Effect of Xuefu Zhuyu Capsules on Polarization of Macrophages in Mice with Atherosclerosis / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Xuefu Zhuyu capsules against atherosclerosis via regulating polarization of macrophages based on Notch1/jagged canonical Notch ligand 1（Jagged1）/Hes family BHLH transcription factor 1（Hes1） signaling pathway. MethodThe mouse models with atherosclerosis were prepared by feeding the mice with an ApoE-/- high-fat diet for four weeks， and they were randomly divided into the model group， Xuefu Zhuyu capsule group， and atorvastatin group. C57BL/6 mice were fed as a normal group. The Xuefu Zhuyu capsule group was intragastrically given Xuefu Zhuyu capsules （0.728 g·kg-1·d-1）， and the atorvastatin group was intragastrically given atorvastatin tablet （6.07 mg·kg-1·d-1）. The normal group and the model group were given equal volume of the deionized water by intragastric administration， and the intervention lasted for 12 weeks. Aortic plaque morphology was observed by hematoxylin-eosin （HE） staining， and aortic plaque area and lipid deposition were observed by oil red O staining. The positive expression levels of CD86 and CD206 in aortic tissue were detected by immunohistochemistry， and serum levels of tumor necrosis factor （TNF）-α， interleukin（IL）-1β， transforming growth factor （TGF）-β1， and IL-10 were detected by enzyme-linked immunosorbent assay （ELISA）. The relative mRNA expressions of inducible nitric oxide synthase （iNOS）， arginase-1 （Arg-1）， Notch1， Jagged1， and Hes1 in aortic tissue were detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The relative protein expression of iNOS， Arg-1， Notch1， Jagged1， and Hes1 in aortic tissue was detected by Western blot. ResultCompared with the normal group， the model group had significant aortic plaque and lipid deposition， and the expression levels of pro-inflammatory cytokines TNF-α and IL-1β were increased （P<0.01）. The expression level of anti-inflammatory cytokine TGF-β1 showed a downward trend， but the difference was not statistically significant. The mRNA and protein expressions of iNOS were increased （P<0.01）. The protein expression of Arg-1 was decreased （P<0.01）， and the mRNA expression of related pathway molecule Jagged1， as well as the protein expressions of Notch1， Jagged1， and Hes1 were increased in the model group （P<0.05， P<0.01）. Compared with those in the model group， the plaque area and lipid deposition had a decreasing trend in the Xuefu Zhuyu capsule group， and the expressions of TNF-α and IL-1β showed a downward trend. The expression of TGF-β1 was increased （P<0.05）， and the expression of macrophage marker CD86 was decreased. The mRNA and protein expressions of iNOS were decreased （P<0.01）. The mRNA and protein expressions of Arg-1 were increased （P<0.05， P<0.01）. Furthermore， the mRNA and protein expressions of Notch1， Jagged1， and Hes1 were decreased （P<0.01）. ConclusionXuefu Zhuyu capsules can reduce aortic plaque area and lipid deposition in mice with atherosclerosis， alleviate inflammation， inhibit M1 macrophages， and promote the expression of M2 macrophages， and the mechanism may be related to the regulation of Notch1/Jagged1/Hes1 signaling pathway.

Research progress of Notch1/Jagged1 signaling pathway in related pathogenesis of chronic heart failure immune inflammation / 重庆医学

Heart failure is a clinical syndrome caused by various causes of myocardial damage and cardi-ac function decrease that cannot meet the body's metabolic needs.The proinflammatory cytokines play an im-portant role in the process of occurrence and development of heart failure.The Notch1/Jagged1 signaling path-way is related to the proinflammatory cytokines,inflammatory responses and immune responses.This paper discusses the involvement of the Notch1/Jagged1 signaling pathway in the development and development of chronic heart failure by modulating immune inflammation.

Effect and mechanism of miR-34a-5p targeting Notchl onH/R-induced apoptosis of human cardiomyocytes / 安徽医科大学学报

Objective@#To investigate the improvement of endoplasmic reticulum stress mediated by microRNANotchl)signaling axis on hypoxia/reoxygenation(H/R)human(miR)-34a-5p/transmembrane fusion protein 1 (es were randomly divided into Control group, H/R group, mimiccardiomyocytes. @*Methods@#Human cardiomyocytNC group, mimic group, inhibitor NC group andinhibitor group. Except the Control group, H/R injury model wasof miR-34a-5p and Notchl were detected by quantitative real.established in other groups. The expression levesurvival rate was detected by thiazolyl blue ( MTT), cell apopto-time polymerase chain reaction(qRT-PCR), celtargeting relationship between miR-34a-5p and Notchl was detec-sis rate was detected by flow cytometry, and theexpressions of transcriptional activator 6(ATF6), inositol demandted by dual luciferase gene reporting method. Thesmic reticulum kinase (PERK) and glucose regulatory protein 78 (GRP78)were detected by Western blot. @*Results@#miR-34a-5p targeted Notchl(P<0.05);compared with Con-apoptosis rate and protein expressions of ATF6, IREl, PERK andtrol group, the expression level of miR-34a-5pGRP78 in H/R group increased, while the cellsurvival rate and Notchl mRNA and protein expressions decreased(P<0.05). Compared with H/R group and minic NC group, miR-34a-5p expression, apoptosis rate , and proteinexpressions of ATF6, IRE1, PERK and GRP78in mimic group increased, while cell survival rate and Notchl mRNA and protein expressions decreased (P <0. 05).Compared with H/R group and inhibitor NC group, the exprespressions of ATF6 , IREl , PERK and GRP78 decreased in inhibi-sion of miR-34a-5p, apoptosis rate and protein etor group,while cell survival rate and Notch1 mINA and protein expressions increased ( P <0.05). @*Conclusion@#miR-34a-5p can inhibit the apoptosis of H/R human cardiomyocytes, possibly through the targeted inhibition ofNotchl-mediated endoplasmic reticulum stress.

