复合益生菌制剂缓解cuprizone诱导的小鼠神经脱髓鞘

[目的]探索复合益生菌制剂对双环己酮草酰二腙(cuprizone,CPZ)诱导的小鼠脑内神经脱髓鞘的作用.[方法]将27只小鼠随机分为正常组(NC)、模型组(CPZ)和益生菌处理组(probiotics).NC组小鼠正常饲喂,CPZ组和probiotics组小鼠饲喂含0.2％CPZ的饲料且每天分别灌饲0.2 mL生理盐...     

PLX3397 inhibits the accumulation of intra‐tumoral macrophages and improves bromodomain and extra‐terminal inhibitor efficacy in melanoma

Bromodomain and extra‐terminal inhibitors (BETi) delay tumor growth, in part, through tumor cell intrinsic alterations and initiation of anti‐tumor CD8+ T‐cell responses. By contrast, BETi effects on pro‐tumoral immune responses remain unclear. Here, we show that the next‐generation BETi, PLX51107, delayed tumor growth to differing degrees in Braf V600E melanoma syngeneic mouse models. These differential responses were associated with the influx of tumor‐associated macrophages during BETi treatment. Tumors that were poorly responsive to PLX51107 showed increased influx of colony‐stimulating factor‐1 receptor (CSF‐1R)‐positive tumor‐associated macrophages. We depleted CSF‐1R+ tumor‐associated macrophages with the CSF‐1R inhibitor, PLX3397, in combination with PLX51107. Treatment with PLX3397 enhanced the efficacy of PLX51107 in poorly responsive Braf V600E syngeneic melanomas in vivo. These findings suggest that tumor‐associated macrophage accumulation limits BETi efficacy and that co‐treatment with PLX3397 can improve response to PLX51107, offering a potential novel combination therapy for metastatic melanoma patients.     

Microsomal prostaglandin E synthase-1 aggravates inflammation and demyelination in a mouse model of multiple sclerosis

Microsomal prostaglandin synthetase-1 (mPGES-1) is an inducible terminal enzyme required for prostaglandin E₂ (PGE₂) biosynthesis. In this study, we examined the role of mPGES-1 in the inflammation and demyelination observed in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We induced EAE with myelin oligodendrocyte glycoprotein_35–55 peptide in mPGES-1-deficient (mPGES-1^−/−) and wild-type (WT) mice. First, we examined the histopathology in the early and late phases of EAE progression. Next, we measured the concentration of PGE₂ in the spinal cord and investigated the expression of mPGES-1 using immunohistochemistry. In addition, we examined the progression of the severity of EAE using an EAE score to investigate a correlation between pathological features and paralysis. In this paper, we demonstrate that WT mice showed extensive inflammation and demyelination, whereas mPGES-1^−/− mice exhibited significantly smaller and more localized changes in the perivascular area. The mPGES-1 protein was induced in vascular endothelial cells and microglia around inflammatory foci, and PGE₂ production was increased in WT mice but not mPGES-1^−/− mice. Furthermore, mPGES-1^−/− mice showed a significant reduction in the maximum EAE score and improved locomotor activity. These results suggest that central PGE₂ derived from non-neuronal mPGES-1 aggravates the disruption of the vessel structure, leading to the spread of inflammation and local demyelination in the spinal cord, which corresponds to the symptoms of EAE. The inhibition of mPGES-1 may be useful for the treatment of human MS.     

CSF1 gene associated with aggressive periodontitis in the Japanese population

Aggressive periodontitis (AgP) is characterized by the early onset of the rapid and progressive destruction of the alveolar bone. We investigated the correlation of single nucleotide polymorphisms (SNPs) in candidate genes with AgP in the Japanese population in order to determine the genetic risk factors for this complex disease. Among 11 genes related to bone formation and resorption, 43 known SNPs were tested in 98 case and 88 control samples for association with AgP by using SNP genotyping techniques. Among these, three polymorphisms located in the colony stimulating factor 1 (CSF1) gene showed a positive association with AgP. This is the first case of an association between a CSF1 polymorphism and a human disease.     

