WIN55212-2 inhibits glycolysis and attenuates acute lung injury in septic mice by regulating mTOR/HIF-1α/PFKFB3 signaling pathway / 中国病理生理杂志

AIM:To investigate the effects of cannabinoid receptor agonist WIN55212-2(WIN)on acute lung injury(ALI)in septic mice,and to explore its potential mechanisms through glycolysis.METHODS:A mouse model of septic ALI was established by intraperitoneal injections of lipopolysaccharide(LPS).Male C57BL/6J mice were randomly divided into 4 groups(n=6):(1)control group;(2)LPS group,receiving intraperitoneal injections of LPS at 10 mg/kg;(3)LPS+WIN group,receiving 1 mg/kg WIN intraperitoneally 30 min prior to LPS injection;(4)LPS+WIN+MHY1485[mammalian target of rapamycin(mTOR)activator]group,receiving 10 mg/kg MHY1485 intraperitoneally 1 d before LPS injection and 1 mg/kg WIN plus 10 mg/kg MHY1485 30 min before LPS injection.Tissues were collected 24 h after modeling for analysis.Lung indexes were calculated,and histopathological changes of lung tissues were observed via he-matoxylin-eosin(HE)staining.Inflammatory cytokines interleukin-1β(IL-1β)and IL-10 in lung tissues,and lactic acid and lactate dehydrogenase A(LDHA)in serum were quantified using ELISA.The levels of mTOR/hypoxia-inducible fac-tor-1α(HIF-1α)/6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3(PFKFB3)signaling pathway-related proteins were assessed by Western blot.RESULTS:Compared with control group,the LPS group exhibited an increased lung in-dex,significant lung tissue damage,an increase in IL-1β levels(P＜0.05),a decrease in IL-10 levels(P＜0.05),and el-evated expressions of lactate and LDHA(P＜0.05),along with increased levels of phosphorylated mTOR(p-mTOR),HIF-1α and PFKFB3 proteins(P＜0.05).The LPS+WIN group showed improvements with a reduced lung index(P＜0.05),lessened lung injury,decreased IL-1β levels(P＜0.05),increased IL-10 levels(P＜0.05),and lower levels of lactic acid,LDHA,p-mTOR,HIF-1α,and PFKFB3(P＜0.05).Conversely,the LPS+WIN+MHY1485 group displayed increased lung indexes and lung tissue damage,elevated IL-1β levels(P＜0.05),reduced IL-10 levels(P＜0.05),and higher expressions of lactic acid,LDHA,p-mTOR,HIF-1α and PFKFB3(P＜0.05)compared to the LPS+WIN group.CONCLUSION:WIN55212-2 mitigates sepsis-induced ALI,potentially by modulating the mTOR/HIF-1α/PFKFB3 sig-naling pathway,thereby inhibiting glycolysis and alleviating inflammatory responses.

Construction of HIF-1α gene knockout plasmid and functional verification in naked mole rats based on CRISPR/Cas9 system / 中国实验动物学报

Objective To construct plasmids and knock out HIF-1α gene expression in an naked mole rat skin fibroblasts(NSF)cell line using CRISPR/Cas9 genomic editing technology,to provide an in vitro cell model for studying the mechanism of hypoxia tolerance and the occurrence and development of hypoxia-related diseases in naked mole rats.Methods We designed four pairs of single guide RNA(sgRNA)sequences targeting exons 1～4 of the NSF HIF-1αgene and successfully constructed an expression plasmid.The plasmid with the optimal sgRNA was identified and transfected into 293T cells,and the supernatant was used for detecting the virus titer.Lentivirus particles carrying sgRNAs of HIF-1α were transfected into NSF cells which express Cas9 protein,based on a previous protocol.After transfection,fluorescence signals were observed under a fluorescence microscope,and HIF-1α expression in NSF cells was detected by Western Blot and T7 endonuclease 1(T7E1)analysis.Results Sanger sequencing showed that the designed sgRNA was successfully inserted into pX459 and pKLV2-U6-sgRNA2 vectors,demonstrating successful construction of a recombinant plasmid for transfection.T7E1 digestion successfully removed three bands and the target efficiency of sgRNA was 54％.Western Blot showed that the HIF-1α gene was successfully knocked out and its protein level was significantly reduced in NSF cells from naked mole rats(P=0.0019).There were no obvious morphological changes in HIF-1α-knockout cells under the microscope,and gene knockout had no obvious effect on cell proliferation.Conclusions We successfully constructed an HIF-1α-knockout cell line using CRISPR/Cas9 technology,to provide an experimental basis for further studies of the biological function of HIF-1α,as well as the mechanism of hypoxia tolerance in naked mole rats.The result also provide a theoretical foundation for the prevention and treatment of hypoxia-related diseases.

