1)) down-regulated the OX-induced the expression of CDK5(vs OX,n=3,P<0.01) and P25/35(vs OX,n=3,P<0.01)in isolated MSNs.CONCLUSION M4 receptor may play an important role in antagonistic regulation D1 dependent signaling,in which CDK5 is required for suppressing D1-DARPP-32 Thr34 phosphorylation in isolated medium spiny neurons.展开更多
The inositol 1,4,5-trisphosphate (lnsP3) receptor was purified from bovine cerebellum and reconstituted in liposomes composed of phosphatidylcholine (PC) and phosphatidylethanola-mine (PE) (1:1) successfully. No effec...The inositol 1,4,5-trisphosphate (lnsP3) receptor was purified from bovine cerebellum and reconstituted in liposomes composed of phosphatidylcholine (PC) and phosphatidylethanola-mine (PE) (1:1) successfully. No effect of Ca2+ concentration on [3H]-lnsP3 binding to unreconsti-tuted lnsP3 receptor could be observed either at 4℃ or at 25℃, whereas the effect of [Ca2+] on reconstituted lnsP3 receptor depended on the temperature. The Ca2+ concentration outside the proteolipsome ([Ca2+]o) had no detectable effect on lnsP3 binding to lnsP3 receptor at 4℃. In contrast, with increase of [Ca2+]o from 0 to 100 nmol/L at 25℃, the lnsP3 binding activity increased gradually. Then the lnsP3 binding activity was decreased drastically at higher [Ca2+]0 and inhibited entirely at 50 nmol/L [Ca2+]. Conformational studies on intrinsic fluorescence of the reconstituted lnsP3 receptor and its quenching by Kl and HB indicated that the global conformation of reconstituted lnsP3 receptor could not be affected by [Ca2+]o展开更多
Opioids,such as morphine,are the most potent drugs used to treat pain.Long-term use results in high tolerance to morphine.High mobility group box-1(HMGB1) has been shown to participate in neuropathic or inflammatory p...Opioids,such as morphine,are the most potent drugs used to treat pain.Long-term use results in high tolerance to morphine.High mobility group box-1(HMGB1) has been shown to participate in neuropathic or inflammatory pain,but its role in morphine tolerance is unclear.In this study,we established rat and mouse models of morphine tolerance by intrathecal injection of morphine for 7 consecutive days.We found that morphine induced rat spinal cord neurons to release a large amount of HMGB1.HMGB1 regulated nuclear factor κB p65 phosphorylation and interleukin-1β production by increasing Toll-like receptor 4receptor expression in microglia,thereby inducing morphine tolerance.Glycyrrhizin,an HMGB1 inhibito r,markedly attenuated chronic morphine tole rance in the mouse model.Finally,compound C(adenosine 5’-monophosphate-activated protein kinase inhibitor) and zinc protoporphyrin(heme oxygenase-1 inhibitor)alleviated the morphine-induced release of HMGB1 and reduced nuclear factor κB p65 phosphorylation and interleukin-1β production in a mouse model of morphine tolerance and an SH-SY5Y cell model of morphine tole rance,and alleviated morphine tolerance in the mouse model.These findings suggest that morphine induces HMGB1 release via the adenosine 5’-monophosphate-activated protein kinase/heme oxygenase-1 signaling pathway,and that inhibiting this signaling pathway can effectively reduce morphine tole rance.展开更多
