Inhibiting ceramide synthase 5 expression in microglia decreases neuroinflammation after spinal cord injury

Microglia,the resident monocyte of the central nervous system,play a crucial role in the response to spinal cord injury.However,the precise mechanism remains unclear.To investigate the molecular mechanisms by which microglia regulate the neuroinflammatory response to spinal cord injury,we performed single-cell RNA sequencing dataset analysis,focusing on changes in microglial subpopulations.We found that the MG1 subpopulation emerged in the acute/subacute phase of spinal cord injury and expressed genes related to cell pyroptosis,sphingomyelin metabolism,and neuroinflammation at high levels.Subsequently,we established a mouse model of contusive injury and performed intrathecal injection of siRNA and molecular inhibitors to validate the role of ceramide synthase 5 in the neuroinflammatory responses and pyroptosis after spinal cord injury.Finally,we established a PC12-BV2 cell co-culture system and found that ceramide synthase 5 and pyroptosis-associated proteins were highly expressed to induce the apoptosis of neuron cells.Inhibiting ceramide synthase 5 expression in a mouse model of spinal cord injury effectively reduced pyroptosis.Furthermore,ceramide synthase 5-induced pyroptosis was dependent on activation of the NLRP3 signaling pathway.Inhibiting ceramide synthase 5 expression in microglia in vivo reduced neuronal apoptosis and promoted recovery of neurological function.Pla2g7 formed a“bridge”between sphingolipid metabolism and ceramide synthase 5-mediated cell death by inhibiting the NLRP3 signaling pathway.Collectively,these findings suggest that inhibiting ceramide synthase 5 expression in microglia after spinal cord injury effectively suppressed microglial pyroptosis mediated by NLRP3,thereby exerting neuroprotective effects.

Carboxyl Ester Lipase Protects Against Metabolic Dysfunction-Associated Steatohepatitis by Binding to Fatty Acid Synthase

Carboxyl ester lipase(CEL),a pivotal enzyme involved in lipid metabolism,is recurrently mutated in obese mice.Here,we aimed to elucidate the functional significance,molecular mechanism,and therapeutic potential of CEL in metabolic dysfunction-associated steatohepatitis(MASH).Hepatocyte-specific carboxyl ester lipase gene(Cel)knockout(Cel^(DHEP))and wildtype(WT)littermates were fed with cholinedeficient high-fat diet(CD-HFD)for 16 weeks,or methionine-and choline-deficient diet(MCD)for three weeks to induce MASH.Liquid chromatography–mass spectrometry and co-immunoprecipitation were employed to identify the downstream targets of CEL.CD-HFD/MCD-fed WT mice received intravenous injections of CEL-adeno-associated viral,serotype 8(AAV8)to induce specific overexpression of CEL in the liver.We observed a decrease in CEL protein levels in MASH induced by CD-HFD or MCD in mice.Cel^(DHEP) mice fed with CD-HFD or MCD exhibited pronounced hepatic steatosis,inflammation,lipid peroxidation,and liver injury compared to WT littermates,accompanied by increased hepatic nuclear factor kappa-light-chain-enhancer of activated B cell(NF-jB)activation.Consistently,Cel knockdown in mouse primary hepatocytes and AML12 cells aggravated lipid accumulation and inflammation,whereas CEL overexpression exerted the opposite effect.Mechanistically,CEL directly bound to fatty acid synthase(FASN),resulting in reduced FASN SUMOylation,which in turn promoted FASN degradation through the proteasome pathway.Furthermore,inhibition of FASN ameliorated hepatocyte lipid accumulation and inflammation induced by Cel knockdown in vivo and in vitro.Hepatocyte-specific CEL overexpression using AAV8-Cel significantly mitigated steatohepatitis in mice fed with CD-HFD or MCD.CEL protects against steatohepatitis development by directly interacting with FASN and suppressing its expression for de novo lipogenesis.CEL overexpression confers a therapeutic benefit in steatohepatitis.