Research progress of glycolysis induced islet β cell dedifferentiation based on HIF-1α/PFKFB3 signaling pathway / 中国糖尿病杂志

Islet β cell dedifferentiation is one of the important reasons leading to insulin secretion defect or insulin resistance in patients with type 2 diabetes mellitus(T2DM).HIF-1α/PFKFB3 signaling pathway is a newly discovered biological pathway related to T2DM,which is involved in the induction of islet β cells dedifferentiation by anaerobic glycolysis under high glucose environment.This article reviews the research progress of the role of HIF-1α/PFKFB3 signaling pathway in glycolysis induced islet β cell dedifferentiation.

Tripartite motif-containing protein 59 inhibits inflammation and lung injury by regulating HIF-1α-mediated lactic acid secretion and IL-10 expression in macrophages / 中华微生物学和免疫学杂志

Objective:To investigate the effect of tripartite motif-containing protein 59 (TRIM59) on glucose metabolism in macrophages and its role in regulating hypoxia-inducible factor-1α (HIF-1α)/IL-10 axis in macrophages under inflammatory conditions.Methods:The differentially expressed genes between macrophages with high expression of TRIM59 and control cells transfected with empty TRIM59 plasmid were analyzed by GO and KEGG. The expression of HIF-1α by RAW264.7 macrophages with high expression of TRIM59 was detected at different time points after lipopolysaccharide (LPS) stimulation by RT-qPCR and Western blot. Bone marrow was isolated from TRIM59-cKO and TRIM59 flox/flox mice and induced to differentiate into bone marrow-derived macrophages (BMDMs). These BMDMs were stimulated with LPS and the supernatants of cell culture were collected at 3, 6, 12 and 24 h after stimulation to detect IL-10 level by ELISA. In addition, mouse models of cecal ligation and puncture (CLP) were established, and bronchoalveolar lavage fluid (BALF) samples were collected at the same time points to detect IL-10 level by ELISA. Histopathological changes in lung tissues were observed after HE staining. Results:There was a significant change in glucose metabolism-related genes in macrophages with high expression of TRIM59, and the content of lactic acid increased significantly. Compared with the control group, the expression of HIF-1α at mRNA level in BMDMs from TRIM59-cKO mice decreased after LPS stimulation ( P<0.05); the level of IL-10 increased at 3 h and 24 h in the TRIM59-cKO group, but there was no significant difference in IL-10 level at 6 h or 12 h between the two groups. In the TRIM59-cKO mouse model of CLP, the levels of IL-10 in the BALF samples increased with time, but decreased at 24 h. The level of IL-10 was higher in the TRIM59-cKO mouse model group than that in the control group at each time point ( P<0.05 or P<0.01). Conclusions:TRIM59 can inhibit inflammation and lung injury by decreasing HIF-1α-mediated lactate secretion and IL-10 expression in macrophages. This study provides a new idea for developing novel anti-sepsis drugs based on TRIM59.

Effect of Xiaoyu Xiezhuo Drink on Podocyte Apoptosis and Expression of HIF1α in Diabetic Kidney Disease Mice / 浙江中医药大学学报

[Objective]To observe the effect of Xiaoyu Xiezhuo Drink on podocyte apoptosis and hypoxia inducible factor 1 alpha(HIF1α)expression in db/db diabetic kidney disease(DKD)model mice.[Methods]Six db/m mice were chosen as negative control group,eighteen db/db mice were chosen and divided into DKD model group,low dosage Xiaoyu Xiezhuo Drink group,high dosage Xiaoyu Xiezhuo Drink group,with six mice in each group.After gastric irrigation for twelve weeks,the urine was collected to test the levels of protein,β2-microglobulin(β2-MG),albumin/creatinine ratio(Acr),β2-microglobulin/creatinine ratio(β2-MG/Ucr);blood was collected to test the level of triglyceride(TG),total cholesterol(TC),albumin(Alb),blood urea nitrogen(BUN),serum creatinine(Scr);the kidney tissue was collected to observe the pathological change by light and electron microscope,and to test HIF1α,nephrin mRNA by Real-time polymerase chain reaction(RT-PCR).[Results]Compared with negative control group,the levels of urine protein,β2-MG,Acr,β2-MG/Ucr,serum TG,TC,BUN,Scr were increased(P＜0.01),serum Alb was decreased(P＜0.01);glomerular volume increased,capillary loops lobed,mesangial cells and matrix hyperplasia,interstitial inflammation and fibrosis increased,foot process fusion increased,basement membrane thickened;podocyte apoptosis was increased;expression of HIF1α mRNA was elevated(P＜0.01),and nephrin mRNA was descended in kidney tissue of DKD model group(P＜0.01).Compared with DKD model group,the level of urine protein,β2-MG,Acr,β2-MG/Ucr,serum TG,TC,BUN,Scr were decreased(P＜0.01),serum Alb was incresed(P＜0.01);the pathological changes of the kidney was improved;the apoptosis of podocyte was reduced;the expression of HIF1α mRNA was decreased(P＜0.01),and nephrin mRNA was incresed(P＜0.01)in the kidney tissue of varied dosage Xiaoyu Xiezhuo Drink groups.There was no statistical significance in the level of urine protein,β2-MG,Acr,β2-MG/Ucr,serum TG,TC,BUN,Scr,Alb,podocyte apoptosis,and HIF1α,nephrin mRNA in the kidney tissue between different dosage Xiaoyu Xiezhuo Drink groups(P＞0.05).[Conclusion]Xiaoyu Xiezhuo Drink could improve urinary protein,renal function,renal structure lesion and podocyte apoptosis in DKD mice,which perhaps by regulating the expression of HIF1α.

