Ab-Induced Insulin Resistance and the Effects of Insulin on the Cholesterol Synthesis Pathway and Ab Secretion in Neural Cells

Alzheimer＇s disease（AD） is characterized by amyloid-b（Ab） toxicity,tau pathology,insulin resistance,neuroinflammation,and dysregulation of cholesterol homeostasis,all of which play roles in neurodegeneration.Insulin has polytrophic effects on neurons and may be at the center of these pathophysiological changes.In this study,we investigated possible relationships among insulin signaling and cholesterol biosynthesis,along with the effects of Ab42 on these pathways in vitro.We found that neuroblastoma 2a（N2a） cells transfected with the human gene encoding amyloid-b protein precursor（Ab PP）（N2aAb PP） produced Ab and exhibited insulin resistance by reduced p-Akt and a suppressed cholesterol-synthesis pathway following insulin treatment,and by increased phosphorylation of insulin receptor subunit-1 at serine 612（p-IRS-S612） as compared to parental N2 a cells.Treatment of human neuroblastoma SH-SY5 Y cells with Ab42 also increased p-IRS-S612,suggesting that Ab42 is responsible for insulin resistance.The insulin resistance was alleviated when N2a-Ab PP cells were treated with higher insulin concentrations.Insulin increased Ab release from N2 aAb PP cells,by which it may promote Ab clearance.Insulin increased cholesterol-synthesis gene expression in SHSY5 Y and N2 a cells,including 24-dehydrocholesterol reductase（DHCR24） and 3-hydroxy-3-methyl-glutaryl-Co A reductase（HMGCR） through sterol-regulatory element-binding protein-2（SREBP2）.While Ab42-treated SH-SY5 Y cells exhibited increased HMGCR expression and c-Jun phosphorylation as pro-inflammatory responses,they also showed down-regulation of neuro-protective/antiinflammatory DHCR24.These results suggest that Ab42 may cause insulin resistance,activate JNK for c-Jun phosphorylation,and lead to dysregulation of cholesterol homeostasis,and that enhancing insulin signaling may relieve the insulin-resistant phenotype and the dysregulated cholesterol-synthesis pathway to promote Ab release for clearance from neural cells.

Characteristics and expression of the TCP transcription factors family in Allium senescens reveal its potential roles in drought stress responses

Allium senescens,is an important economic and ecological grassland plant with drought-resistant characteristics.A TCP protein transcription factor is important in the regulation of plant development and adverse responses.However,the mechanism by which TCP transcription functions in drought resistance in Allium senescens is still not clear.Here,we obtained a total of 190,305 transcripts with 115,562 single gene clusters based on RNA-Seq sequencing of Allium senescens under drought stress.The total number of bases was 97,195,096 bp,and the average length was 841.06 bp.Furthermore,we found that there were eight genes of the TCP family that showed an upregulated expression trend under drought stress in Allium senescens.We carried out an investigation to determine the evolution and function of the AsTCP family and how they produce an effect in drought resistance.The 14 AsTCP genes were confirmed and divided into class I and class II containing CIN and CYC/TBI subfamilies,respectively.We also found that the expression of AsTCP17 was remarkably upregulated with drought treatment.Besides,the transformation of AsTCP17 in Arabidopsis revealed that the protective enzymes,namely polyphenol oxidase(POD)and superoxide dismutase(SOD),were increased by 0.4 and 0.8 times,respectively.Chlorophyll content was also increased,while the H2O2 and malondialdehyde(MDA)contents were decreased.Staining assays with 3,3′-diaminobenzidine(DAB)also suggested that the AsTCP17 downregulates reactive oxygen species(ROS)accumulation.In addition,overexpression of the AsTCP17 affected the accumulation of drought-related hormones in plants,and the synthesis of ABA.The expression of AtSVP and AtNCED3,related ABA synthesis pathway genes,indicated that the level of expression of AtSVP and AtNCED3 was obviously enhanced,with the overexpression of line 6 showing a 20.6-fold and 7.0-fold increase,respectively.Taken together,our findings systematically analyze the AsTCPs family at the transcriptome expression level in Allium senescens,and we also demonstrated that AsTCP17 protein,as a positive regulator,was involved in drought resistance of Allium senescens.In addition,our research contributes to the comprehensive understanding of the drought stress defense mechanism in herbaceous plants.