Effect of Buzhong Yiqitang on Th17/Treg Immune Imbalance and Notch1 Signaling Pathway in AIT Mice / 中国实验方剂学杂志

ObjectiveTo explore the effect of Buzhong Yiqitang on the immune imbalance of helper T cell 17 （Th17）/regulatory T cell （Treg） and Notch1 signaling pathway in mice with autoimmune thyroiditis （AIT）. MethodA total of 60 8-week-old NOD.H-2h4 mice were randomly divided into the normal group， model group， western medicine group （selenium yeast tablet， 32.5 mg·kg-1）， and low-dose （4.78 g·kg-1·d-1）， middle-dose （9.56 g·kg-1·d-1）， and high-dose （19 g·kg-1·d-1） Buzhong Yiqitang groups， with 10 mice in each group. The normal group was fed with distilled water， and the other groups were fed with water containing 0.05% sodium iodide for eight weeks. After the animal model of AIT was formed spontaneously， the mice were killed under anesthesia after intragastric administration for eight weeks. Serum anti-thyroglobulin antibodies （TGAb）， thyroid-stimulating hormone （TSH）， free triiodothyronine （FT3）， and free thyroid hormone （FT4） were detected by enzyme-linked immunosorbent assay （ELISA）， and thyroid tissue changes were observed by hematoxylin-eosin （HE） staining. The mRNA and protein expressions of retinoid-related orphan receptor-γt （RORγt）， interleukin （IL）-17， forkhead box P3 （FoxP3）， IL-10， Notch1， and hair division-related enhancer 1 （Hes1） in thyroid tissue were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. ResultCompared with the normal group， the thyroid structure of the model group was severely damaged， and lymphocytes were infiltrated obviously. The levels of serum TGAb， FT3， and FT4 contents were significantly increased， and TSH content was significantly decreased （P<0.01）. The mRNA and protein expression levels of RORγt， IL-17， Notch1， and Hes1 were significantly increased， while those of FoxP3 and IL10 were significantly decreased in the model group （P<0.01）. Compared with the model group， thyroid structural damage and lymphocyte infiltration were improved in the treatment groups， and serum TGAb， FT3， and FT4 contents were significantly decreased. TSH content was increased， and mRNA and protein expression levels of RORγt， IL-17， Notch1， and Hes1 were decreased. mRNA and protein expression levels of FoxP3 and IL-10 were increased to different degrees （P<0.05， P<0.01）， and the middle-dose Buzhong Yiqitang group had the most significant intervention effect. ConclusionBuzhong Yiqitang can alleviate the thyroid structural damage in AIT mice， and its mechanism may be related to improving the abnormal differentiation of Th17/Treg immune cells and inhibiting the activation of the Notch1 signaling pathway.

Triptolide inhibits the proliferation and induces ferroptosis of osteosarcoma U2OS cells via the miR-34b-5p/Notch1 axis / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探究雷公藤甲素（TPL）通过miR-34b-5p调控Notch1表达对骨肉瘤U2OS细胞铁死亡影响的机制。方法：常规培养U2OS细胞，将其分为对照组、TPL（10 μmol/L）组、TPL（10 μmol/L）+Fer-1（铁死亡抑制剂，20 μmol/L） 组、miR-NC组、miR-34b-5p组、miR-34b-5p+Fer-1（20 μmol/L）组、TPL（10 μmol/L）+anti-miR-34b-5p组、anti-miR-34b-5p+Fer-1（20 μmol/L）组。qPCR法、CCK-8法、铁离子检测试剂、DHE-荧光探针和WB法分别检测各组U2OS细胞中miR-34b-5p的表达、增殖能力、Fe2+水平、ROS水平以及铁死亡相关蛋白（GPX4、SLC7A11及Notch1蛋白）的表达，双萤光素酶报告基因实验验证miR-34b-5p与Notch1的靶向结合关系。结果： TPL可促进U2OS细胞中miR-34b-5p表达，Fer-1和anti-miR-34b-5p则抑制miR-34b-5p的表达（均P<0.05）。TPL明显抑制U2OS细胞的增殖、GPX4、SLC7A11、Notch1蛋白的表达、增加细胞中Fe2+和ROS的含量，Fer-1可逆转TPL对U2OS细胞的作用（均P<0.05）。过表达miR-34b-5p与TPL对U2OS细胞的作用相似（均P<0.05）。miR-34b-5p可靶向结合Notch1（均P<0.05）。miR-34b-5p抑制剂可明显抑制TPL对U2OS细胞的影响，Fer-1可增强miR-34b-5p抑制剂的作用（均P<0.05）。结论：TPL可抑制U2OS细胞的增殖能力并促进其铁死亡，其作用机制可能与miR-34a-5p靶向调节Notch1表达有关。

A molecular approach to triple-negative breast cancer: targeting the Notch signaling pathway