Applying a stimulating current to acupoints through acupuncture needles–known as electroacupuncture–has the potential to produce analgesic effects in human subjects and experimental animals. When acupuncture was app...Applying a stimulating current to acupoints through acupuncture needles–known as electroacupuncture–has the potential to produce analgesic effects in human subjects and experimental animals. When acupuncture was applied in a rat model, adenosine 5-triphosphate disodium in the extracellular space was broken down into adenosine, which in turn inhibited pain transmission by means of an adenosine A1 receptor-dependent process. Direct injection of an adenosine A1 receptor agonist enhanced the analgesic effect of acupuncture. The analgesic effect of acupuncture appears to be mediated by activation of A1 receptors located on ascending nerves. In neuropathic pain, there is upregulation of P2X purinoceptor 3 (P2X3) receptor expression in dorsal root ganglion neurons. Conversely, the onset of mechanical hyperalgesia was diminished and established hyperalgesia was significantly reversed when P2X3 receptor expression was downregulated. The pathways upon which electroacupuncture appear to act are interwoven with pain pathways, and electroacupuncture stimuli converge with impulses originating from painful areas. Electroacupuncture may act via purinergic A1 and P2X3 receptors simultaneously to induce an analgesic effect on neuropathic pain.展开更多
Homer1a是由中枢神经系统即刻早期基因(immediate early genes,IEG)编码的支架蛋白质,广泛的临床及基础研究表明Homer1a不仅与多种神经精神疾病的发病有关,也是多种抗抑郁治疗方案的关键蛋白质。抑郁症的发病与1型代谢型谷氨酸受体(meta...Homer1a是由中枢神经系统即刻早期基因(immediate early genes,IEG)编码的支架蛋白质,广泛的临床及基础研究表明Homer1a不仅与多种神经精神疾病的发病有关,也是多种抗抑郁治疗方案的关键蛋白质。抑郁症的发病与1型代谢型谷氨酸受体(metabotropic glutamate receptor,mGluR1/5)的表达和可用性降低,N-甲基-D-天冬氨酸受体(N-methyl-D-aspartate receptor,NMDAR)的功能障碍所致的突触可塑性失调相关。Homer1a对mGluR1/5的作用具有高度特异性。Homer1a过表达可通过增加突触后膜mGluR1/5的表达和可用性,调节突触后膜的离子型谷氨酸受体NMDAR和α-氨基-3-羟基-5-甲基-4-异噁唑丙酸受体(α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor,AMPAR),调节突触可塑性,最终发挥抗抑郁作用。Homer1a在突触活动的调节中发挥中心作用,因此了解Homer1a发挥作用的机制能为研究靶向Homer1a蛋白的抗抑郁药物提供新思路。展开更多
目的:观察白介素-6(IL-6)对N-甲基-D-天门冬氨酸(NMDA)损伤小脑颗粒神经元NMDA受体亚单位1(NR1)和三磷酸肌醇受体1(IP3R1)蛋白表达的影响。方法:取出生后8 d SD大鼠小脑进行小脑颗粒神经元(CGNs)体外培养。在培养液中加入IL-6(40或120 n...目的:观察白介素-6(IL-6)对N-甲基-D-天门冬氨酸(NMDA)损伤小脑颗粒神经元NMDA受体亚单位1(NR1)和三磷酸肌醇受体1(IP3R1)蛋白表达的影响。方法:取出生后8 d SD大鼠小脑进行小脑颗粒神经元(CGNs)体外培养。在培养液中加入IL-6(40或120 ng/mL),培养8 d后,用NMDA(100μmol/L)损伤神经元30 min,建立神经元损伤模型。Western Blot法检测NR1和IP3R1蛋白的表达。结果:IL-6下调NR1的蛋白表达,并抑制NMDA诱导的IP3R1的蛋白表达增高。结论:IL-6通过抑制NMDA受体和IP3受体实现神经保护作用。展开更多