Mechanism of Wenyang Shengji Ointment in treating diabetic wounds based on network pharmacology and animal experiments / 数字中医药（英文）

Objective @#To explore the mechanism of Wenyang Shengji Ointment (温阳生肌膏, WYSJO) in the treatment of diabetic wounds from the perspective of network pharmacology, and to verify it by animal experiments.@*Methods@#The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and related literature were used to screen active compounds in WYSJO and their corresponding targets. GeneCards, Online Mendelian Inheritance in Man (OMIM), DrugBank, PharmGkb, and Therapeutic Target Database (TTD) databases were employed to identify the targets associated with diabetic wounds. Cytoscape 3.9.0 was used to map the active ingredients in WYSJO, which was the diabetic wound target network. Search Tool for the Retrieval of Interaction Gene/Proteins (STRING) platform was utilized to construct protein-protein interaction (PPI) network. Kyoto Encyclopedia of Genes and Genomes (KEGG) andGene Ontology (GO) enrichment analyses were performed to identify signaling pathways between WYSJO and diabetic wounds. AutoDock 1.5.6 was used for molecular docking of core components in WYSJO to their targets. Eighteen rats were randomly divided into control, model, and WYSJO groups (n = 6). The model and WYSJO groups were used to prepare the model of refractory wounds in diabetes rats. The wound healing was observed on day 0, 5, 9, and 14 after treatment, and the wound tissue morphology was observed by hematoxylin-eosin(HE) staining. The expression levels of core genes were detected by quantitative real-timepolymerase chain reaction (qPCR).@*Result@#A total of 76 active compounds in WYSJO, 206 WYSJO drug targets, 3 797 diabetic wound targets, and 167 diabetic wound associated WYSJO targets were screened out through network pharmacology. With the use of WYSJO-diabetic wound target network, core targets of seven active compounds encompassing quercetin, daidzein, kaempferol, rhamnetin, rhamnocitrin, strictosamide, and diisobutyl phthalate (DIBP) in WYSJO were found. GO enrichment analysis showed that the treatment of diabetes wounds with WYSJO may involve lipopolysaccharide, bacteria-derived molecules, metal ions, foreign stimuli, chemical stress, nutrient level, hypoxia, and oxidative stress in the biological processes. KEGG enrichment analysis showed that the treatment of diabetes wounds with WYSJO may involve advanced glycation end products (AGE-RAGE), p53, interleukin (IL)-17, tumor necrosis factor (TNF),hypoxia inducible factor-1 (HIF-1), apoptosis, lipid, atherosclerosis, etc. The results of animal experiments showed that WYSJO could significantly accelerate the healing process of diabetic wounds (P < 0.05), alleviate inflammatory response, promote the growth of granulation tissues, and down-regulate the expression levels of eight core genes [histone crotonyltransferase p300 (EP300), protoc gene-oncogene c-Jun (JUN), myelocytomatosis (MYC), hypoxia inducible factor 1A (HIF1A), mitogen-activated protein kinase 14 (MAPK14), specificity protein 1 (SP1), tumor protein p53 (TP53), and estrogen receptor 1 (ESR1)] predicted by the network pharmacology (P < 0.05).@*Conclusion@#The mechanism of WYSJO in treating diabetes wounds may be closely related to AGE-RAGE, p53, HIF-1, and other pathways. This study can provide new ideas for the pharmacological research of WYSJO, and provide a basis for its further transformation and application.