Post-translational modifications of prostaglandin-endoperoxide synthase 2 in colorectal cancer:An update

The biosynthesis of prostanoids is involved in both physiological and pathological processes. The expression of prostaglandin-endoperoxide synthase 2(PTGS2; also known as COX-2) has been traditionally associated to the onset of several pathologies, from inflammation to cardiovascular, gastrointestinal and oncologic events. For this reason, the search of selective PTGS2 inhibitors has been a focus for therapeutic interventions. In addition to the classic non-steroidal anti-inflammatory drugs, selective and specific PTGS2 inhibitors, termed coxibs, have been generated and widely used. PTGS2 activity is less restrictive in terms of substrate specificity than the homeostatic counterpart PTGS1, and it accounts for the elevated prostanoid synthesis that accompanies several pathologies. The main regulation of PTGS2 occurs at the transcription level. In addition to this, the stability of the mRNA is finely regulated through the interaction with several cytoplasmic elements, ranging from specificmicroR NAs to proteins that control mR NA degradation. Moreover, the protein has been recognized to be the substrate for several post-translational modifications that affect both the enzyme activity and the targeting for degradation via proteasomal and non-proteasomal mechanisms. Among these modifications, phosphorylation, glycosylation and covalent modifications by reactive lipidic intermediates and by free radicals associated to the proinflammatory condition appear to be the main changes. Identification of these post-translational modifications is relevant to better understand the role of PTGS2 in several pathologies and to establish a correct analysis of the potential function of this protein in diseases progress. Finally, these modifications can be used as biomarkers to establish correlations with other parameters, including the immunomodulation dependent on molecular pathological epidemiology determinants, which may provide a better frame for potential therapeutic interventions.

Fatty Acid Synthase Inhibitors from Plants and Their Potential Application in the Prevention of Metabolic Syndrome

Fatty acid synthase （FAS） attracts more and more attention recently as a potential target for metabolic syndrome,such as cancer, obesity, diabetes and cerebrovascular disease. FAS inhibitors are widely existed in plants, consisting of diversiform compounds. These inhibitors exist not only in herbs also in many plant foods, such as teas, allium vegetables and some fruits. These effective components include gallated catechins, theaflavins,flavonoids, condensed and hydrolysable tannins, thioethers,pentacyclic triterpenes, stilbene derivatives, etc, and they target at the different domains of FAS, showing different inhibitory mechanisms. Interestingly, these FAS inhibitor-contained herbs and plant foods and their effective components are commonly related to the prevention of metabolic syndromes including fatreducing and depression of cancer. From biochemical angle,FAS can control the balance between energy provision and fat production. Some studies have shown that the effects of those effective components in plants on metabolic syndromes are mediated by inhibiting FAS. This suggests that FAS plays a critical role in the regulation of energy metabolism, and the FAS inhibitors from plants have significant potential application value in the treatment and prevention of metabolic syndromes.

Expression of inducible nitric oxide synthase and cyclooxygenase-2 in pancreatic adenocarcinoma:Correlation with microvessel density

AIM:Cydooxygenases (COX) are key enzymes for conversion of arachidonic acid to prostaglandins.Nitric oxide synthase (NOS) is the enzyme responsible for formation of nitric oxide. Both have constitutive and inducible isoforms.The inducible isoforms (iNOS and COX-2) are of great interest as regulators of tumor angiogenesis,tumorigenesis and inflammatory processes.This study was to clarify their role in pancreatic adenocarcinomas. METHODS:We investigated the immunohistochemical iNOS and COX-2 expression in 40 pancreatic ductal adenocardnomas of different grade and stage.The results were compared with microvessel density and dinicopathological data. RESULTS:Twenty-one (52.5%) of the cases showed iNOS expression,15 (37.5%) of the cases were positive for COX-2. The immunoreaction was heterogeneously distributed within the tumors.Staining intensity was different between the tumors.No correlation between iNOS and COX-2 expression was seen.There was no relationship with microvessel density. However,iNOS positive tumors developed more often distant metastases and the more malignant tumors showed a higher COX-2 expression.There was no correlation with other clinicopathological data. CONCLUSION:Approximately half of the cases expressed iNOS and COX-2.These two enzymes do not seem to be the key step in angiogenesis or carcinogenesis of pancreatic adenocarcinomas.Due to a low prevalence of COX-2 expression,chemoprevention of pancreatic carcinomas by COX-2 inhibitors can only achieve a limited success.