小鼠 CYP2R1的异源表达及其对癌细胞增殖的差异作用

细胞色素P450 (cytochrome P450, CYP450)是一类血红素氧化酶。细胞色素P450家族2亚家族R成员1（cytochrome P450 family 2 subfamily R member 1, CYP2R1）是一种主要在肝组织中表达的维生素D羟化酶。目前,对于小鼠CYP2R1蛋白质的结构、物化特性和病理机制的理了解仍十分有限。本研究结合基因克隆和生物信息学分析,获得小鼠CYP2R1基因CDS序列及其生物学特征。随后,利用pcDNA3.1-CYP2R1真核表达载体,通过细胞划痕、MTT分析、real-time qPCR方法检测异源表达CYP2R1对肺癌细胞A549、胃癌细胞7901、肝癌细胞HepG2以及正常细胞HEK293T细胞的迁移、增殖和细胞周期基因表达的影响,探明其对癌细胞增殖的作用。结果显示,由C57BL/6小鼠肝组织的CYP2R1基因,序列长度1 506 bp,其中,CD_S 468 bp。其编码的155个氨基酸,与其他11个物种间的同源性均较高,其三级结构与人CYP2R1略有不同。构建的pcDNA3.1-CYP2R1真核表达载体,可在体外培养细胞中提高CYP2R1基因mRNA表达105倍以上,蛋白质水平提高约30倍。值得注意的是,异源表达CYP2R1在癌细胞增殖中的作用具有差异性,其中,CYP2R1通过显著降低细胞周期蛋白基因CyclinD1(P<0.05)和Caspase3(P<0.01),而抑制7901细胞的增殖。该研究结果为进一步探索CYP2R1的生物学作用,特别是在分析其对癌细胞增殖方面提供了基础研究数据,并为进一步明确CYP2R1在癌症相关治疗中的重要意义奠定了理论基础。     

Involvement of purinergic P2Y1R in antidepressant-like effects of electroacupuncture treatment on social isolation stress mice

Depression is a common neuropsychiatric disorder with high incidence and disability. Electroacupuncture (EA) is effective in the treatment of depression. However, the underlying mechanisms are not fully understood. Social isolation stress during post-weaning period can impair purinergic signaling in the brain of rodents and has emerged as a major risk factor for depression. The purpose of this study was to investigate the involvement of P2Y1 receptor (P2Y1R) in the antidepressant-like effects of EA. In this study, C57BL/6 mice were randomly assigned to group-housed (GH) or social isolated (SI) groups at post-natal day 21. After 6 weeks of social isolation, EA was performed on acupoints “Bai-hui” (GV20) and “Yin-tang” (GV29), or non-acupoints for 4 weeks. The SI mice received either intracerebroventricular injection of a selective P2Y1R agonist, MRS2365 (1 nmol); or a selective P2Y1R antagonist, MRS2179 (2 μmol), before and after EA. We found that SI mice exhibited depression-like behaviors accompanied with anxiety-like behaviors. The expressions of P2Y1R were well co-localized with GFAP-positive astrocytes and increased in the prefrontal cortex and hippocampus of SI mice. After treated with MRS2179, the depression-like behaviors of SI mice were attenuated, but not with MRS2365. Meanwhile, we found that EA could attenuate social isolation caused depression- and anxiety-like behaviors, and inhibited the up-regulation of P2Y1R in the prefrontal cortex and hippocampus of SI mice. Notably, the positive effects of EA on depression-like behaviors of SI mice could be reversed by MRS2365, while MRS2365 had no effect on the anxiolytic-like effects of EA. Therefore, we provide new evidence that EA could ameliorate depression- and anxiety-like behaviors in social isolation stress mice, and P2Y1R was involved in the antidepressant-like effects of EA.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11302-021-09827-1.     

6-苄氨基嘌呤对叶绿体偶联因子功能的影响

可溶性偶联因子经6-BA修饰后,明显促进Mg~(2 )-ATPase活力。从6-BA处理的叶绿体上洗脱下来的偶联因子,其Mg~(2 )-及Ca~(2 )-ATP酶活力都比对照有明显的增加。从~3H-6BA处理叶绿体上洗脱下来的偶联因子等蛋白,经聚丙烯酰胺电泳分析,~3H-6BA除与偶联因子结合外,还与RuBP羧化酶及其他蛋白结合。用6-BA处理提纯的β亚单位,能明显促进其Mg~(2 )-ATPase活力,表明6-BA至少有一个结合位点是在CF_1的β亚单位上并可影响其能量转换反应。     

Chromosomal localization of HTLV-1 viral integration sites using in situ hybridization: detection of a novel IL2R fragment