Determination of Traditional Chinese Medicine Syndrome of Rat Model of Follicular Dysplasia Induced by Tripterygium Glycosides Based on Prescriptions via AMPK/mTOR/HIF-1/VEGF Pathway / 中国实验方剂学杂志

ObjectiveTo determine the syndrome of a rat model of follicular dysplasia induced by Tripterygium glycosides based on prescriptions and investigate the mechanism of traditional Chinese medicine intervention via the adenosine monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR）/hypoxia-inducible factor-1 （HIF-1）/vascular endothelial growth factor （VEGF） pathway. MethodForty-eight rats with regular estrous cycles were randomly assigned into a normal group （n=8） and a modeling group （n=40）. The rats in the modeling group were administrated with Tripterygium glycoside suspension （75 mL·kg-1） by gavage for 30 days. The modeled rats were assigned into model， Siwutang （3.69 g·kg-1）， Youguiyin （3.11 g·kg-1）， Zuoguiyin （7.29 g·kg-1）， and Guishenwan （10.35 g·kg-1） groups， with 8 rats in each group. The drug intervention lasted for 14 days. The changes of estrous cycle were detected by Pap staining， and a stereoscope was used to observe the morphology of the ovarian tissue. Hematoxylin-eosin staining was employed to observe the pathological changes and follicle count in the ovarian tissue. Enzyme-related immunosorbent assay （ELISA） was used to measure the levels of follicle-stimulating hormone （FSH）， luteinizing hormone （LH）， and estradiol （E2） in the serum. Real-time fluorescence quantitative polymerase chain reaction and Western blot were employed to determine the mRNA and protein levels， respectively， of AMPK， mTOR， HIF-1， and VEGF in the ovarian tissue. ResultCompared with the normal group， the model group had a disordered estrous cycle， reduced secondary and mature follicles， increased atretic follicles， elevated FSH and LH levels， lowered E2 level， up-regulated mRNA and protein levels of AMPK， and down-regulated mRNA and protein levels of mTOR， HIF-1， and VEGF （P<0.01）. Compared with the model group， Guishenwan increased secondary and mature follicles， decreased atretic follicles， lowered the FSH and LH levels， elevated the E2 level， down-regulated the mRNA and protein levels of AMPK， and up-regulated the mRNA and protein levels of mTOR， HIF-1， and VEGF （P<0.01）. Compared with Guishenwan group， Siwutang， Youguiyin， and Zuoguiyin decreased mature follicles， increased atretic follicles （P<0.01）， elevated the LH （P<0.01） and FSH （P<0.05） levels， and lowered the E2 level （P<0.05）. In addition， Youguiyin up-regulated the protein level of AMPK （P<0.05） and down-regulated the mRNA levels of mTOR and HIF-1 （P<0.01） as well as the mRNA and protein levels of VEGF （P<0.01）. Siwutang down-regulated the mRNA levels of mTOR and HIF-1 as well as the mRNA and protein levels of VEGF （P<0.05）. Zuoguiyin down-regulated the mRNA level of mTOR and the protein and mRNA levels of VEGF （P<0.05）. ConclusionGuishenwan may improve the ovarian function and promote follicle maturation in a rat model of follicular dysplasia by inhibiting the AMPK/mTOR/HIF-1/VEGF pathway， with the therapeutic effect superior to Zuoguiyin， Youguiyin， and Siwutang. It was hypothesized that this model presented the syndrome of kidney-essence deficiency.