AMPK-associated signaling to bridge the gap between fuel metabolism and hepatocyte viability

The adenosine monophosphate-activated protein kinase (AMPK) and p70 ribosomal S6 kinase-1 pathway may serve as a key signaling flow that regulates energy metabolism; thus, this pathway becomes an attractive target for the treatment of liver diseases that result from metabolic derangements. In addition, AMPK emerges as a kinase that controls the redox-state and mitochondrial function, whose activity may be modulated by antioxidants. A close link exists between fuel metabolism and mitochondrial biogenesis. The relationship between fuel metabolism and cell survival strongly implies the existence of a shared signaling network, by which hepatocytes respond to challenges of external stimuli. The AMPK pathway may belong to this network. A series of drugs and therapeutic candidates enable hepatocytes to protect mitochondria from radical stress and increase cell viability, which may be associated with the activation of AMPK, liver kinase B1, and other molecules or components. Consequently, the components downstream of AMPK may contribute to stabilizing mitochondrial membrane potential for hepatocyte survival. In this review, we discuss the role of the AMPK pathway in hepatic energy metabolism and hepatocyte viability. This information may help identify ways to prevent and/or treat hepatic diseases caused by the metabolic syndrome. Moreover, clinical drugs and experimental therapeutic candidates that directly or indirectly modulate the AMPK pathway in distinct manners are discussed here with particular emphasis on their effects on fuel metabolism and mitochondrial function.

Role of abnormal lipid metabolism in development,progression,diagnosis and therapy of pancreatic cancer

There is growing evidence that metabolic alterations play an important role in cancer development and progression.The metabolism of cancer cells is reprogrammed in order to support their rapid proliferation.Elevated fatty acid synthesis is one of the most important aberrations of cancer cell metabolism.An enhancement of fatty acids synthesis is required both for carcinogenesis and cancer cell survival,as inhibition of key lipogenic enzymes slows down the growth of tumor cells and impairs their survival.Based on the data that serum fatty acid synthase(FASN),also known as oncoantigen 519,is elevated in patients with certain types of cancer,its serum level was proposed as a marker of neoplasia.This review aims to demonstrate the changes in lipid metabolism and other metabolic processes associated with lipid metabolism in pancreatic ductal adenocarcinoma(PDAC),the most common pancreatic neoplasm,characterized by high mortality.We also addressed the influence of some oncogenic factors and tumor suppressors on pancreatic cancer cell metabolism.Additionally the review discusses the potential role of elevated lipid synthesis in diagnosis and treatment of pancreatic cancer.In particular,FASN is a viable candidate for indicator of pathologic state,marker of neoplasia,as well as,pharmacological treatment target in pancreatic cancer.Recent research showed that,in addition to lipogenesis,certain cancer cells can use fatty acids from circulation,derived from diet(chylomicrons),synthesized in liver,or released from adipose tissue for their growth.Thus,the interactions between de novo lipogenesis and uptake of fatty acids from circulation by PDAC cells require further investigation.

Suppression of glutamate synthase genes significantly affects carbon and nitrogen metabolism in rice (Oryza sativa L.)

Rice (Oryza sativa) glutamate synthase (GOGAT,EC 1.4.1.14) enzymes have been proposed to have great potential for improving nitrogen use efficiency,but their functions in vivo and their effects on carbon and nitrogen metabolism have not been systematically explored.In this research,we analyzed transcriptional profiles of rice GOGAT genes using a genome-wide microarray database,and investigated the effects of suppression of glutamate synthase genes on carbon and nitrogen metabolism using GOGAT co-suppressed rice plants.Transcriptional profiles showed that rice GOGAT genes were expressed differently in various tissues and organs,which suggested that they have different roles in vivo.Compared with the wild-type,tiller number,total shoot dry weight,and yield of GOGAT co-suppressed plants were significantly decreased.Physiological and biochemical studies showed that the contents of nitrate,several kinds of free amino acids,chlorophyll,sugars,sugar phosphates,and pyridine nucleotides were significantly decreased in leaves of GOGAT co-suppressed plants,but the contents of free ammonium,2-oxoglutarate,and isocitrate in leaves were increased.We conclude that GOGATs play essential roles in carbon and nitrogen metabolism,and that they are indispensable for efficient nitrogen assimilation in rice.