BACKGROUND NLRP3-mediated pyroptosis is recognized as an essential modulator of renal disease pathology.Long noncoding RNAs(lncRNAs)are active participators of diabetic nephropathy(DN).X inactive specific transcript(X...BACKGROUND NLRP3-mediated pyroptosis is recognized as an essential modulator of renal disease pathology.Long noncoding RNAs(lncRNAs)are active participators of diabetic nephropathy(DN).X inactive specific transcript(XIST)expression has been reported to be elevated in the serum of DN patients.AIM To evaluate the mechanism of lncRNA XIST in renal tubular epithelial cell(RTEC)pyroptosis in DN.METHODS A DN rat model was established through streptozotocin injection,and XIST was knocked down by tail vein injection of the lentivirus LV sh-XIST.Renal metabolic and biochemical indices were detected,and pathological changes in the renal tissue were assessed.The expression of indicators related to inflammation and pyroptosis was also detected.High glucose(HG)was used to treat HK2 cells,and cell viability and lactate dehydrogenase(LDH)activity were detected after silencing XIST.The subcellular localization and downstream mechanism of XIST were investigated.Finally,a rescue experiment was carried out to verify that XIST regulates NLR family pyrin domain containing 3(NLRP3)/caspase-1-mediated RTEC pyroptosis through the microRNA-15-5p(miR-15b-5p)/Toll-like receptor 4(TLR4)axis.RESULTS XIST was highly expressed in the DN models.XIST silencing improved renal metabolism and biochemical indices and mitigated renal injury.The expression of inflammation and pyroptosis indicators was significantly increased in DN rats and HG-treated HK2 cells;cell viability was decreased and LDH activity was increased after HGtreatment. Silencing XIST inhibited RTEC pyroptosis by inhibiting NLRP3/caspase-1. Mechanistically,XIST sponged miR-15b-5p to regulate TLR4. Silencing XIST inhibited TLR4 by promotingmiR-15b-5p. miR-15b-5p inhibition or TLR4 overexpression averted the inhibitory effect ofsilencing XIST on HG-induced RTEC pyroptosis.CONCLUSIONSilencing XIST inhibits TLR4 by upregulating miR-15b-5p and ultimately inhibits renal injury inDN by inhibiting NLRP3/caspase-1-mediated RTEC pyroptosis.展开更多
目的探索FKBP12.6基因敲除对葡聚糖硫酸钠诱导小鼠急性结肠炎模型结肠动力的影响及其初步机制。方法分别提取FKBP12.6基因敲除(knock-out,KO)组小鼠及野生小鼠(wild-type,WT)组结肠平滑肌m RNA,并利用反转录、RTPCR检测,证明FKBP12.6在...目的探索FKBP12.6基因敲除对葡聚糖硫酸钠诱导小鼠急性结肠炎模型结肠动力的影响及其初步机制。方法分别提取FKBP12.6基因敲除(knock-out,KO)组小鼠及野生小鼠(wild-type,WT)组结肠平滑肌m RNA,并利用反转录、RTPCR检测,证明FKBP12.6在野生型小鼠结肠平滑肌中表达。选择8~12周龄、雌雄比4∶9的野生型小鼠及与其年龄、性别相匹配同窝对照FKBP12.6基因敲除小鼠各26只,分别将其随机分为对照组及结肠炎组,即野生型对照组、野生型结肠炎组、基因敲除对照组、基因敲除结肠炎组,每组各13只。两结肠炎组小鼠均通过饮用3%葡聚糖硫酸钠(DSS)溶液7 d诱导急性结肠炎模型。对比两结肠炎组小鼠临床表现、体质量减轻程度、结肠长度。测定4组小鼠离体结肠环形肌条静息状态下收缩情况。利用蛋白印迹方法检测结肠平滑肌钙通路相关蛋白(三磷酸肌醇受体1、受磷蛋白、大电导钙激活钾通道)表达。结果 KO对照组与WT对照组相比,结肠长度无差异。WT结肠炎组及KO结肠炎组临床表现、体质量减轻程度、结肠长度无差异。静息状态下,WT对照组及KO对照组结肠收缩频率无差异;WT结肠炎组较KO结肠炎组结肠自发收缩频率明显加快[(15.22±2.90)次/5 min vs(2.10±0.38)次/5 min,n=9,P<0.01];WT结肠炎组较WT对照组结肠自发收缩频率加快[(15.22±2.90)次/5 min vs(3.78±1.64)次/5 min,n=9,P<0.01];而KO结肠炎组较KO对照组结肠收缩频率无明显变化。蛋白印迹结果表明WT对照组与KO对照组IP3R1、PLB、BKCa无差异。在两结肠炎组中,KO组较WT组IP3R1表达减少(0.56±0.14 vs 0.84±0.09,P<0.05),BKCa表达上调(0.97±0.14 vs 0.53±0.13,P<0.05),PLB表达无统计学差异。结论 FKBP12.6敲除对DSS诱导小鼠结肠炎模型临床表现、体质量及结肠长度变化程度无影响。FKBP12.6敲除后DSS诱导小鼠结肠炎模型结肠平滑肌收缩频率接近野生对照组小鼠。展开更多