Effect of Gualou Xiebai Banxiatang on Myocardial Microangiogenesis and HIF-1α/VEGF-related Pathways in Myocardial Ischemia Model Rats / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Gualou Xiebai Banxiatang on cardiac function and myocardial histopathological changes in rats with ischemic myocardial injury， and to observe the effect of myocardial microvascular density （MVD）， phosphatidylinositol 3-kinase （PI3K）， mammalian target of rapamycin （mTOR）， hypoxia-inducible factor-1 alpha （HIF-1α）， and vascular endothelial growth factor （VEGF） signaling pathways on myocardial microangiogenesis. MethodSeventy male SD rats were randomly selected， with six rats in the normal group. The remaining rats were fed a high-fat diet and injected with isoproterenol hydrochloride （ISO，80 mg·kg-1·d-1， 2 d） to induce a hyperlipidemia-based ischemic heart disease model. After successful modeling， the rats were randomly divided into the model group， high， medium， and low dose groups of Gualou Xiebai Banxiatang， and the metoprolol group. The high， medium， and low dose groups of Gualou Xiebai Banxiatang were given Gualou Xiebai Banxiatang at 10.42， 5.21， 2.61 g·kg-1·d-1， respectively， while the metoprolol group was given metoprolol at 2.6 mg·kg-1·d-1. Both the normal and model groups were given an equivalent volume of physiological saline for 28 days. After the intervention， relevant tests were conducted， and serum was collected to measure heart function-related indicators. Hematoxylin-eosin （HE） and Masson staining were performed on ventricular tissue to observe pathological changes under a light microscope. Immunohistochemistry （IHC） was used to detect the positive expression of platelet endothelial cell adhesion molecule （CD31）. Enzyme-linked immunosorbent assay （ELISA） was used to detect the expression of N-terminal pro-brain natriuretic peptide （NT-proBNP） and VEGF. Western blot was used to detect the protein expression levels of PI3K/mTOR/HIF-1α/VEGF. ResultCompared with the normal group， the model group showed significantly increased serum levels of LDH， CK， CK-MB， NT-proBNP， and VEGF （P<0.01）， significantly increased collagen volume fraction （CVF） （P<0.01）， significantly decreased MVD （P<0.01）， and elevated protein expression levels of PI3K， mTOR， HIF-1α， and VEGF （P<0.05， P<0.01）. Compared with the model group， the metoprolol group had significantly lower serum levels of LDH， CK， CK-MB， and NT-proBNP （P<0.01）， significantly higher VEGF levels （P<0.01）， significantly decreased CVF （P<0.01）， significantly increased MVD （P<0.01）， and significantly increased protein expression levels of PI3K， mTOR， and VEGF （P<0.01）， with no statistically significant change in HIF-1α protein expression. Compared with the model group， the high and medium dose groups of Gualou Xiebai Banxiatang had decreased serum levels of LDH， CK， CK-MB， and NT-proBNP （P<0.05， P<0.01）， increased VEGF levels （P<0.05， P<0.01）， significantly reduced CVF （P<0.01）， increased MVD （P<0.05， P<0.01）， and significantly increased protein levels of PI3K， mTOR， HIF-1α， and VEGF （P<0.01）. In the low dose group of Gualou Xiebai Banxiatang， compared with the model group， serum levels of LDH and NT-proBNP were decreased （P<0.05）， VEGF was increased （P<0.05）. Moreover， CVF was decreased （P<0.05）， and the protein expression levels of PI3K， mTOR， HIF-1α， and VEGF were significantly increased （P<0.01）. ConclusionGualou Xiebai Banxiatang can improve cardiac function， reduce myocardial pathological damage， enhance endothelial cell function， promote myocardial microvascular formation， and upregulate the expression of PI3K， mTOR， HIF-1α， and VEGF proteins in myocardial tissue in rats with ischemic myocardial injury.

Effect of Jianpi Bushen Huoxue Prescription on Rat Brain Microvascular Endothelial Cells Based on HIF-1α/VEGF Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effects of Jianpi Bushen Huoxue prescription （JPBSHX） on rat brain microvascular endothelial cells （RBMECs） based on hypoxia-inducible factor-1α （HIF-1α）/vascular endothelial growth factor （VEGF） signaling pathway， aiming to provide a theoretical basis for the treatment of ischemic stroke. MethodTwelve 8-week-old male SPF-grade SD rats were selected. Eight of them were randomly chosen and given 3.25 g·mL-1 JPBSHX solution by gavage at a dose of 10 mL·kg-1 for 5 consecutive days to prepare the medicated serum， which was then preserved for later use. The remaining four rats were given the same volume of normal saline. Follow-up operations were the same as those of the above eight rats. Normal rat serum was collected and stored for later use. RBMECs were revived， cultured， passaged， and randomly divided into five groups： normal group （20% normal rat serum+80% high glucose DMEM）， model group （hypoxia-reoxygenation injury） （20% normal rat serum+80% glucose-free DMEM）， medicated serum group （20% JPBSHX-medicated serum+80% glucose-free DMEM）， medicated serum+HIF-1α inhibitor group （20% JPBSHX-medicated serum+HIF-1α inhibitor 1 mg +80% glucose-free DMEM）， and medicated serum+VEGF inhibitor group （20% JPBSHX-medicated serum +VEGF inhibitor 1 mg+80% glucose-free DMEM）. The relative protein expression levels of Claudin-1 and Claudin-5 in RBMECs， the expression levels of HIF-1α and VEGF in RBMEC culture supernatants， the repair ability of RBMECs， and the number of nodes， microvessels， and their lengths after 72 h of culture were observed in each group. ResultAfter 24 h of reoxygenation， the scratch healing rate in the model group was significantly lower than in the normal group （P<0.01）. Compared with the result in the model group， the scratch healing rates significantly improved in the medicated serum group， medicated serum+HIF-1α inhibitor group， and medicated serum+VEGF inhibitor group （P<0.05）. However， the healing rates in the medicated serum+HIF-1α inhibitor group and medicated serum+VEGF inhibitor group were significantly lower than that in the medicated serum group （P<0.05）. The number of nodes， microvessels， and total length of microvessels in the model group were significantly lower than those in the normal group （P<0.01）. These indicators significantly improved in the medicated serum group， medicated serum+HIF-1α inhibitor group， and medicated serum+VEGF inhibitor group compared with those in the model group （P<0.05）， but were significantly lower in the medicated serum+HIF-1α inhibitor group and medicated serum+VEGF inhibitor group compared with those in medicated serum group （P<0.05）. The relative expression levels of Claudin-1 and Claudin-5 proteins were significantly lower in the model group than in the normal group （P<0.01）. These levels were significantly higher in medicated serum group， medicated serum+HIF-1α inhibitor group， and medicated serum+VEGF inhibitor group than those in the model group （P<0.05）， but were significantly lower in the medicated serum+HIF-1α inhibitor group and medicated serum+VEGF inhibitor group than those in the medicated serum group （P<0.05）. The expression levels of HIF-1α and VEGF in the RBMEC culture supernatants were significantly lower in the model group than those in the normal group （P<0.01）. These levels were significantly higher in the medicated serum group， medicated serum+HIF-1α inhibitor group， and medicated serum+VEGF inhibitor group than those in the model group （P<0.05）， but were significantly lower in the medicated serum+HIF-1α inhibitor group and medicated serum+VEGF inhibitor group than those in the medicated serum group （P<0.05）. ConclusionJPBSHX can promote the proliferation， migration， and angiogenesis， such as tubule formation， of RBMECs damaged by hypoxia-reoxygenation injury， and this effect may be achieved through the regulation of the HIF-1α/VEGF signaling pathway.