Acyl-CoA synthase ACSL4:an essential target in ferroptosis and fatty acid metabolism

Long-chain acyl-coenzyme A(CoA)synthase 4(ACSL4)is an enzyme that esterifies CoA into specific polyunsaturated fatty acids,such as arachidonic acid and adrenic acid.Based on accumulated evidence,the ACSL4-catalyzed biosynthesis of arachidonoyl-CoA contributes to the execution of ferroptosis by triggering phospholipid peroxidation.Ferroptosis is a type of programmed cell death caused by iron-dependent peroxidation of lipids;ACSL4 and glutathione peroxidase 4 positively and negatively regulate ferroptosis,respectively.In addition,ACSL4 is an essential regulator of fatty acid(FA)metabolism.ACSL4 remodels the phospholipid composition of cell membranes,regulates steroidogenesis,and balances eicosanoid biosynthesis.In addition,ACSL4-mediated metabolic reprogramming and antitumor immunity have attracted much attention in cancer biology.Because it facilitates the cross-talk between ferroptosis and FA metabolism,ACSL4 is also a research hotspot in metabolic diseases and ischemia/reperfusion injuries.In this review,we focus on the structure,biological function,and unique role of ASCL4 in various human diseases.Finally,we propose that ACSL4 might be a potential therapeutic target.

Molecular Regulation of the Metabolic Pathways of the Medicinal Plants: <i>Phyla dulcis</i>

Phyla (Lippia) dulcis contains hernundulcin sesquiterpene zero-caloric sweetener that is about a thousand times sweeter than sucrose, and also bitter constituents including camphor and limonene. There is yet no simple method to remove the undesirable constituents. The yield of sweetener hernundulcin is very low, and there is no simple method to maximize its composition. The aim of the project was to characterize the mRNA targets that regulate the primary and terpenoid metabolic enzymes of P. dulcis. Restriction fragment differential display polymerase chain reaction of P. dulcis glutamate dehydrogenase-synthesized RNA showed that many mRNAs encoding β-caryophyllene, (+)-epi-α-bisabolol, bicyclogermacrene, bifunctional sesquiterpene, and geraniol synthases shared sequence homologies with ribulose-1,5-bisphophatase carboxylase, granule-bound starch synthase, pyruvate kinase, glucose-6-phosphate dehydrogenase, and phosphoenol pyruvate carboxylase. Sequence similarities between mRNAs encoding primary metabolic enzymes and terpene synthases suggested that photosynthesis could regulate terpenoid metabolism in order to increase the yield of sweetener hernundulcin.

脂代谢相关蛋白在恶性肿瘤中的表达及关系研究

脂代谢紊乱和肥胖是乳腺癌、结直肠癌、卵巢癌等多种恶性肿瘤的高危因素。脂质能为癌细胞的增殖、迁移及侵袭提供生物膜合成的原料、营养成分、信号分子、能量支持。脂代谢过程中关键酶表达异常,是脂代谢重编程的主要表现。本文综述脂肪酸从头合成相关酶、脂肪酸摄取转运蛋白、脂质水解和动员相关酶及脂肪酸氧化相关酶的临床研究进展,从代谢层面探索恶性肿瘤的发病机制、寻找治疗新靶点。