ABSTRACT Introduction Triple-negative breast cancer is an aggressive subtype of breast cancer characterized by the absence of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression. This phenotype renders triple-negative breast cancer cells refractory to conventional therapies, resulting in poor clinical outcomes and an urgent need for novel therapeutic approaches. Recent studies have implicated dysregulation of the Notch receptor signaling pathway in the development and progression of triple-negative breast cancer. Objective This study aimed to conduct a comprehensive literature review to identify potential therapeutic targets of the Notch pathway. Our analysis focused on the upstream and downstream components of this pathway to identify potential therapeutic targets. Results Modulating the Notch signaling pathway may represent a promising therapeutic strategy to treat triple-negative breast cancer. Several potential therapeutic targets within this pathway are in the early stages of development, including upstream (such as Notch ligands) and downstream (including specific molecules involved in triple-negative breast cancer growth). These targets represent potential avenues for therapeutic intervention in triple-negative breast cancer. Comments Additional research specifically addressing issues related to toxicity and improving drug delivery methods is critical for the successful translation of these potential therapeutic targets into effective treatments for patients with triple-negative breast cancer.

LncRNA GNAS-AS1 participates in the proliferation and migration of gastric cancer cells by regulating the miR-449a/Notch1 axis / 实用医学杂志

Objective To explore the impacts of long non-coding RNA(LncRNA)GNAS antisense RNA1(GNAS-AS1)on the proliferation and migration of gastric cancer(GC)cells by regulating the miR-449a/Notch1 axis.Method Tumor tissue and adjacent tissue samples were collected from 30 patients diagnosed with GC at Sichuan Provincial People's Hospital from September 2013 to September 2017;GC cells AGS were randomly divided into Control group,si-NC group,si-GNAS-AS1 group,si-GNAS-AS1+inhibitor NC group,and si-GNAS-AS1+miR-449a inhibitor group.Real-time fluorescence quantitative PCR method was applied to detect the expres-sion of GNAS-AS1,miR-449a,and Notch1 mRNA;MTT experiments and plate cloning experiments were applied to detect the proliferation;wound healing test was applied to detect cell migration;Transwell experiment was applied to detect cell invasion.Western Blot was applied to detect the expression of Notch1,E-cadherin,Vimentin,and N-cadherin proteins.Double Luciferase reporter gene experiment was applied to verify the relationship between GNAS-AS1 and miR-449a,between miR-449a and Notch1,respectively.Results Compared with adjacent tissues,the expression of GNAS-AS1 and Notch1 mRNA in tumor tissue was increased,the expression of miR-449a was reduced(P<0.05).Compared with the Control group and si-NC group,the expression of GNAS-AS1,OD490 value,number of clones formed,scratch healing rate,number of cell invasions,and the expression of Notch1,Vimentin,and N-cadherin proteins in AGS cells in the si-GNAS-AS1 group reduced,the expression of miR-449a and E-cadherin protein increased(P<0.05).Compared with the si-GNAS-AS1 group and the si-GNAS-AS1+inhibitor NC group,the OD490 value,scratch healing rate,number of cell invasions,Notch1,Vimentin,and N-cadherin expression in the si-GNAS-AS1+miR-449a inhibitor group increased,the expression of miR-449a and E-cadherin protein reduced(P<0.05).GNAS-AS1 targeted and negatively regulated miR-449a expression,while miR-449a targeted and negatively regulated Notch1 expression.Conclusion Silencing GNAS-AS1 may inhibit the expression of Notch1 protein by up-regulating miR-449a,thereby inhibiting the proliferation,migration,and invasion pro-cesses of GC cells.

Expression of nicastrin,N1ICD and hes1 proteins in normal and cancerous liver tissues of mice / 实用医学杂志

Objective To detecting the expression of nicastrin,N1ICD and hes1 proteins in normal liver tissues and liver cancer tissues of C57BL/6 mice.Methods Twelve 6-week-old male C57BL/6 mice were ran-domly equally divided into the control group and model group.In the model group,the in situ liver cancer model was established with injection of Hepa1-6 cells into the liver,and the control group was treated with injection of the same amount of normal saline.Liver cancer was verified by HE staining.The expression of nicastrin,N1ICD and hes1 proteins in the normal and cancerous liver tissues was detected by IHC and Western blot.Results IHC results showed that nicastrin,N1ICD and hes1 proteins were localized in the hepatic sinusoidal endothelial cells in the normal hepatocytes,but not expressed in the hepatocytes.The model group showed higher expression of nicas-trin protein but lower expression of N1ICD and hes1 protein in the cancer cells compared with the control group.Conclusion NCSTN gene may play a carcinogenic role in mouse hepatocellular carcinoma,while notch1 and hes1 may play a carcinostasis role in the carcinoma.

Variation in renal function of type 2 diabetic rats undergoing aerobic exercise / 中国组织工程研究