Human ESC-derived vascular cells promote vascular regeneration in a HIF-1α dependent manner

Hypoxia-inducible factor (HIF-1α), a core transcription factor responding to changes in cellular oxygen levels, is closely associated with a wide range of physiological and pathological conditions. However, its differential impacts on vascular cell types and molecular programs modulating human vascular homeostasis and regeneration remain largely elusive. Here, we applied CRISPR/Cas9-mediated gene editing of human embryonic stem cells and directed differentiation to generate HIF-1α-deficient human vascular cells including vascular endothelial cells, vascular smooth muscle cells, and mesenchymal stem cells (MSCs), as a platform for discovering cell type-specific hypoxia-induced response mechanisms. Through comparative molecular profiling across cell types under normoxic and hypoxic conditions, we provide insight into the indispensable role of HIF-1α in the promotion of ischemic vascular regeneration. We found human MSCs to be the vascular cell type most susceptible to HIF-1α deficiency, and that transcriptional inactivation of ANKZF1, an effector of HIF-1α, impaired pro-angiogenic processes. Altogether, our findings deepen the understanding of HIF-1α in human angiogenesis and support further explorations of novel therapeutic strategies of vascular regeneration against ischemic damage.

Adiponectin restores the obesity-induced impaired immunomodulatory function of mesenchymal stromal cells via glycolytic reprogramming

Obesity has been known to negatively modulate the life-span and immunosuppressive potential of mesenchymal stromal cells (MSC). However, it remains unclear what drives the compromised potency of obese MSC. In this study, we examined the involvement of adiponectin, an adipose tissue-derived hormone, in obesity-induced impaired therapeutic function of MSC. Diet-induced obesity leads to a decrease in serum adiponectin, accompanied by impairment of survival and immunomodulatory effects of adipose-derived MSC (ADSC). Interestingly, priming with globular adiponectin (gAcrp) improved the immunomodulatory potential of obese ADSC. Similar effects were also observed in lean ADSC. In addition, gAcrp potentiated the therapeutic effectiveness of ADSC in a mouse model of DSS-induced colitis. Mechanistically, while obesity inhibited the glycolytic capacity of MSC, gAcrp treatment induced a metabolic shift toward glycolysis through activation of adiponectin receptor type 1/p38 MAPK/hypoxia inducible factor-1α axis. These findings suggest that activation of adiponectin signaling is a promising strategy for enhancing the therapeutic efficacy of MSC against immune-mediated disorders.