肌醇代谢在植物响应非生物胁迫中的作用

非生物胁迫制约了植物的生长和发育,降低作物的产量,严重时导致植物死亡。为了应对非生物胁迫,植物在进化过程中形成了一系列胁迫响应机制,包括肌醇(MI,myo-inositol)代谢途径。肌醇为一类化学性质稳定的极性小分子,植物可通过积累其糖苷类衍生物参与渗透调节途径,从而响应非生物胁迫。肌醇-1-磷酸合酶(MIPS,myo-inositol-1-phosphate synthase)、肌醇单磷酸酶(IMP,inositol monophosphtease)和肌醇加氧酶(MIOX,myo-inositol oxygenase)在肌醇的生物合成或分解过程中发挥作用,它们通过调控植物中肌醇的含量,以及后续一系列复杂的转化途径,参与L-抗坏血酸(L-AsA,Lascorbic acid)和部分细胞壁多糖的合成,响应盐、干旱、碱和低温等非生物胁迫。本文综述了肌醇的结构、生物学作用、肌醇代谢途径相关酶和肌醇衍生物在植物响应非生物胁迫中的研究进展,并对未来的研究方向进行了展望,旨在为利用肌醇代谢增强植物对非生物胁迫的抗性,培育抗逆植物品种提供理论基础。

不同成熟果色辣椒中PSY1基因的差异表达分析

为了研究PSY1基因在辣椒不同成熟果色形成过程中的作用机理,以Y15016、Y15016-2、SP01、SP02、Z1为材料,克隆PSY1基因并进行时空表达分析,结合部分生物信息学方法,对PSY蛋白功能性质及辣椒不同果色材料中PSY1基因的表达情况进行研究分析。结果表明:5个品种辣椒中均能克隆到PSY1全长基因,且序列无差异;基因结构分析表明,辣椒PSY1基因包含6个外显子、5个内含子,全长为2 844 bp,其CDS全长为1 260 bp,编码419个氨基酸。序列比对和进化树分析表明,辣椒PSY蛋白与同科的番茄、烟草同源PSY蛋白在亲缘关系上最为接近。qRT-PCR分析结果显示:PSY1基因在试验选用的5个品种辣椒根、茎、叶、花组织中均显示表达,在根组织中表达量最低,叶组织中的表达量最高;在橙色突变体Y15016-2的表达量总体高于其在野生型Y15016中的表达量,而在黄色突变体SP02中,其表达量明显低于野生型SP01;在果实不同发育阶段,PSY1基因除Ⅲ期表达有所降低外,其表达量随果实发育总体呈现上升趋势,且在不同果色材料的成熟期(Ⅳ—Ⅴ期)达到最大值。PSY1基因启动子分析结果显示,供试材料中其序列未见差异。结果表明,在不同果色辣椒的果色形成过程中,PSY1基因的差异表达可能起到了较为重要的作用。

光响应基因BcCfaS通过脂质代谢调控灰葡萄孢菌的光形态建成及致病力

Light is a fundamental environmental factor for living organisms on earth—not only as a primary energy source but also as an informational signal.In fungi,light can be used as an indicator for both time and space to control important physiological and morphological responses.Botrytis cinerea(B.cinerea)is a devastating phytopathogenic fungus that exploits light cues to optimize virulence and the balance between conidiation and sclerotia development,thereby improving its dispersal and survival in ecosystems.However,the components and mechanisms underlying these processes remain obscure.Here,we identify a novel light-signaling component in B.cinerea,BcCfaS,which encodes a putative cyclopropane fatty-acyl-phospholipid synthase.BcCfaS is strongly induced by light at the transcriptional level and plays a crucial role in regulating photomorphogenesis.Deletion of BcCfaS results in reduced vegetative growth,altered colony morphology,impaired sclerotial development,and enhanced conidiation in a lightdependent manner.Moreover,the mutant exhibits serious defects in stress response and virulence on the host.Based on a lipidomics analysis,a number of previously unknown fungal lipids and many BcCfaS-regulated lipids are identified in B.cinerea,including several novel phospholipids and fatty acids.Importantly,we find that BcCfaS controls conidiation and sclerotial development by positively regulating methyl jasmonate(MeJA)synthesis to activate the transcription of light-signaling components,revealing for the first time the metabolic base of photomorphogenesis in fungi.Thus,we propose that BcCfaS serves as an integration node for light and lipid metabolism,thereby providing a regulatory mechanism by which fungi adapt their development to a changing light environment.These new findings provide an important target for antifungal design to prevent and control fungal disease.