BACKGROUND:Type 2 diabetes is often accompanied by renal dysfunction.Increasing studies have shown that exercise can alleviate metabolic disorders and renal dysfunction in diabetic patients.However,the specific mechanism underlying the renal protective effect of exercise in patients with type 2 diabetes is rarely reported. OBJECTIVE:To investigate whether aerobic exercise can improve renal function in type 2 diabetic rats by inhibiting transforming growth factor β1/Notch1 pathway. METHODS:Male Sprague-Dawley rats were randomly divided into normal control group and diabetes model group.After successful modeling,they were randomly divided into diabetes control group and diabetes exercise group.Rats in the diabetes exercise group were subjected to an 8-week aerobic exercise.Samples were collected after exercise,and the relevant indexes of glucose and lipid metabolism and renal function were detected by automatic biochemical analyzer and ELISA.The microscopic structure of renal cortex was observed by electron microscope.ELISA and RT-PCR were used to detect the expression of related proteins and genes in rat kidney tissue. RESULTS AND CONCLUSION:Compared with the normal control group,fasting blood glucose,total cholesterol,and triglyceride levels and insulin resistance index were significantly increased in the diabetic control group(P＜0.05).Aerobic exercise could significantly reduce fasting blood glucose and triglyceride levels(P＜0.05).Compared with the normal control group,the diabetic control group had significantly increased contents of urinary microalbumin,serum urea nitrogen and serum creatinine(P＜0.01),thickened renal basement membrane,mesangial matrix hyperplasia,accompanied by a certain degree of foot process fusion,and obvious lesion of the kidney.Aerobic exercise could significantly down-regulate the overexpressions of urinary microalbumin,serum urea nitrogen and serum creatinine in type 2 diabetic rats(P＜0.01),and significantly improve the pathological changes of the kidney in diabetic rats.Compared with the normal control group,the protein and gene expression levels of transforming growth factor β1,Notch1,Jagged1 and Hes1 in rat kidney tissue were significantly increased in the diabetic control group(P＜0.01).Aerobic exercise had a highly significant inhibitory effect on the overexpression of transforming growth factor β1,Notch1 and Jagged1 proteins and genes(P＜0.01)and also significantly inhibited the overexpression of Hes1 protein(P＜0.05).In conclusion,aerobic exercise can protect renal function and delay the pathological progression of the kidney in diabetic rats,which may be achieved by inhibiting the overexpression of transforming growth factor β1/Notch1 signaling pathway.

Effects of aerobic exercise on the expression of Notch1 and Caspase-3 in the hippocampus of Alzheimer's disease mice / 中国组织工程研究

BACKGROUND:β-amyloid protein and Tau protein have adverse effects on the cognitive function of Alzheimer's disease patients,and Notch1 and Caspase-3 can regulate the expression of β-amyloid protein and Tau protein.It is not clear whether Notch1 and Caspase-3 mediate the process of aerobic exercise to improve the cognitive ability of Alzheimer's disease patients.At present,there is a lack of studies on the effect of long-term aerobic exercise on the expression of Notch1 and Caspase-3 in the hippocampus of Alzheimer's disease mice. OBJECTIVE:To observe the expression of Notch1 and Caspase-3 in the hippocampus of Alzheimer's disease mice undergoing long-term aerobic exercise and to investigate the effects of Notch1 and Caspase-3 in Alzheimer's disease mice. METHODS:Wild type and APP/PS1 double-transgenic Alzheimer's disease mice aged 3 months were randomly divided into four groups:wild control group,wild exercise group,Alzheimer's disease control group and Alzheimer's disease exercise group,with 20 mice in each group.Mice in the control groups were not subjected to exercise,while those in the exercise groups received aerobic exercise intervention for 5 months.After the exercise intervention,Morris water maze was used to detect the spatial learning and memory ability of mice.Real-time PCR,immunofluorescence and western blot were used to detect the expressions of Aβ1-42,Tau,Notch1 and Caspase-3 in the hippocampal tissues of mice in each group. RESULTS AND CONCLUSION:The spatial learning and memory ability of Alzheimer's mice was significantly worse than that of wild-type mice(P＜0.05).The spatial learning and memory ability of mice in the exercise groups were significantly better than that in the corresponding control groups(P＜0.05).The expressions of Aβ1-42,Tau,Notch1 and Caspase-3 in the hippocampus were significantly higher in the Alzheimer's disease control group than the wild control group(P＜0.05)and were significantly lower in the Alzheimer's disease exercise group than the Alzheimer's disease control group(P＜0.05).To conclude,long-term aerobic exercise can improve the spatial learning and memory ability of Alzheimer's disease mice,which may be related to the decreased expression of Notch1,Caspase-3,Aβ1-42 and Tau protein in the hippocampus of Alzheimer's disease mice.

Notch1-mediated aerobic exercise promotes hippocampal nerve cell proliferation in Alzheimer's disease mice / 中国组织工程研究

BACKGROUND:Abnormal Notch1 signaling pathway is mostly found in the brain of Alzheimer's disease patients,but the role of these signaling pathways in the pathogenesis of Alzheimer's disease has not been fully clarified.Long-term aerobic exercise can alter the expression of Notch1 by affecting the methylation rate of factors related to the Notch1 signaling pathway.However,it is not clear whether aerobic exercise affects hippocampal nerve cell proliferation and histopathological features of Alzheimer's disease mice through the Notch1 signaling pathway. OBJECTIVE:To observe the effects of aerobic exercise on the proliferation and histopathological features of hippocampal nerve cells in Alzheimer's disease mice after DAPT inhibited the Notch1 signaling pathway. METHODS:APP/PS1 double transgenic Alzheimer's disease mice aged 3 months were randomly divided into four groups:control group,exercise control group,inhibitor group,and exercise inhibitor group,with 20 mice in each group.The control group was fed naturally,and the exercise group received aerobic exercise intervention.Both natural feeding and exercise intervention lasted for 20 weeks.The mice were injected with solvent or Notch1 inhibitor at week 18.After 20 weeks,the brain tissue was collected,and Aβ1-42,Tau,Ki67,and Notch1 expression levels were detected by real-time PCR,immunofluorescence,and western blot assay. RESULTS AND CONCLUSION:Compared with the control group,the expressions of Ki67 and Notch1 in the dentate gyrus region of the hippocampus were significantly decreased in the inhibitor group(P<0.05),but there were no significant differences in Aβ1-42 and Tau.The expression of Ki67 in the dentate gyrus region of the hippocampus in the exercise control group was significantly higher than that in the control group,while the expressions of Aβ1-42,Tau,and Notch1 were significantly lower than those in the control group(P<0.05).The expressions of Aβ1-42,Tau,Ki67,and Notch1 in the dentate gyrus region of the hippocampus of the exercise inhibitor group were not significantly different from those of the inhibitor group.In conclusion,the Notch1 signaling pathway may mediate exercise to improve the proliferation and histopathological features of hippocampal nerve cells in Alzheimer's disease mice.