5-HT1A receptor is associated with a variety of pathophysiology of neuropsychiatric disorders. Accordingly, we have synthesized a new 5-HT1A receptor ligand (HYNIC-MPP4) and labeled it with 99mTc using N-(2-hydroxyeth...5-HT1A receptor is associated with a variety of pathophysiology of neuropsychiatric disorders. Accordingly, we have synthesized a new 5-HT1A receptor ligand (HYNIC-MPP4) and labeled it with 99mTc using N-(2-hydroxyethyl)ethylenediaminetriacetic acid (HEDTA) as coligand. 99mTc-HEDTA/HYNIC-MPP4 was prepared under pH 6 at room temperature. Biodistribution of 99mTc-HEDTA/HYNIC-MPP4 in normal mice showed that this complex had moderate brain uptake (0.60% ID·g-1 at 2 min p.i.) and good retention. The hippocampus had the highest radioactivity uptake at 2 min p.i. (1.84% ID·g-1). The ratio of Hipp/CB was 3.1 at 2 min p.i. and increased to 4.4 at 60 min p.i. After blocking with 8-hydroxy-2-(dipropylamino) tetralin, the uptake of hippocampus was decreased significantly from 1.84% ID·g-1 to 0.53% ID·g-1 at 2 min p.i., while the cerebellum had no significant decrease. This 99mTc complex could be a potent agent for 5-HT1A receptor imaging.展开更多
基金Supported by National Natural Science Foundation of China, No. 30270607
文摘AIM: To delineate the mechanisms of renal vasocon- striction in hepatorenal syndrome (HRS), we investigated the expression of typeⅠinositol 1, 4, 5-triphosphate receptors (IP3RⅠ) of kidney in mice with fulminant hepatic failure (FHF). METHODS: FHF was induced by lipopolysaccharide (LPS) in D-galactosamine (GalN) sensitized BALB/c mice. There were 20 mice in normal saline (NS)-treated group, 20 mice in LPS-treated group, 20 mice in GalN- treated group, and 60 mice in GalN/LPS-treated group (FHF group). Liver and kidney tissues were obtained at 2, 6, and 9 h after administration. The liver and kidney specimens were stained with hematoxylin-eosin for studying morphological changes under light microscope. The expression of IP3RⅠin kidney tissue was tested by immunohistochemistry, Western blot and reverse transcription (RT)-PCR. RESULTS: Kidney tissues were morphologically normal at all time points in all groups. IP3RⅠproteins were found localized in the plasma region of glomerular mesangial cells (GMC) and vascular smooth muscle cells (VSMC) in kidney by immunohistochemical staining. In kidney of mice with FHF at 6 h and 9 h IP3RⅠstaining was up- regulated. Results from Western blot demonstrated consistent and significant increment of IP3RⅠexpression in mice with FHF at 6 h and 9 h (t = 3.16, P < 0.05; t = 5.43, P < 0.01). Furthermore, we evaluated IP3RⅠ mRNA expression by RT-PCR and observed marked up- regulation of IP3RⅠmRNA in FHF samples at 2 h, 6 h and 9 h compared to controls (t = 2.97, P < 0.05; t = 4.42, P < 0.01; t = 3.81, P < 0.01). CONCLUSION: The expression of IP3RⅠprotein increased in GMC and renal VSMC of mice with FHF, possibly caused by up-regulation of IP3RⅠmRNA.