Effect of lncRNA HIF1A-AS1 on chemotherapy sensitivity of vincristine-resistant in retinoblastoma cells / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To investigate the effect of long non-coding RNA-HIF1A-AS1(lncRNA HIF1A-AS1)on the chemotherapy sensitivity of vincristine(VCR)-resistant in retinoblastoma(RB)cells by regulating the expression of hypoxia-inducible factor-1α(HIF-1α).METHODS: The human RB VCR-resistant cell line SO-RB50/VCR was established, expression of lncRNA HIF1A-AS1 in SO-RB50 and SO-RB50/VCR cells were detected by reverse transcription-quantitative real-time PCR(RT-qPCR); inhibition of lncRNA HIF1A-AS1 expression or simultaneous overexpression of HIF-1α in SO-RB50/VCR cells, and then median inhibitory concentration(IC50)of VCR and cell proliferation and apoptosis were detected in SO-RB50/VCR cells; the protein expressions of HIF-1α, multidrug resistance associate protein(MRP)and P-glycoprotein(P-gp)were measured by Western blot.RESULTS: Compared with SO-RB50 cells, the expression levels of lncRNA HIF1A-AS1 and HIF-1α protein in SO-RB50/VCR cells were increased(P&#x003C;0.05); after inhibiting the expression of lncRNA HIF1A-AS1 in SO-RB50/VCR cells, the apoptosis rate was significantly increased(P&#x003C;0.05), optical density(OD450), the IC50 value of VCR on cells and the expression levels of HIF-1α, MRP and P-gp proteins were significantly reduced(P&#x003C;0.05); overexpression of HIF-1α attenuates the inhibitory effect of down-regulated lncRNA HIF1A-AS1 expression on drug resistance in SO-RB50/VCR cells.CONCLUSION: The lncRNA HIF1A-AS1 was highly expressed in SO-RB50/VCR cells, and inhibition of lncRNA HIF1A-AS1 expression reduced VCR resistance in SO-RB50/VCR cells by down-regulating HIF-1α expression.

Huanglian Jiedutang Regulates HIF-1α/VEGF Signaling Pathway to Improve Learning and Memory Abilities of APP/PS1 Transgenic Mice / 中国实验方剂学杂志

ObjectiveTo reveal the effects of Huanglian Jiedutang （HLJDT） on the learning and memory abilities of APP/PS1 transgenic mice via hypoxia-inducible factor-1α （HIF-1α）/vascular endothelial growth factor （VEGF） signaling pathway. MethodForty 5-month-old β-amyloid precursor protein （APP）/presenilin 1（PS1） mice were randomized into the model， donepezil （0.001 g·kg-1·d-1）， and low-， medium-， and high-dose （1.5， 3， 6 g·kg-1·d-1， respectively） HLJDT groups， and 8 C57BL/6 mice were taken as the normal group. After 45 days of continuous administration， Morris water maze test was conducted， and the organ indexes were calculated. The morphological structure of cerebral vascular endothelial cells in mice was observed under a transmission electron microscope. Western blot was employed to measure the protein levels of APP， HIF-1α， VEGF，VEGFA， and brain-derived neurotrophic factor （BDNF） in the hippocampus. The mRNA levels of APP， HIF-1α， and VEGF were determined by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the normal group， the model group showed prolonged escape latency （P<0.05）， reduced distance and time around the target platform （P<0.05）， decrease brain and spleen indexes （P<0.05）， vascular endothelial cells with karyopyknosis and not abundant cytoplasm， up-regulated protein levels of APP， HIF-1α， VEGF， and VEGFA （P<0.05）， down-regulated protein level of BDNF （P<0.05）， and up-regulated mRNA levels of APP， HIF-1α， and VEGF （P<0.05） in the hippocampus. Compared with the model group， high-dose HLJDT shortened the escape latency （P<0.05）， increased the distance and time around the target platform （P<0.05）， raised the brain and spleen indexes （P<0.05）， repaired the organelles of vascular endothelial cells， down-regulated the protein levels of APP， HIF-1α， VEGF， and VEGFA （P<0.05）， up-regulated the protein level of BDNF （P<0.05）， and down-regulated the mRNA levels of APP， HIF-1α， and VEGF （P<0.05） in the hippocampus. ConclusionHLJDT can improve the learning and memory abilities of mice by reducing the expression of HIF-1α and VEGF， thus protecting the nerves.