Astragaloside inhibits astrocyte activation and inflammatory response induced by inflammation / 中国组织工程研究

BACKGROUND:Astrocytes play an important role in the pathology of central nervous system diseases.The phenotypic and functional changes in astrocytes suggest that it may be an effective therapeutic target for central nervous system diseases.Our previous studies have confirmed that astragaloside can inhibit the lipopolysaccharide-induced astrocyte inflammatory response.Whether astragaloside can regulate the phenotype and function of astrocytes through Notch-1 and its downstream signaling pathway remains unclear. OBJECTIVE:To explore the effect of astragaloside on astrocyte activation and inflammatory response induced by inflammation and its possible mechanism. METHODS:Cerebral cortex astrocytes derived from neonatal C57BL/6 mouse were cultured in vitro.CCK-8 assay was used to determine the optimum concentration of astragaloside and Notch active inhibitor DAPT.The astrocytes were divided into five groups:PBS group,lipopolysaccharide group,lipopolysaccharide + astragaloside group,lipopolysaccharide + DAPT group and lipopolysaccharide + DAPT + astragaloside group.The secretion level of inflammatory factors was detected by ELISA,and the level of nitric oxide was detected by Griess method.The astrocytes and splenic mononuclear cells were co-cultured in Transwell chamber to observe the migration of CD4T cells.The expression of astrocyte activation marker GFAP,A1 marker C3 and A2 marker S100A10 as well as Notch 1 and Jag-1 was detected by immunofluorescence staining.The expressions of CFB,C3,S100A10,PTX3,Notch-1,Jag-1,and Hes were detected by western blot assay. RESULTS AND CONCLUSION:(1)According to the results of CCK8 assay,the final concentration of astragaloside was selected as 25 μmol/L and the final concentration of DAPT was 50 μmol/L for follow-up experiments.(2)Compared with PBS group,interleukin-6,interleukin-12 and nitric oxide secretion levels in the lipopolysaccharide group were significantly increased(P<0.05,P<0.05,P<0.01).Compared with the lipopolysaccharide group,interleukin-6(all P<0.05),interleukin-12(P>0.05,P<0.05,P<0.05)and nitric oxide(P<0.05,P<0.01,P<0.01)secretion significantly reduced in the lipopolysaccharide + astragaloside group,lipopolysaccharide +DAPT group,lipopolysaccharide + DAPT + astragaloside group.(3)Compared with the PBS group,the expression of GFAP that is the marker of activated astrocytes and the migration of CD4 T cells were significantly increased in the lipopolysaccharide group(P<0.01).Compared with the lipopolysaccharide group,astrocyte activation was significantly inhibited and CD4 T cell migration was significantly reduced in the lipopolysaccharide + astragaloside,lipopolysaccharide +DAPT,lipopolysaccharide + DAPT + astragaloside group(P<0.05,P<0.05,P<0.01).(4)Compared with the PBS group,the expressions of A1 markers C3 and CFB in the lipopolysaccharide group were increased(P<0.01,P<0.05),and the expressions of A2 markers S100A10 and PTX3 were decreased(P<0.01,P<0.05).Compared with the lipopolysaccharide group,C3(all P<0.01)and CFB(both P<0.05)were significantly reduced and S100A10(all P<0.01)and PTX3(P<0.05,P<0.05 and P>0.05)were increased in the lipopolysaccharide + astragaloside,lipopolysaccharide +DAPT,lipopolysaccharide + DAPT + astragaloside group.(5)Compared with the PBS group,the expressions of Jag-1,Notch-1 and Hes in the lipopolysaccharide group were significantly increased(all P<0.01).Compared with the lipopolysaccharide group,the expressions of Jag-1(all P<0.01),Notch-1(all P<0.01)and Hes(P<0.05,P<0.01 and P<0.01)were significantly reduced in the lipopolysaccharide + astragaloside,lipopolysaccharide +DAPT,lipopolysaccharide + DAPT + astragaloside group.(6)The results indicate that astragaloside can promote the transformation of astrocytes from A1 to A2 by regulating Notch-1 signaling pathway,reduce the secretion of inflammatory factors and the migration of CD4 T cells,and thus inhibit astrocyte activation and inflammatory response.