Fasn involved in the nephrotoxicity induced by polystyrene nanoplastics and the intervention of melatonin through intestinal microbiota-mediated lipid metabolism disorder

Nanoplastics(NPs)can accumulate in the kidney and cause kidney injury,but the multi-organ interaction mechanism and preventive measures of kidney injury are still unclear.In this study,in vivo(60μg/day,42 days)and in vitro(0.4μg/μL,24 h)exposure models of polystyrene nanoplastics(PS-NPs,80 nm)in mice and human kidney cortex proximal tubule epithelial cells(HK-2 cells)were established,respectively.Our study revealed that PS-NPs caused obvious pathological changes and impaired renal function in mice,which were related to lipid metabolism disorders mediated by intestinal flora.Desulfovibrionales-fatty acid synthase(Fasn)-docosahexaenoic acid(DHA)pathway may be one of the mechanisms of kidney injury in mice.Importantly,we also found that melatonin attenuates PS-NPs-induced nephrotoxicity by modulating lipid metabolism disorders(represented by DHA)and affects Fasn expression.In conclusion,our study revealed the important role of intestinal flora-mediated lipid metabolism in PS-NPs-induced nephrotoxicity and preliminarily provided potential key gene targets and effective preventive measures for PS-NPs-induced nephrotoxicity.

Filamentation and inhibition of prokaryotic CTP synthase with ligands

Cytidine triphosphate synthase(CTPS)plays a pivotal role in the de novo synthesis of cytidine triphosphate(CTP),a fundamental building block for RNA and DNA that is essential for life.CTPS is capable of directly binding to all four nucleotide triphosphates:adenine triphosphate,uridine triphosphate,CTP,and guanidine triphosphate.Furthermore,CTPS can form cytoophidia in vivo and metabolic filaments in vitro,undergoing regulation at multiple levels.CTPS is considered a potential therapeutic target for combating invasions or infections by viral or prokaryotic pathogens.Utilizing cryo-electron microscopy,we determined the structure of Escherichia coli CTPS(ecCTPS)filament in complex with CTP,nicotinamide adenine dinucleotide(NADH),and the covalent inhibitor 6-diazo-5-oxo-L-norleucine(DON),achieving a resolution of 2.9A.We constructed a phylogenetic tree based on differences in filament-forming interfaces and designed a variant to validate our hypothesis,providing an evolutionary perspective on CTPS filament formation.Our computational analysis revealed a solvent-accessible ammonia tunnel upon DON binding.Through comparative structural analysis,we discern a distinct mode of CTP binding of ecCTPS that differs from eukaryotic counterparts.Combining biochemical assays and structural analysis,we determined and validated the synergistic inhibitory effects of CTP with NADH or adenine on CTPS.Our results expand our comprehension of the diverse regulatory aspects of CTPS and lay a foundation for the design of specific inhibitors targeting prokaryotic CTPS.

Association of Methionine Synthase rs1805087 Polymorphism With Arsenic-Related Skin Pigmentary Changes:A Population-Based Case-Control Study

Objective:Chronic arsenic exposure causes skin lesions including skin cancers,pigmentary changes,and keratosis.Genetic polymorphism in arsenic metabolism may increase susceptibility to the development of arsenic-related skin lesions.This study was performed to determine whether arsenic metabolism-related gene variants are associated with arsenic-related pigmentary changes.Methods:This case-control study involved 189 patients with arsenic-related pigmentary changes and 103 controls.Thirty-eight polymorphisms in 10 genes determined by mass spectrometry assay served as candidate drivers of arsenic-induced pigmentary changes.Urine and plasma arsenic levels were determined by inductively coupled plasma mass spectrometry.Hair arsenic concentrations were measured by nondispersive atomic fluorescence spectrometry.Arsenic metabolites in urine were determined using high-performance liquid chromatography with inductively coupled plasma mass spectrometry.Serum folate was measured using a folate radio assay kit.Analysis of variance,nonparametric test,or the chi-square test was selected according to the data distribution.Spearman correlation analysis was used to determine the correlation between two parameters.Logistic regression was used to estimate the effect of single-nucleotide polymorphisms.Results:The arsenic concentrations in urine,plasma,and hair and the urine arsenic species were not significantly different between patients and controls.Logistic regression revealed that among the polymorphisms,the methionine synthase(MTR)rs1805087 polymorphism showed a protective effect against arsenic-related pigmentary changes.In the codominant model,the adjusted odds ratio for age,sex,and ethnicity was 0.41(95%confidence interval[CI],0.21-0.80;P=0.008)for the AG genotype and 0.11(95%CI,0.02-0.60;P=0.012)for the GG genotype.Conclusion:MTR polymorphism showed a protective effect against arsenic-related pigmentary changes in the logistic regression model.The effect of MTR rs1805087 might be independent of arsenic metabolism and one-carbon metabolism.More studies are needed to clarify the biological function of MTR rs1805087 and its relationship with the etiology of arsenic-related pigmentary changes.

