Thrombin increases the expression of cholesterol 25-hydroxylase in rat astrocytes after spinal cord injury

Astrocytes are important cellular centers of cholesterol synthesis and metabolism that help maintain normal physiological function at the organism level.Spinal cord injury results in aberrant cholesterol metabolism by astrocytes and excessive production of oxysterols,which have profound effects on neuropathology.25-Hydroxycholesterol(25-HC),the main product of the membrane-associated enzyme cholesterol-25-hydroxylase(CH25H),plays important roles in mediating neuroinflammation.However,whether the abnormal astrocyte cholesterol metabolism induced by spinal cord injury contributes to the production of 25-HC,as well as the resulting pathological effects,remain unclear.In the present study,spinal cord injury-induced activation of thrombin was found to increase astrocyte CH25H expression.A protease-activated receptor 1 inhibitor was able to attenuate this effect in vitro and in vivo.In cultured primary astrocytes,thrombin interacted with protease-activated receptor 1,mainly through activation of the mitogen-activated protein kinase/nuclear factor-kappa B signaling pathway.Conditioned culture medium from astrocytes in which ch25h expression had been knocked down by siRNA reduced macrophage migration.Finally,injection of the protease activated receptor 1 inhibitor SCH79797 into rat neural sheaths following spinal cord injury reduced migration of microglia/macrophages to the injured site and largely restored motor function.Our results demonstrate a novel regulatory mechanism for thrombin-regulated cholesterol metabolism in astrocytes that could be used to develop anti-inflammatory drugs to treat patients with spinal cord injury.

基于12导联动态心电图的ST段联合卵磷脂胆固醇脂酰基转移酶、 脂蛋白a对冠心病的诊断价值

目的探讨12导联动态心电图的ST段联合血清卵磷脂胆固醇脂酰基转移酶、脂蛋白a检测对冠心病患者的诊断价值。方法选取2020年4月至2020年12月广东医科大学附属医院收治的120例冠心病患者进行研究,根据冠状动脉造影结果分为CAG阳性组(n=66)和CAG阴性组(n=54)。检测并比较两组患者的血清磷脂胆固醇酰基转移酶(LCAT)、脂蛋白a(LPa)水平及心肌缺血ST段压低发作时间。比较单独及联合检测的诊断效能。以冠状动脉造影术(CAG)结果作为诊断金标准。结果CAG阳性组患者的血清LPa、LCAT水平高于CAG阴性组,差异有统计学意义(P<0.05);两组各个时间段的心肌缺血ST段压低发作时间比较,差异无统计学意义(P>0.05);三者联合检测的特异度、准确度高于单独检测。结论12导联动态心电图的ST段联合血清LPa、LCAT检测可有效提高对冠心病的诊断准确率。

Distribution of cholesterol 24-hydroxylase in the monkey brain

Objective Cholesterol 24-hydroxylase catalyzes the conversion of cholesterol to 24-hydroxycholesterol,which is a major pathway for cholesterol elimination from the brain,since 24-hydroxycholesterol can readily cross the blood brain barrier.The present study aimed to elucidate the distribution of cholesterol 24-hydroxylase in the monkey brain.Methods The distribution of cholesterol 24-hydroxylase in the monkey brain was examined using Western blot and immunohistochemistry methods,and was observed under light microscopy and electron microscopy.Results High levels of cholesterol 24-hydroxylase were observed in projection neurons and neuropil in structures derived from telencephalon,including the cerebral neocortex,hippocampus,amygdala,nucleus basalis of Meynert,and striatum.Electron microscopy revealed that the enzyme was localized in the axon terminals.One the other hand,cholesterol 24-hydroxylase was expressed at a lower level in the thalamus,globus pallidus and brainstem.Conclusion The high level of cholesterol 24-hydroxylase in the telencephalon possibly reflects a high rate of cholesterol turnover in this part of brain.

Mitochondrial function and regulation of macrophage sterol metabolism and inflammatory responses

The aim of this review is to explore the role of mitochondria in regulating macrophage sterol homeostasis and inflammatory responses within the aetiology of atherosclerosis.Macrophage generation of oxysterol activators of liver X receptors(LXRs),via sterol 27-hydroxylase,is regulated by the rate of flux of cholesterolto the inner mitochondrial membrane,via a complex of cholesterol trafficking proteins.Oxysterols are key signalling molecules,regulating the transcriptional activity of LXRs which coordinate macrophage sterol metabolism and cytokine production,key features influencing the impact of these cells within atherosclerotic lesions.The precise identity of the complex of proteins mediating mitochondrial cholesterol trafficking in macrophages remains a matter of debate,but may include steroidogenic acute regulatory protein and translocator protein.There is clear evidence that targeting either of these proteins enhances removal of cholesterol via LXRα-dependent induction of ATP binding cassette transporters(ABCA1,ABCG1) and limits the production of inflammatory cytokines; interventions which influence mitochondrial structure and bioenergetics also impact on removal of cholesterol from macrophages.Thus,molecules which can sustain or improve mitochondrial structure,the function of the electron transport chain,or increase the activity of components of the protein complex involved in cholesterol transfer,may therefore have utility in limiting or regressing atheroma development,reducing the incidence of coronary heart disease and myocardial infarction.