Puerarin inhibits the differentiation of Raw264.7 cells into osteoclasts through the Notch signaling pathway / 中国组织工程研究

BACKGROUND:Previous studies have shown that puerarin can inhibit the differentiation of osteoclasts,and the expression of Notch signaling pathway-related proteins such as Notch1,HES1,and Jagged1 is decreased.However,the specific mechanism of the Notch1 signaling pathway for the inhibition of osteoclast differentiation by puerarin is not clear. OBJECTIVE:To explore the effect of Notch signaling pathway on puerarin inhibiting the differentiation of mouse macrophage Raw264.7 into osteoclasts. METHODS:Raw264.7 cells were divided into seven groups for intervention culture.Blank control group was cultured in high-sugar DMEM medium;the osteoclast induction group was cultured in osteoclast induction medium;the puerarin intervention group was cultured with 50 μmol/L puerarin at the same time of osteoclast induction;Notch1 siRNA control group,Notch1 siRNA group,Notch1 overexpression control group and Notch1 overexpression group were transfected with Notch1 siRNA control sequence,Notch1 siRNA,Notch1 overexpression control plasmid and Notch1 overexpression plasmid,respectively,and then cultured with osteoclast induction medium and puerarin.The number and size of osteoclasts were observed by tartrate-resistant acid phosphatase staining,the skeleton formation of osteoclasts was observed by F-actin staining,and the gene expression level of osteoclast formation markers was detected by RT-PCR. RESULTS AND CONCLUSION:Tartrate-resistant acid phosphatase staining results showed that puerarin intervention could inhibit the generation of osteoclasts,Notch1 silencing could further reduce the number of osteoclasts,while the number of osteoclasts in the osteoclast-induced group increased significantly after Notch1 overexpression.The results of F-actin showed that Raw264.7 cells could form a well-defined F-actin ring after osteoclast induction.Puerarin intervention would inhibit the formation of cytoskeleton,and Notch1 silencing could aggravate the inhibitory effect of cytoskeleton formation,while Notch1 overexpression could alleviate this inhibitory effect of puerarin.RT-PCR results showed that puerarin could inhibit the mRNA expression levels of tartrate-resistant acid phosphatase,Cathepsin K and c-Fos,the expression of the above-mentioned three factors decreased significantly after Notch1 gene silencing,and Notch1 overexpression could upregulate the expression of these factors.These finding indicate that puerarin inhibits the differentiation of Raw264.7 cells into osteoclasts through the Notch signaling pathway.

Mechanism by which Notch1 interference regulates the apoptosis of pulmonary microvascular endothelial cells / 中国基层医药

Objective:To investigate the mechanism of how Notch1 interference regulates the apoptosis of pulmonary vascular endothelial cells.Methods:During January to December 2022, human pulmonary microvascular endothelial cells were transfected with Notch1 siRNA, and the cell viability in each group was evaluated using the Cell Counting Kit-8 assay. The level of reactive oxygen species was determined using flow cytometry, while cell apoptosis was assessed using the same technique. After treatment with Notch1 siRNA, the protein expression levels of Notch1, Bcl-2, and Caspase-3 in the human pulmonary microvascular endothelial cells were detected using western blot assay.Results:The expression level of Notch1 mRNA in human pulmonary microvascular endothelial cells was significantly lower in the blank control and si-Notch1 groups than that in the siNC group ( t = 11.25, 9.47, both P < 0.05). Additionally, the optical density value and Bcl-2 protein expression level in the lipopolysaccharide (LPS) + Notch1 siRNA group were significantly higher than those in the LPS and LPS + siRNA groups ( t = 11.26, 11.68, both P < 0.05). The level of reactive oxygen species and the apoptosis rate of cells were significantly lower in the LPS + Notch1 siRNA group compared with the LPS and LPS + siRNA groups ( t = 11.68, 11.87, both P < 0.05). Furthermore, the protein expression levels of Notch1 and Caspase-3 were also significantly lower in the LPS + Notch1 siRNA group compared with the LPS and LPS + siRNA groups ( t = 5.08, 6.60, 3.84, 5.83, all P < 0.05). Conclusion:Notch1 interference may interference in the apoptosis of human pulmonary microvascular endothelial cells through regulating the level of reactive oxygen species, downregulating the protein expression of Notch1 and Caspase-3, and upregulating the protein expression of Bcl-2. These actions may contribute to the treatment of chronic obstructive pulmonary disease.

Effects of 5-Aza-CdR on hippocampal neurogenesis in mice via the Notch1 pathway / 中国比较医学杂志

Objective To investigate the effect of 5-Aza-CdR on Notch1 pathway and neural regeneration and to explore the effects of 5-Aza-CdR on learning memory ability in mice by exploring active avoidance behavior.Methods Sixty 6～8-week-old SPF-grade ICR male mice were divided into two groups.5-Aza-CdR was administered to one group of mice via lateral ventricular injection,while the control group was injected with bovine serum albumin.Notch1 and HES1 mRNA and protein expression levels were detected by Real-time PCR and Western blot 24 hours after injection;5-bromo-2'-deoxyuridine-positive cells were observed by laser confocal microscopy,and Notch1 expression in hippocampal dentate gyrus was viewed with laser confocal microscopy.Notch1 methylation changes were detected by ethylation-specific PCR,and learning and memory behaviors of mice were assessed by passive avoidance tests and shuttle avoidance assays.Results Injection of 5-Aza-CdR increased hippocampal Notch1 pathway activity,promoted neuronal regeneration in the DG region,decreased methylation levels in the Notch1 promoter region,and enhanced the ability of mice to perform active avoidance behavior.Conclusions The effect of 5-Aza-CdR on active avoidance behavior may be related to the influence of hippocampal neural regeneration through the Notch 1 pathway.