Summary The presence of human T-cell leukemia virus (HTLV-1) in patients with adult T-cell leukemia (ATL) was investigated by Southern blotting and in situ hybridization. In all seven patients, HTLV-1 provirus was detected. A large and variable number of labeled restriction fragments were observed, indicating multiple integrations. Two of the patients analyzed by in situ hybridization had two, while the third patient had three, sites of viral integration on six different chromosomes, suggesting random integration. A single site of integration was shared by two patients, which was on chromosome 10 at bands p11p15. One of these sites was on an apparently normal chromosome 10 and the other was on a derivative chromosome 10,t(10;14)(p12;g32). The interleukin 2 receptor (IL2R) has previously been localized to this region (10p14p15). The -chain of the IL2R is continuously expressed on affected T-cells in this disease. Southern blotting with pIL2R showed the presence of a novel 3.5 kb fragment in five out of the seven patients. This novel fragment has not been previously reported. No direct correlation was found between the novel 3.5 kb fragment, present in patients both cytogenetically normal and abnormal, and viral integration in the 10p11p15 region in two patients. Therefore, it is suggested that the presence of the 3.5 kb fragment and the numerous chromosomal breaks associated with this disease may not be direct results of viral integration.     

Renal protective effects of arjunolic acid in a cisplatin-induced nephrotoxicity model

Cisplatin is the first platinum-containing anti-cancer drugs. Cisplatin notable side effect of nephrotoxicity limits its use in clinic. Meanwhile, arjunolic acid possesses anti-inflammatory properties and plays protective roles against chemically induced organ pathophysiology. This study was conducted to find out whether arjunolic acid could attenuate kidney damage in rats, and to elucidate its possible mechanism of action. Fifty rats were treated with cisplatin (10 mg/kg) in the presence/absence of 100 or 250 mg/kg arjunolic acid. Arjunolic acid is given 1 h after cisplatin. Morphological changes were assessed in kidney sections stained with Hematoxylin/Eosin and Masson Trichrome. Kidney samples were used for measurements of transforming growth factor (TGF)-β1 and its type 1 receptor (TGF-βR1), tumor necrosis factor (TNF)-α and interleukin (IL)-1β by ELISA. Gene expression NFκB was determined by real time-PCR. Kidney tissue apoptosis was assessed by measuring the activities of caspase-3/8/9. The renal protective effect of arjunolic acid was confirmed by approximately normal appearance of renal tissue and the relatively unaffected serum creatinine and urea levels. Furthermore, arjunolic acid showed dose dependent reduction in cisplatin-induced elevation in renal levels of TGF-βR1, TGF-β1, TNF-α, IL-1β and caspases. These findings demonstrated that arjunolic acid attenuates cisplatin nephrotoxicity either indirectly by enhancing body antioxidant activity or directly through several mechanisms, including inhibition of pro-inflammatory cytokines, blocking activation of TGF-β1, and anti-apoptotic effects.     

Effect of roscovitine,a selective cyclin B-dependent kinase 1 inhibitor,on assembly of the nucleolus in mitosis

It is well known that at the beginning of mitosis the nucleolus disassembles but then reassembles at the end of mitosis. However, the mechanisms of these processes are still unclear. In the present work, we show for the first time that selective inhibition of cyclin B-dependent kinase 1 (CDK1) by roscovitine induces premature assembly of the nucleolus in mammalian cells in metaphase. Treatment of metaphase cells with roscovitine induces formation of structures in their cytoplasm that contain major proteins of the mature nucleolus participating in rRNA processing, such as B23/nucleophosmin, C23/nucleolin, fibrillarin, Nop52, as well as partially processed (immature) 46-45S pre-rRNA. This effect is reproducible in cells of various types; this indicates that general mechanisms regulate early stages of the nucleolus reassembly with CDK1 participation in mammalian cells. Based on our and literature data, we suggest that inactivation of the CDK1-cyclin B complex at the end of mitosis results in dephosphorylation of B23/nucleophosmin and C23/nucleolin; this facilitates their interaction with pre-rRNA and leads to formation of insoluble supramolecular complexes--nucleolus-derived foci.     

Differential expression of inflammation- and apoptosis-related genes in spinal cords of a mutant SOD1 transgenic mouse model of familial amyotrophic lateral sclerosis

Familial amyotrophic lateral sclerosis (FALS)-linked mutations in copper-zinc superoxide dismutase (SOD1) cause motor neuron death through one or more acquired toxic properties. We analyzed the molecular mechanism underlying motor neuron degeneration in the transgenic mouse model expressing the SOD1 gene with G93A mutation. Using cDNA microarray, the differentially expressed genes were identified in the spinal cords of G93A mice, 30 being elevated and seven decreased. cDNA microarray analysis to monitor gene expression during neurodegeneration revealed an up-regulation of genes related to an inflammatory process, such as the tumor necrosis factor-alpha (TNF-alpha) gene, resulting from glial cell activation, together with the change in apoptosis-related gene expression, such as caspase-1. The increased expression of the inflammation- and apoptosis-related genes occurred at 11 weeks of age in the presymptomatic stage prior to motor neuron death. These results suggest a mechanism of neurodegeneration that includes an inflammatory response as an important component. Thus, ALS has paralleled other neurodegenerative disorders, such as Alzheimer's and prion diseases, in which the inflammatory process is believed to participate directly in neuronal death.     