二氧化硅对MLE-12细胞脂质沉积及PI3K/AKT/mTOR通路的影响

[背景]脂质代谢失衡与多种疾病的发生发展密切相关,磷脂酰肌醇-3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶点(PI3K/AKT/mTOR)信号通路是脂质代谢的重要调控通路,矽肺与脂质代谢异常是否有关尚待探索。[目的]观察二氧化硅(SiO_(2))染毒后,肺泡Ⅱ型上皮细胞MLE-12细胞内脂质沉积情况,胆固醇及PI3K、AKT、mTOR磷酸化蛋白表达的变化,探讨SiO_(2)是否通过该通路调控脂质变化。[方法](1)采用50 mg·LSiO_(2)混悬液染毒MLE-12细胞,分为对照组和12、24、48 h SiO_(2)染毒组。(2)根据PI3K抑制剂LY294002对细胞增殖影响的实验结果,选择5μmol·L^(-1)进行后续实验。将细胞分为对照、50 mg·L-1 SiO_(2)、50 mg·LSiO_(2)+5μmol·L^(-1)LY294002、5μmol·L^(-1)LY294002四组,均染毒细胞48 h。采用酶法试剂盒检测各组细胞内总胆固醇、游离胆固醇、胆固醇酯(总胆固醇与游离胆固醇的差值)、甘油三酯含量,采用油红“O”染色法观察细胞内脂质沉积状况,采用Western blotting检测PI3K、AKT、mTOR磷酸化蛋白的表达水平。[结果](1)50 mg·L的SiO_(2)染毒后,与对照组相比,随着染毒时间的延长:细胞内总胆固醇、游离胆固醇和胆固醇酯的含量呈现增加的趋势,24、48 h组均明显升高,在48 h组,总胆固醇由对照组的(2.242±0.181)mg·g升高到(5.148±0.544)mg·g,游离胆固醇从(1.923±0.158)mg·g升高至(4.168±0.433)mg·g,胆固醇酯也从(0.318±0.067)mg·g升至(0.978±0.134)mg·g(均P<0.01);与对照组相比,甘油三酯含量无明显变化(P>0.05);细胞中的橙红色染色颗粒沉积增加,48 h橙红色颗粒细胞内沉积增加最明显(P<0.01);p-PI3K、p-AKT、p-mTOR蛋白表达呈现增高的趋势,在染毒48 h时达到高峰(均P<0.01)。(2)与SiO_(2)染毒组相比,SiO_(2)+LY294002组细胞内总胆固醇、游离胆固醇和胆固醇酯均降低(均P<0.01),细胞内橙红色脂质染色颗粒沉积减少,细胞p-PI3K、p-AKT、p-mTOR蛋白表达降低(均P<0.01)。[结论]SiO_(2)可能通过激活PI3K/AKT/mTOR信号通路诱导MLE-12细胞内胆固醇成分增加,促进细胞内脂质沉积。

BmCH25H,a vertebrate interferon-stimulated gene(ISG)homolog,inhibits BmNPV infection dependent on its hydroxylase activity in Bombyx mori

Cholesterol-25-hydroxylase(CH25H)has been identified as an interferon-stimulated gene(ISG)in mammals that exerts its antiviral effects by catalyzing the conversion of cholesterol to 25-hydroxycholesterol(25HC).However,invertebrates lack an antiviral system homologous to vertebrate interferons(IFNs)because the genomes of invertebrates do not encode IFN-like cytokines.Nevertheless,CH25H is present in insect genomes and it therefore deserves further study of whether and by which mechanism it could exert an antiviral effect in invertebrates.In this study,the Bombyx mori CH25H(BmCH25H)gene,of which the encoded protein has high homology with other lepidopteran species,was identified and located on chromosome 9.Interestingly,we found that the expression of BmCH25H was significantly upregulated in B.mori nucleopolyhedrovirus(BmNPV)-infected BmN cells and silkworm(B.mori)larvae at the early infection stage.The inhibitory effect of BmCH25H on BmNPV replication was further demonstrated to depend on its catalytic residues to convert cholesterol to 25HC.More importantly,we demonstrated that during BmNPV infection,BmCH25H expression was increased through the Janus kinase–signal transducer and activator of transcription(JAK–STAT)pathway,similar to the induction of ISGs following virus infection in vertebrates.This is the first report that CH25H has antiviral effects in insects;the study also elucidates the regulation of its expression and its mechanism of action.