Forkhead Box M1 Regulates the Proliferation,Invasion,and Drug Resistance of Gastric Cancer Cells via circ_NOTCH1 / 中国医学科学院学报

Objective To investigate the impacts of forkhead box M1(FOXM1)on the proliferation,invasion,and drug resistance of gastric cancer cells by regulating the circular RNA circ_NOTCH1.Methods Western blotting and real-time quantitative PCR were performed to determine the expression of FOXM1 protein and circ_NOTCH1,respectively,in the gastric cancer tissue,para-carcinoma tissue,human normal gastric mucosa epithelial cell line GES-1 and gastric cancer cell lines MGC-803,HGC-27,and BGC-823.BGC-823 cells were classified into the following groups:control,short hairpin RNA FOXM1(sh-FOXM1)and negative control(sh-NC),small interfering RNA circ_NOTCH1(si-circ_NOTCH1)and negative control(si-NC),and sh-FOXM1+circ_NOTCH1 overexpression plasmid(sh-FOXM1+pcDNA-circ_NOTCH1)and sh-FOXM1+negative control(sh-FOXM1+pcDNA).CCK-8 assay and clone formation assay were employed to measure the cell proliferation,and Transwell assay to measure cell invasion.After treatment with 1.0 mg/L adriamycin for 48 h,the cell resistance in each group was analyzed.Western blotting was employed to determine the expression levels of FOXM1,proliferating cell nuclear antigen(PCNA),Bax,multi-drug resistance-associated protein 1(MRP1),and multi-drug resistance gene 1(MDR1).RNA pull-down and RNA immunoprecipitation were employed to examine the binding of circ_NOTCH1 to FOXM1 protein.Results Compared with those in the para-carcinoma tissue,the expression levels of FOXM1 protein and circ_NOTCH1 in the gastric cancer tissue were up-regulated(all P<0.001).Compared with GES-1 cells,MGC-803,HGC-27,and BGC-823 cells showed up-regulated expression levels of FOXM1 protein and circ_NOTCH1(all P<0.001).Compared with the control group and sh-NC group,the sh-FOXM1 group with down-regulated expression of FOXM1 protein and circ_NOTCH1 showed decreased optical density value,clone formation rate,cell invasion number,and cell viability,down-regulated expression of PCNA,MRP1,and MDR1,and up-regulated expression of Bax protein in BGC-823 cells(all P<0.001).Compared with the control group and the si-NC group,the si-circ_NOTCH1 group with down-regulated expression of circ_NOTCH1 showed decreased optical density value,clone formation rate,cell invasion number,and cell viability,down-regulated expression of PCNA,MRP1,and MDR1,and up-regulated expression of Bax protein in BGC-823 cells(all P<0.001).Compared with sh-FOXM1 group and sh-FOXM1+pcDNA group,the sh-FOXM1+pcDNA-circ_NOTCH1 group with up-regulated expression of circ_NOTCH1 showed increased optical density value,clone formation rate,cell invasion number,and cell viability,up-regulated expression of PCNA,MRP1,and MDR1,and down-regulated expression of Bax protein(all P<0.001).FOXM1 protein was able to interact with circ_NOTCH1.Conclusion Interference with FOXM1 may inhibit the proliferation,invasion,and drug resistance of gastric cancer cells by silencing circ_NOTCH1 expression.

The repair effect of extremely low frequency electromagnetic field on neurological function in ischemic stroke was investigated based on miRNA23/Nocth signaling pathway / 中风与神经疾病杂志

@#Objective To study the mechanism of extremely low frequency electromagnetic field (ELF EMF) promoting the recovery of neural function loss in ischemic stroke by regulating the miRNA23 (miR-23)/Loch signal pathway. Methods An adult male SD rat model of ischemic stroke was established. ELF EMF intervention was performed and NC miR sequence or miR-23 inhibitor was injected into the lateral ventricle. The degree of neurological deficit,the volume of cerebral infarction,the expression level of miR-23 and the expression level of Notch1,Jagged1 and Hes1 proteins were evaluated. Results Compared with the sham operation group,the Zea Longa score,cerebral infarction volume and apoptosis rate of the model group were increased,and the expression levels of miR-23,Notch1,Jagged1 and Hes1 were decreased (P<0.05);Compared with the model group,the Zea Longa score,cerebral infarction volume and apoptosis rate of ELF EMF group were decreased,and the expression levels of miR-23,Notch1,Jagged1 and Hes1 increased (P<0.05);Compared with miR-NC+ELF EMF group,the Zea Longa score,cerebral infarction volume and apoptosis rate of miR-23+ELF EMF group were increased,and the expression levels of miR-23,Notch1,Jagged1 and Hes1 were decreased (P<0.05). Conclusion ELF EMF can promote the recovery of neurological deficit in ischemic stroke rats,which is related to the increase of miR-23 expression and activation of downstream Notch1 pathway.

Baicalin Antagonizes Prostate Cancer Stemness via Inhibiting Notch1/NF-κB Signaling Pathway / 中国结合医学杂志