Experimental study on the synergistic inhibition of malignant biological behavior of hepatocellular carcinoma cells by the combination of DMDD and sorafenib

Objective:To investigate the effects and possible mechanisms of the combination of DMDD(2-dodecyl-6-methoxycyclohexa-2-5-diene-1-4-dione),a traditional Chinese medicine monomer,and sorafenib on the malignant biological behavior of human hepatocellular carcinoma Huh7 cells.Methods:The experiment was divided into four groups:Huh7 cells control group,DMDD group,sorafenib group and DMDD and sorafenib combination group.The CCK-8 assay was used to measure the viability of Huh7 cells,and the Kim's formula was used to determine the synergistic effect.The plate cloning experiment was conducted to test colony formation ability of Huh7 cells.The scratch and Transwell experiments were performed to evaluate the migration ability and the invasion ability of Huh7 cells.The cell cycle of Huh7 cells was detected by flow cytometry.RT-qPCR and Western blot were used to measure the mRNA transcription level and protein expression level of PHGDH in the serine synthesis pathway.Results:The plate cloning experiment,scratch experiment,and Transwell migration experiment showed that the combined application of DMDD and Sorafenib significantly enhanced the inhibitory effect on the proliferation,migration,and invasion ability of Huh7 cells compared to the control group,DMDD group,and Sorafenib group(P<0.05).According to the Kim's formula,the combination of DMDD(final concentrations of 2,4,8μmol/L)and Sorafenib(final concentrations of 1,2,4μmol/L)had a synergistic inhibitory effect on the proliferation of Huh7 cells(Q>1.15).6,10μmol/L DMDD combined with 3,5μmol/L Sorafenib showed additive effect.The cell cycle of Huh7 cells was detected by flow cytometry,and the results showed that after 48 hours of drug intervention,the proportion of G2/M phase cells in the control group,DMDD group,Sorafenib group,and combination group were(10.63±0.32)%,(35.77±1.22)%,(30.03±2.22)%,and(38.97±0.60)%,respectively.Compared with the control group,the proportion of G2/M phase cells in the three groups significantly increased(P<0.0001).Compared with the Sorafenib group,the proportion of G2/M phase cells in the combination group significantly increased(P<0.0001).RT-qPCR and Western blot results showed that the combined application of DMDD and Sorafenib significantly inhibited the mRNA transcription level and protein expression level of PHGDH(P<0.05).Conclusion:The combined application of DMDD and Sorafenib has a synergistic effect that can enhance the inhibitory effect on the proliferation,invasion,and migration ability of Huh7 cells.The mechanism of this effect is related to the synergistic inhibition of the gene transcription and protein expression of PHGDH in the serine synthesis pathway.

Identification of a novel PHGDH covalent inhibitor by chemical proteomics and phenotypic profiling

The first rate-limiting enzyme of the serine synthesis pathway(SSP), phosphoglycerate dehydrogenase(PHGDH), is hyperactive in multiple tumors, which leads to the activation of SSP and promotes tumorigenesis. However, only a few inhibitors of PHGDH have been discovered to date, especially the covalent inhibitors of PHGDH. Here, we identified withangulatin A(WA), a natural small molecule,as a novel covalent inhibitor of PHGDH. Affinity-based protein profiling identified that WA could directly bind to PHGDH and inactivate the enzyme activity of PHGDH. Biolayer interferometry and LC-MS/MS analysis further demonstrated the selective covalent binding of WA to the cysteine 295 residue(Cys295)of PHGDH. With the covalent modification of Cys295, WA blocked the substrate-binding domain(SBD)of PHGDH and exerted an allosteric effect to induce PHGDH inactivation. Further studies revealed that with the inhibition of PHGDH mediated by WA, the glutathione synthesis was decreased and intracellular levels of reactive oxygen species(ROS) were elevated, leading to the inhibition of tumor proliferation.This study indicates WA as a novel PHGDH covalent inhibitor, which identifies Cys295 as a novel allosteric regulatory site of PHGDH and holds great potential in developing anti-tumor agents for targeting PHGDH.