文摘OBJECTIVE Previous studies have demonstrated acetylcholine muscarinic 4(M4) receptor regulates DARPP-32 phosphorylation at Thr75 in isolated medium spiny neurons(MSNs),indicating antagonistic mechanism with D1 dependent signal cascade,but the exact molecular mechanisms remain unclearly.In this study,we investigated the roles of M4 receptor in modulation D1 dependent signal to integrate striatal DA inputs in isolated MSNs.METHODS(1)Lentivirus technology was employed to genetically knock down the M4 receptor of MSNs;(2) Apomorphine(APO),acts as a dopamine receptor agonist,while SCH23390,acts as a selective antagonist for D1,were used to study the pharmacologically profiles with D1 receptor stimulation or blockade,respectively.Then the no subtype-selective muscarinic agonist oxotremorine M(OX) were used to show that mAchRs activation,in order to dissect the particular function of M4,a selective M4 antagonist,MT3 was used;(3) Intracellular cAMP production of MSNs was measured by using time resolved fluorescence resonance energy transfer detection method;(4) Laser confocal was used to explore the expression of M4 and D1 in MSNs;(5) Immunofluorescence cytochemistry and Western blotting were used to confirm the alteration of signaling molecular including P-CREB,DARPP-32 P-Thr34,DARPP-32 P-Thr75,cyclin-dependent kinase 5(CDK5) as wel as p25/35,which are involved in DA-dependent signaling modulations.RESULTS Firstly,TR-FRET assay revealed APO(10-2 mol·L^(-1))significantly increased the level of intracellular cAMP(vs control,n=3,P<0.01),also Western blotting results showed that APO(10-6 mol · L^(-1))increased DARPP-32 Thr34 phosphorylation(vs control,n=3,P<0.01),and these effect were reversed by D1 receptor antagonist SCH23390(vs APO,n=3,P<0.01).Interestingly,we confirmed that OX(10-6 mol · L^(-1)) down-regulated APO-induced DARPP-32 Thr34 phosphorylation(vs APO,n=3,P<0.01),due to its effects on DARPP-32 phosphorylation at Thr75.The results presented the antagonistic mechanism of mAchRs stimulation with D1 dependent signal cascade in MSNs.Meanwhile,OX(10-7,10-6 and10^(-5) mol·L^(-1)) stimulated DARPP-32 phosphorylation at Thr75,and simultaneously up regulated P25/35 and CDK5 activity(vs control,n=3,P<0.01) by using Western blotting assay.Furthermore,roscovitine(10^(-5) mol · L^(-1)),acts as a CDK5 inhibitor,suppressed CDK5 activity(vs control,n=10,P<0.01),and fully inhibited OX-induced DARPP-32 Thr75 phosphorylation(vs OX,n=10,P<0.01).More important,pretreated with roscovitine(10^(-5) mol·L^(-1)),the effect of APO on DARPP-32 Thr34 phosphorylation was potentiated(vs APO,n=3,P<0.05).The result presented CDK5 is required in suppression of APO on DARPP-32 Thr34 phosphorylation mediated through mAchRs stimulation.In addition,laser confocal results showed that the CDK5 up-regulation was mostly confined to MSNs co-expressing M4,which means that M4 participated in CDK5-mediated phosphorylation of DARPP-32 at Thr75.Consistently,immunofluorescence and Western blotting results confirmed that both genetic knockdown and pharmacologic inhibition of M4 receptors with MT3(10-7 mol · L^(-1)) down-regulated the OX-induced the expression of CDK5(vs OX,n=3,P<0.01) and P25/35(vs OX,n=3,P<0.01)in isolated MSNs.CONCLUSION M4 receptor may play an important role in antagonistic regulation D1 dependent signaling,in which CDK5 is required for suppressing D1-DARPP-32 Thr34 phosphorylation in isolated medium spiny neurons.
文摘The inositol 1,4,5-trisphosphate (lnsP3) receptor was purified from bovine cerebellum and reconstituted in liposomes composed of phosphatidylcholine (PC) and phosphatidylethanola-mine (PE) (1:1) successfully. No effect of Ca2+ concentration on [3H]-lnsP3 binding to unreconsti-tuted lnsP3 receptor could be observed either at 4℃ or at 25℃, whereas the effect of [Ca2+] on reconstituted lnsP3 receptor depended on the temperature. The Ca2+ concentration outside the proteolipsome ([Ca2+]o) had no detectable effect on lnsP3 binding to lnsP3 receptor at 4℃. In contrast, with increase of [Ca2+]o from 0 to 100 nmol/L at 25℃, the lnsP3 binding activity increased gradually. Then the lnsP3 binding activity was decreased drastically at higher [Ca2+]0 and inhibited entirely at 50 nmol/L [Ca2+]. Conformational studies on intrinsic fluorescence of the reconstituted lnsP3 receptor and its quenching by Kl and HB indicated that the global conformation of reconstituted lnsP3 receptor could not be affected by [Ca2+]o
基金supported by the National Natural Science Foundation of ChinaNos.81971047 (to WTL) and 82073910 (to XFW)+2 种基金the Natural Science Foundation of Jiangsu Province,No.BK20191253 (to XFW)Key R&D Program (Social Development) Project of Jiangsu Province,No.BE2019 732 (to WTL)Jiangsu Province Hospital (the First Affiliated Hospital of Nanjing Medical University) Clinical Capacity Enhancement Project,No.JSPH-511B2018-8 (to YBP)。