Effect of Dangfei Liganning capsule(当飞利肝宁胶囊) on liver X receptor α/steroid regulatory element binding protein-1/fatty acid synthase signal pathway in rats with metabolic-associated fatty liver disease

OBJECTIVE: To study the mechanism of Dangfei Liganning capsule(当飞利肝宁胶囊) in the treatment of rats with metabolic associated fatty liver disease(MAFLD). METHODS: Totally 48 specific pathogen free SpragueDawley male rats were randomly divided into normal Group, model group, Dangfei Liganning high, moderate, and low-dose groups and Essentiale group which were fed with high fat diet for 8 weeks, and gavage and molding were carried out simultaneously. Dangfei Liganning high, middle and low-dose group were given 0.27, 0.135 and 0.0675 g·kg-1·d-1 respectively by gavage, Essentiale group was given 0.123 g·kg-1·d-1 by gavage, the same amount of distilled water was given by gavage in the normal group and the model group. The rats were weighed at the 0th week, 2nd week, 4th week, 6th week and 8th weekend respectively. The rats were sacrificed at the end of the 8th week. Serum levels of alanine aminotransferase(ALT), alanine aminotransferase(AST),triglyceride(TG), total cholesterol(CHO), high-density lipoprotein cholesterol(HDL-C), low-density lipoprotein (LDL-C), total protein(TP), albumin(Alb), globulin(GLB), total bilirubin(TBIL), direct bilirubin(DBIL), tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) were measured. The levels of liver tumor necrosis factor-α(TNF-α), interleukin-6(IL-6) and liver pathology [hematoxylin and eosin(HE) staining, oil red O staining] were detected. The expression levels of liver X receptor α(LXRα), steroid regulatory element binding protein-1(SREBP-1) and fatty acid synthase(FAS) were detected by immunohistochemistry, Western blot and reverse transcription-polymerase chain reaction reverse transcription-polymerase chain reaction. RESULTS: From the beginning to the 8th week, the growth rate of body weight in the Dangfei Liganning highdose group was slower than all other groups. There was no significant difference in ALB level in all groups(P > 0.05). Compared with the model group, the levels of ALT, AST, LDL-C, TG, CHO, TP, GLB, TBIL, DBIL, IL-6, TNF-α were significantly decreased and HDL-C were significantly increased in Dangfei Liganning high-dose group(P < 0.01, < 0.05). HE and oil red O staining showed that the fatty lesions in rat liver were alleviated, while the expressions of LXRα, SREBP-1, FAS m RNA and protein were significantly decreased(P < 0.01). CONCLUSIONS: Dangfei Liganning capsule can slow down the increase of body weight of MAFLD rats, reduce the levels of transaminase, Lipid and inflammatory factors in MAFLD rats, promote the synthesis of liver protein and bile metabolism, and improve the liver fatty lesion of MAFLD rats, among which the Dangfei Liganning highdose group is more effective. The mechanism of action may be through blocking LXR-SREBP-1-FAS signal pathway.