Insights into function and regulation of small heat shock protein 25 (HSPB1) in a mouse model with targeted gene disruption

The mammalian small heat shock protein (sHSPs) family is comprised of 10 members and includes HSPB1, which is proposed to play an essential role in cellular physiology, acting as a molecular chaperone to regulate diverse cellular processes. Whilst differential roles for sHSPs are suggested for specific tissues, the relative contribution of individual sHSP family members in cellular and organ physiology remains unclear. To address the function of HSPB1 in vivo and determine its tissue-specific expression during development and in the adult, we generated knock-in mice where the coding sequence of hspb1 is replaced by a lacZ reporter gene. Hspb1 expression marks myogenic differentiation with specific expression first confined to developing cardiac muscles and the vascular system, and later in skeletal muscles with specific expression at advanced stages of myoblast differentiation. In the adult, hspb1 expression was observed in other tissues, such as stratified squamous epithelium of skin, oronasal cavity, tongue, esophagus, and uterine cervix but its expression was most prominent in the musculature. Interestingly, in cardiac muscle hsbp1 expression was down-regulated during the neonatal period and maintained to a relatively low steady-level throughout adulthood. Despite this widespread expression, hspb1-/- mice were viable and fertile with no apparent morphological abnormalities in tissues under physiological conditions. However, at the cellular level and under stress conditions (heat challenge), HSPB1 act synergistically with the stress-induced HSPA1 (HSP70) in thermotolerance development, protecting cells from apoptosis. Our data thus indicate a nonessential role for HSPB1 in embryonic development and for maintenance of tissues under physiological conditions, but also shows that it plays an important role by acting synergistically with other HSPs during stress conditions to exert cytoprotection and anti-apoptotic effects.     

The Administration of an α-MSH Analogue Reduces the Serum Release of IL-1α and TNFα Induced by the Injection of a Sublethal Dose of Lipopolysaccharides in the BALB/c Mouse

The injection of α-MSH or of one of its analogues ([Nle₄-D.Phe₇] α-MSH_4–10) reduced, in vivo, the release of two cytokines (IL-1α and TNFα) involved in inflammation. The inflammatory state was induced in BALB/c mice by intraperitoneal injection of a sublethal dose of lipopolysaccharides (LPS). The assay of these cytokines by ELISA showed a reduction of 20% with α-MSH and between 30 and 60% with the α-MSH analogue. The α-MSH or the analogue was administered in one of two ways: intravenously or subcutaneously. The most efficient method seemed to be the subcutaneous one because it improved the activity 10,000 times more than the intravenous method. Moreover, the analogue induced a regression of mortality in the animals treated by the intravenous method. Our results show that α-MSH and one of its analogues inhibit IL-1α and TNFα, and can be used as anti-inflammatory molecules.     

Temporal patterns of cytokine and apoptosis-related gene expression in spinal cords of the G93A-SOD1 mouse model of amyotrophic lateral sclerosis

Familial amyotrophic lateral sclerosis (FALS) is often caused by gain-of-function mutations in Cu,Zn-superoxide dismutase (SOD1). Multiprobe ribonuclease protection assays (RPAs) were used to investigate expression of 36 different cytokines and apoptosis-related genes in spinal cords of mice that ubiquitously express human SOD1 bearing a glycine (r) alanine substitution at residue 93 (G93A-SOD1). Mice were studied at late presymptomatic stage (80 days), and at 120 days when the animals experience severe hindlimb paralysis and accumulation of oxidatively modified proteins. Spinal cord tissue from G93A-SOD1 mice expressed a selective subset of macrophage-typical cytokines (monokines) including interleukin (IL)1alpha, IL1beta and IL1RA at 80 days increasing by 120 days. Contrastingly, T-cell derived cytokines (lymphokines) including IL2, IL3 and IL4 were detected at low levels in non-transgenic mice but these were not elevated in G93A-SOD1 mice even at 120 days. Apoptosis-related genes were generally unaffected at 80 days but multiple caspases and death receptor components were up-regulated at 120 days; the only exceptions being FADD and the tumor necrosis factor (TNF)alpha receptor p55 which was up-regulated at 80 days and increased further at 120 days. These data indicate that in the G93A-SOD1 mouse: (i) cytokine expression changes precede bulk protein oxidation and apoptosis gene expression; (ii) lymphocyte contributions to cytokine expression in FALS are likely minor; and (iii) TNFalpha and its receptors may link inflammation to apoptosis in ALS.     