文摘Opioids,such as morphine,are the most potent drugs used to treat pain.Long-term use results in high tolerance to morphine.High mobility group box-1(HMGB1) has been shown to participate in neuropathic or inflammatory pain,but its role in morphine tolerance is unclear.In this study,we established rat and mouse models of morphine tolerance by intrathecal injection of morphine for 7 consecutive days.We found that morphine induced rat spinal cord neurons to release a large amount of HMGB1.HMGB1 regulated nuclear factor κB p65 phosphorylation and interleukin-1β production by increasing Toll-like receptor 4receptor expression in microglia,thereby inducing morphine tolerance.Glycyrrhizin,an HMGB1 inhibito r,markedly attenuated chronic morphine tole rance in the mouse model.Finally,compound C(adenosine 5’-monophosphate-activated protein kinase inhibitor) and zinc protoporphyrin(heme oxygenase-1 inhibitor)alleviated the morphine-induced release of HMGB1 and reduced nuclear factor κB p65 phosphorylation and interleukin-1β production in a mouse model of morphine tolerance and an SH-SY5Y cell model of morphine tole rance,and alleviated morphine tolerance in the mouse model.These findings suggest that morphine induces HMGB1 release via the adenosine 5’-monophosphate-activated protein kinase/heme oxygenase-1 signaling pathway,and that inhibiting this signaling pathway can effectively reduce morphine tole rance.
文摘Applying a stimulating current to acupoints through acupuncture needles–known as electroacupuncture–has the potential to produce analgesic effects in human subjects and experimental animals. When acupuncture was applied in a rat model, adenosine 5-triphosphate disodium in the extracellular space was broken down into adenosine, which in turn inhibited pain transmission by means of an adenosine A1 receptor-dependent process. Direct injection of an adenosine A1 receptor agonist enhanced the analgesic effect of acupuncture. The analgesic effect of acupuncture appears to be mediated by activation of A1 receptors located on ascending nerves. In neuropathic pain, there is upregulation of P2X purinoceptor 3 (P2X3) receptor expression in dorsal root ganglion neurons. Conversely, the onset of mechanical hyperalgesia was diminished and established hyperalgesia was significantly reversed when P2X3 receptor expression was downregulated. The pathways upon which electroacupuncture appear to act are interwoven with pain pathways, and electroacupuncture stimuli converge with impulses originating from painful areas. Electroacupuncture may act via purinergic A1 and P2X3 receptors simultaneously to induce an analgesic effect on neuropathic pain.
基金Supported by Natural Science Foundation of Shenzhen University General Hospital (SUGH2020QD011)
文摘BACKGROUND NLRP3-mediated pyroptosis is recognized as an essential modulator of renal disease pathology.Long noncoding RNAs(lncRNAs)are active participators of diabetic nephropathy(DN).X inactive specific transcript(XIST)expression has been reported to be elevated in the serum of DN patients.AIM To evaluate the mechanism of lncRNA XIST in renal tubular epithelial cell(RTEC)pyroptosis in DN.METHODS A DN rat model was established through streptozotocin injection,and XIST was knocked down by tail vein injection of the lentivirus LV sh-XIST.Renal metabolic and biochemical indices were detected,and pathological changes in the renal tissue were assessed.The expression of indicators related to inflammation and pyroptosis was also detected.High glucose(HG)was used to treat HK2 cells,and cell viability and lactate dehydrogenase(LDH)activity were detected after silencing XIST.The subcellular localization and downstream mechanism of XIST were investigated.Finally,a rescue experiment was carried out to verify that XIST regulates NLR family pyrin domain containing 3(NLRP3)/caspase-1-mediated RTEC pyroptosis through the microRNA-15-5p(miR-15b-5p)/Toll-like receptor 4(TLR4)axis.RESULTS XIST was highly expressed in the DN models.XIST silencing improved renal metabolism and biochemical indices and mitigated renal injury.The expression of inflammation and pyroptosis indicators was significantly increased in DN rats and HG-treated HK2 cells;cell viability was decreased and LDH activity was increased after HGtreatment. Silencing XIST inhibited RTEC pyroptosis by inhibiting NLRP3/caspase-1. Mechanistically,XIST sponged miR-15b-5p to regulate TLR4. Silencing XIST inhibited TLR4 by promotingmiR-15b-5p. miR-15b-5p inhibition or TLR4 overexpression averted the inhibitory effect ofsilencing XIST on HG-induced RTEC pyroptosis.CONCLUSIONSilencing XIST inhibits TLR4 by upregulating miR-15b-5p and ultimately inhibits renal injury inDN by inhibiting NLRP3/caspase-1-mediated RTEC pyroptosis.