OBJECTIVE@#To investigate the molecular mechanisms underlying the effect of baicalin on prostate cancer (PCa) progression both in vivo and in vitro.@*METHODS@#The in situ PCa stem cells (PCSCs)-injected xenograft tumor models were established in BALB/c nude mice. Tumor volume and weight were respectively checked after baicalin (100 mg/kg) treatment. Hematoxylin-eosin (HE) staining was used to observe the growth arrest and cell necrosis. mRNA expression levels of acetaldehyde dehydrogenase 1 (ALDH1), CD44, CD133 and Notch1 were determined by reverse transcription-polymerase chain reaction. Protein expression levels of ALDH1, CD44, CD133, Notch1, nuclear factor κB (NF-κB) P65 and NF-κB p-P65 were detected by Western blot. Expression and subcellular location of ALDH1, CD44, CD133, Notch1 and NF-κB p65 were detected by immunofluorescence analysis. In vitro, cell cycle distribution and cell apoptosis of PC3 PCSCs was assessed by flow cytometry after baicalin (125 µmol/L) treatment. The migration and invasion abilities of PCSCs were assessed using Transwell assays. Transmission electron microscopy scanning was utilized to observe the structure and autophagosome formation of baicalin-treated PCSCs. In addition, PCSCs were infected with lentiviruses expressing human Notch1.@*RESULTS@#Compared with the control group, the tumor volume and weight were notably reduced in mice treated with 100 mg/kg baicalin (P<0.05 or P<0.01). Histopathological analysis showed that baicalin treatment significantly inhibited cell proliferation and promoted cell apoptosis. Furthermore, baicalin treatment reduced mRNA and protein expression levels of CD44, CD133, ALDH1, and Notch1 as well as the protein expression of NF-κB p-P65 in the xenograft tumor (P<0.01). In vitro, the cell proliferation of PCSCs was significantly attenuated after treatment with 125 µmol/L baicalin for 72 h (P<0.01). The cell migration and invasion rates were decreased following treatment with baicalin for 48 and 72 h (P<0.01). Baicalin notably induced cell apoptosis and seriously damaged the structure of PCSCs. The mRNA and protein expressions of CD133, CD44, ALDH1 and Notch1 in PCSCs were significantly downregulated following baicalin treatment (P<0.01). Importantly, the inhibitory effects of baicalin on PCa progression and stemness were reversed by Notch1 overexpression (P<0.05 or P<0.01).@*CONCLUSION@#Mechanistically, baicalin exhibited a potential therapeutic effect on PCa via inhibiting the Notch1/NF-κB signaling pathway and its mediated cancer stemness.

Mechanism of the Notch1 signaling pathway regulating osteogenic factor influences lumbar disc calcification / 中国骨伤

OBJECTIVE@#To explore the mechanism of the Notch1 signaling pathway in regulating osteogenic factors and influencing lumbar disc calcification.@*METHODS@#Primary annulus fibroblasts from SD rats were isolated and subcultured in vitro. The calcification-inducing factors bone morphogenetic protein-2 (BMP-2) and basic fibroblast growth factor (b-FGF) were added to separate groups to induce calcification, which were referred to as the BMP-2 group and the b-FGF group, respectively. A control group was also set up, which was cultured in normal medium. Subsequently, cell morphology and fluorescence identification, alizarin red staining, ELISA, and quantitative real-time polymerase chain reaction (QRT-PCR) were performed to determine the effect of calcification induction. Cell grouping was performed again, including the control group, the calcification group (adding the inducer BMP-2), the calcification + LPS group(adding the inducer BMP-2 and the Notch1 pathway activator LPS), and the calcification + DAPT group (adding the inducer BMP-2 and the Notch1 pathway inhibitor DAPT). Alizarin red staining and flow cytometry were used to detect cell apoptosis, ELISA was used to detect the content of osteogenic factors, and Western blot was used to detect the expression of BMP-2, b-FGF, and Notch1 proteins.@*RESULTS@#The induction factor screening results showed that the number of mineralized nodules in fibroannulus cells in BMP-2 group and b-FGF group was significantly increased, and the increase was greater in the BMP-2 group Meanwhile, ELISA and Western blot results showed that BMP-2, b-FGF and mRNA expression levels of BMP-2, b-FGF and Notch1 in the induced group were significantly increased (P<0.01). The results of the mechanism of Notch1 signaling pathway affecting lumbar disc calcification showed that compared to calcified group, the number of fibroannulus cell mineralization nodules, apoptosis rate, BMP-2, b-FGF content, the expression levels of BMP-2, b-FGF, and Notch1 proteins were further increased significantly However, the number of mineralization nodules, apoptosis rate, BMP-2 and b-FGF levels, BMP-2, b-FGF and Notch1 protein expression levels were decreased in the calcified +DAPT group (P<0.05 or P<0.01).@*CONCLUSION@#Notch1 signaling pathway promotes lumbar disc calcification through positive regulation of osteogenic factors.

Expressions and Clinical Significance of Notch1 and Hes1 in Diffuse Large B-Cell Lymphoma / 中国实验血液学杂志

OBJECTIVE@#To investigate the expressions of Notch1 and Hes1 in diffuse large B-cell lymphoma (DLBCL), and their correlations with clinical features.@*METHODS@#Immunohistochemistry (IHC) was performed on DLBCL samples (54 cases) and lymphadenitis tissues (20 cases) to evaluate the expressions of Notch1 and Hes1, and analyze their correlations with clinical characteristics of patients. Based on Oncomine database, the expressions of Notch1 and Hes1 mRNA and DNA were also explored.@*RESULTS@#IHC result showed that the positive expression rates of Notch1 and Hes1 in DLBCL patients were significantly higher than those in the control group (P <0.05). In DLBCL patients, the expression of Notch1 was closely associated with B symptoms, Ann Arbor stage, lymphocyte count and the level of lactate dehydrogenase (P <0.05), while the expression level of Hes1 was significantly higher in patients with B symptoms (P <0.05). Notch+/Hes1+ expression was found in 21 DLBCL tissues (38.9%), and there was a correlation between Notch1 and Hes1 expression (r =0.296, P <0.05). Bioinformatics analysis (Oncomine database) showed that the mRNA expressions of Notch1 and Hes1 in the Brune dataset were significantly higher than those in the control tissues (P <0.05).@*CONCLUSION@#The expressions of Notch1 and Hes1 in DLBCL are significantly higher than those in lymphadenitis, and correlated with B symptoms and Ann Arbor stage, suggesting that Notch1 and Hes1 play important roles in the occurrence and development of DLBCL.