Characterizing the multiple roles of FGF-2 in SOD1G93A ALS mice in vivo and in vitro

We have previously shown that knockout of fibroblast growth factor-2 (FGF-2) and potential compensatory effects of other growth factors result in amelioration of disease symptoms in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). ALS is a rapidly progressive neurological disorder leading to degeneration of cortical, brain stem, and spinal motor neurons followed by subsequent denervation and muscle wasting. Mutations in the superoxide dismutase 1 (SOD1) gene are responsible for approximately 20% of familial ALS cases and SOD1 mutant mice still are among the models best mimicking clinical and neuropathological characteristics of ALS. The aim of the present study was a thorough characterization of FGF-2 and other growth factors and signaling effectors in vivo in the SOD1^G93A mouse model. We observed tissue-specific opposing gene regulation of FGF-2 and overall dysregulation of other growth factors, which in the gastrocnemius muscle was associated with reduced downstream extracellular-signal-regulated kinases (ERK) and protein kinase B (AKT) activation. To further investigate whether the effects of FGF-2 on motor neuron death are mediated by glial cells, astrocytes lacking FGF-2 were cocultured together with mutant SOD1 ^G93A motor neurons. FGF-2 had an impact on motor neuron maturation indicating that astrocytic FGF-2 affects motor neurons at a developmental stage. Moreover, neuronal gene expression patterns showed FGF-2- and SOD1 ^G93A-dependent changes in ciliary neurotrophic factor, glial-cell-line-derived neurotrophic factor, and ERK2, implying a potential involvement in ALS pathogenesis before the onset of clinical symptoms.     

敲减MC4R表达对牛胎儿成纤维细胞CMS系统关键因子的影响

为获得敲减黑素皮质素4受体（melanocortin 4 receptor,MC4R）基因的牛胎儿成纤维细胞,并探讨其在能量平衡神经调节系统中的作用,将构建成功并已鉴定为有效序列的短发夹状RNA (short hairpin RNA, shRNA)真核表达载体pGSH1 GFP MC4R,利用阳离子脂质体转染牛胎儿成纤维细胞并使用G418筛选稳定转染细胞株.利用实时荧光定量和Western印迹检测MC4R及中枢黑素皮质素系统(central melanocortin system, CMS)关键因子的表达水平变化.结果表明,在稳定转染的牛胎儿成纤维细胞系中, MC4R表达显著抑制,瘦蛋白(leptin)和阿黑色素原(POMC)表达下调,黑素皮质素拮抗物agouti相关蛋白(AGRP)和MC3R表达上调,而神经肽Y (NPY)表达无明显改变.综上所述,本研究成功获得了敲减MC4R基因表达的牛胎儿成纤维细胞.相关基因表达水平检测结果提示, MC4R的表达水平对CMS系统中的各关键基因的表达有不同的抑制或促进影响.     

胰高血糖素样肽l对脂多糖诱导的血管内皮细胞炎性反应的影响

目的:探讨胰高血糖素样肽l(glucagon like peptide 1,GLP-1)对脂多糖(1ipopolysaccharide,LPS)诱导的血管内皮细胞(VEC)炎性反应的影响。方法:以体外培养的人动脉VEC为研究模型,将细胞分为四组(对照组、LPS刺激组、LPS+GLP-1组、GLP-1组),Rhodamin-Phalloidin检测肌动蛋白骨架F-actin分布,用苏木素-伊红(HE)染色观察细胞间连接的形态特征,用示踪剂Rhodamine B isothiocyanate-Dextran检测VECs单层通透性变化改变,酶联免疫吸附实验检测细胞分泌白介素(IL)-6和IL-8的变化。结果:GLP-1(100 nM)可减少LPS(1μg/mL)刺激后细胞肌动蛋白骨架F-actin应力纤维的形成,并抑制LPS刺激后细胞间连接的中断。Rhodamine B isothiocyanate-Dextran细胞通透性检测结果显示:GLP-1可明显降低LPS刺激引起的VEC通透性增加[由(2.57±0.19)×10-5cm/s降至(2.10±0.18)×10-5cm/s,P0.05]。此外,GLP-1可抑制LPS刺激后VEC中炎性细胞因子IL-6和IL-8的表达[分别由(42130±6522)pg/ml降至(27478±5096)pg/ml和(18376±1561)pg/ml降至(14414±927)pg/ml,均P0.05]。结论:GLP-1可对抗LPS刺激引起的VEC炎症反应和细胞通透性增加,改善LPS诱导的内皮细胞炎性损伤。