昆虫几丁质合成酶基因转录调控研究进展

几丁质广泛存在于真菌、昆虫和甲壳类动物中,但不存在于植物和脊椎动物中,为昆虫外骨骼、气管及中肠围食膜的重要组分。昆虫蜕皮过程中,富含几丁质的结构需重建以完成虫体扩张,这一过程中需要严格控制几丁质的合成。因此,几丁质生物合成一直是害虫控制的重要靶标。随着昆虫种群耐药性的迅速发展,害虫防控面临新的挑战,需要不断寻找新的害虫防治靶点,以开发新型杀虫剂,实现害虫的有效防控。几丁质合成酶(Chitin synthase,CHS)为昆虫几丁质合成途径的关键酶,在几丁质合成中发挥重要作用。昆虫存在CHS1和CHS2两类几丁质合成酶,CHS1在昆虫外骨骼及气管中表达,催化几丁质合成,CHS2则主要负责中肠围食膜的几丁质合成。昆虫CHS1基因受干扰后可导致虫体表皮产生缺陷,背气管干发育异常,而CHS2基因受到抑制后则往往会导致虫体中肠变短,体重下降。两类CHS基因下调均可引起昆虫大量死亡。昆虫体内具有复杂的CHS转录调控机制以保证其正常生长发育和应对外界刺激。根据国内外研究,该文综述了昆虫CHS基因转录调控研究进展,包括昆虫激素、转录因子、表皮损伤及进食刺激、几丁质代谢相关基因及物质、microRNA以及几丁质合成抑制剂等对昆虫CHS mRNA水平的影响,为将来开发和利用以CHS为靶标的绿色农药防治害虫提供理论依据。

广藿香醇合酶PcPTS互作蛋白的筛选与鉴定分析

【目的】广藿香[Pogostemon cablin(Blanco)Benth.]是临床常用的芳香化湿药,其质量指标成分广藿香醇具有良好的抗病毒、抗炎症等活性。广藿香醇合酶(Patchouli alcohol synthase,PcPTS)在广藿香醇生物合成途径中起关键作用,但其在蛋白互作层面调控广藿香醇合成的机制尚不清楚。旨在筛选PcPTS的互作蛋白,以揭示PcPTS在广藿香醇生物合成途径中的调控机制和功能。【方法】利用酵母双杂交技术和GST pull-down联合液相色谱-串联质谱(LC-MS/MS)技术筛选可能与PcPTS互作的蛋白,利用MASCOT软件和BLAST分析注释互作蛋白,选取有文献报道参与其他蛋白互作、影响蛋白转运、修饰或降解、参与次生代谢调控的蛋白,利用酵母双杂交技术初步验证它们与PcPTS蛋白的互作关系并对互作蛋白进行生物信息学分析。【结果】通过PCR技术成功克隆了PcPTS的cDNA序列,开放阅读框(ORF)为1659 bp,编码522个氨基酸。构建了广藿香cDNA初级文库和酵母杂交文库,初级和次级文库容量分别为7.6×10^(6) CFU和8.6×10^(6) CFU,初级和次级文库的重组率均为100%,且插入片段平均长度均大于800 bp。构建的诱饵载体pGBKT7-PcPTS对酵母菌株不产生毒性,且无自激活活性。利用酵母双杂交筛选得到阳性克隆并进行测序和BLAST分析,获得17个候选蛋白。成功构建了GST-PcPTS表达载体,诱导表达纯化获得GST-PcPTS诱饵蛋白,利用诱饵蛋白从广藿香总蛋白中捕获互作蛋白,共鉴定出98个候选互作蛋白。从候选蛋白中筛选14个可能与PcPTS互作的蛋白,利用酵母双杂交进行点对点验证,结果发现PcC3H6、PcENO3、PcACR11和PcHAD与PcPTS存在相互作用。生物信息分析表明,四者分别属于CCCH锌指蛋白家族、烯醇化酶蛋白、ACR亚家族和HAD-like超家族。【结论】初步筛选验证获得与PcPTS存在相互作用的4个蛋白PcC3H6、PcENO3、PcACR11和PcHAD,有助于揭示PcPTS在广藿香醇生物合成途径中的作用机制,也为进一步研究广藿香醇的合成调控机制奠定了坚实基础。