文摘目的探索FKBP12.6基因敲除对葡聚糖硫酸钠诱导小鼠急性结肠炎模型结肠动力的影响及其初步机制。方法分别提取FKBP12.6基因敲除(knock-out,KO)组小鼠及野生小鼠(wild-type,WT)组结肠平滑肌m RNA,并利用反转录、RTPCR检测,证明FKBP12.6在野生型小鼠结肠平滑肌中表达。选择8~12周龄、雌雄比4∶9的野生型小鼠及与其年龄、性别相匹配同窝对照FKBP12.6基因敲除小鼠各26只,分别将其随机分为对照组及结肠炎组,即野生型对照组、野生型结肠炎组、基因敲除对照组、基因敲除结肠炎组,每组各13只。两结肠炎组小鼠均通过饮用3%葡聚糖硫酸钠(DSS)溶液7 d诱导急性结肠炎模型。对比两结肠炎组小鼠临床表现、体质量减轻程度、结肠长度。测定4组小鼠离体结肠环形肌条静息状态下收缩情况。利用蛋白印迹方法检测结肠平滑肌钙通路相关蛋白(三磷酸肌醇受体1、受磷蛋白、大电导钙激活钾通道)表达。结果 KO对照组与WT对照组相比,结肠长度无差异。WT结肠炎组及KO结肠炎组临床表现、体质量减轻程度、结肠长度无差异。静息状态下,WT对照组及KO对照组结肠收缩频率无差异;WT结肠炎组较KO结肠炎组结肠自发收缩频率明显加快[(15.22±2.90)次/5 min vs(2.10±0.38)次/5 min,n=9,P<0.01];WT结肠炎组较WT对照组结肠自发收缩频率加快[(15.22±2.90)次/5 min vs(3.78±1.64)次/5 min,n=9,P<0.01];而KO结肠炎组较KO对照组结肠收缩频率无明显变化。蛋白印迹结果表明WT对照组与KO对照组IP3R1、PLB、BKCa无差异。在两结肠炎组中,KO组较WT组IP3R1表达减少(0.56±0.14 vs 0.84±0.09,P<0.05),BKCa表达上调(0.97±0.14 vs 0.53±0.13,P<0.05),PLB表达无统计学差异。结论 FKBP12.6敲除对DSS诱导小鼠结肠炎模型临床表现、体质量及结肠长度变化程度无影响。FKBP12.6敲除后DSS诱导小鼠结肠炎模型结肠平滑肌收缩频率接近野生对照组小鼠。
基金Supported by the National Natural Science Foundation of China (Grant No. 20401004)the Analysis and Test fund of Beijing Normal University
文摘5-HT1A receptor is associated with a variety of pathophysiology of neuropsychiatric disorders. Accordingly, we have synthesized a new 5-HT1A receptor ligand (HYNIC-MPP4) and labeled it with 99mTc using N-(2-hydroxyethyl)ethylenediaminetriacetic acid (HEDTA) as coligand. 99mTc-HEDTA/HYNIC-MPP4 was prepared under pH 6 at room temperature. Biodistribution of 99mTc-HEDTA/HYNIC-MPP4 in normal mice showed that this complex had moderate brain uptake (0.60% ID·g-1 at 2 min p.i.) and good retention. The hippocampus had the highest radioactivity uptake at 2 min p.i. (1.84% ID·g-1). The ratio of Hipp/CB was 3.1 at 2 min p.i. and increased to 4.4 at 60 min p.i. After blocking with 8-hydroxy-2-(dipropylamino) tetralin, the uptake of hippocampus was decreased significantly from 1.84% ID·g-1 to 0.53% ID·g-1 at 2 min p.i., while the cerebellum had no significant decrease. This 99mTc complex could be a potent agent for 5-HT1A receptor imaging.