Mechanism of Shugan Huazheng Prescription Against Liver Fibrosis Based on HIF-1α/VEGF/TGF-β1 Pathway / 中国实验方剂学杂志

ObjectiveTo observe the therapeutic effect of Shugan Huazheng prescription on hepatic fibrosis model rats induced by carbon tetrachloride （CCl4） and explore whether it plays its role through hypoxia-induced factor-1α/vascular endothelial growth factor/transforming growth factor-β1 （HIF-1α/VEGF/TGF-β1） pathway. MethodA total of 54 male SPF SD rats were randomly divided into six groups： blank group， model group， colchicine group （0.2 mg·kg-1）， and high-， medium-， and low-dose groups （29.52， 14.76， and 7.38 g·kg-1） of Shugan Huazheng prescription， with nine rats in each group. The molding was conducted three times a week for eight weeks. Administration began the day after the first injection， and the drug intervention was once a day for eight weeks. On the day after the last administration， the rats were deprived of food and water， and they were killed the next day， during which the physiological status of each group of rats was dynamically monitored. The pathological changes in the liver were observed by hematoxylin-eosin （HE） staining， and the content of hydroxyproline （HYP） and angiotensin Ⅱ （AngⅡ） in liver tissue were detected by enzyme-related immunosorbent assay （ELISA）. Real-time fluorescent quantitative PCR （Real-time PCR） was used to determine the mRNA expression levels of HIF-1α， VEGF， and TGF-β1 in liver tissue， and immunohistochemical method （IHC） and Western blot were used to detect the protein expression levels of HIF-1α， VEGF， and TGF-β1 in liver tissue. ResultCompared with the blank group， the overall condition of rats in the model group decreased significantly. The proliferation of connective tissue and the increase in adipose cells between hepatocytes were obvious. The content of HYP and Ang was increased. The mRNA and protein expressions of HIF-1α， VEGF， and TGF-β1 were increased to varying degrees （P<0.05）. Compared with the model group， the proliferation of connective tissue and inflammatory cell infiltration in the liver tissue of colchicine and Shugan Huazheng prescription groups were reduced. The content of HYP and Ang was decreased. The mRNA and protein expression levels of HIF-1α， VEGF， and TGF-β1 were decreased， and the colchicine group and high-dose group of Shugan Huazheng prescription were the most significant （P<0.05）. ConclusionShugan Huazheng prescription has an obvious therapeutic effect on CCl4-induced hepatic fibrosis model rats. Its therapeutic mechanism may be related to the regulation of the HIF-1α/VEGF/TGF-β1 signaling pathway and the improvement of hepatic hypoxia， vascular remodeling， and the syndrome of Qi deficiency and blood stasis in hepatic fibrosis.

Effect and mechanism of Panax notoginseng saponins on wound healing after anal fistula surgery in rats / 中国药房

OBJECTIVE To investigate the effect and mechanism of Panax notoginseng saponins （PNS） on wound healing after anal fistula surgery in rats by regulating the hypoxia-inducible factor-1α （HIF-1α）/vascular endothelial growth factor （VEGF）/ vascular endothelial growth factor receptor-2 （VEGFR2） signaling pathway. METHODS SD rats were selected to establish a postoperative rat model of anal fistula by infecting wound with Escherichia coli. The model rats were randomly grouped into model group， PNS low-dose and high-dose groups （15， 30 mg/cm2）， high-dose of PNS+2-methoxyestradiol （2ME2） group （PNS 30 mg/cm2+HIF-1α inhibitor 2ME2 4 mg/kg）， with 10 rats in each group. Another 10 normal rats were selected for back hair removal treatment as the control group. Each drug group was injected with the corresponding drug solution intramuscularly or （and） intraperitoneally， once a day， for 3 weeks. After the last administration， the wound healing rate （excluding the control group）， microvascular density （MVD）， the expression of collagen Ⅰ and fibronectin （FN） in the wound tissue were detected in each group； the levels of angiogenic factors [VEGF， E-mail：842710813@qq.com angiopoietin-Ⅰ （Ang-Ⅰ）， Ang-Ⅱ] in serum， the levels of inflammatory factors [interleukin-6 （IL-6） and IL-2] in serum binggui7183@163.com and wound tissue as well as the expressions of the related proteins of HIF-1α/VEGF/VEGFR2 signaling pathway in the wound tissue of rats were also detected in each group. RESULTS The MVD， the expression of collagen Ⅰ and FN in the wound tissue， and the levels of IL-6 and IL-2 in serum and wound tissue of rats increased significantly in the model group， compared to the control group （P＜0.05）， while the serum levels of VEGF， Ang- Ⅰ and Ang-Ⅱ decreased significantly （P＜0.05）. The wound healing rate， the MVD in wound tissue， the serum levels of VEGF， Ang-Ⅰ and Ang-Ⅱ， the expressions of collagen Ⅰ and FN in the wound tissue， and protein expressions of HIF-1α， VEGF and VEGFR2 in the PNS low-dose and high-dose groups increased significantly， compared to the model group （P＜0.05）， while the levels of IL-6 and IL-2 in serum and wound tissue decreased significantly （P＜0.05）； the high-dose PNS had a stronger effect （P＜ 0.05）. 2ME2 could weaken the effect of PNS on above indicators of rats after anal fistula surgery （P＜0.05）. CONCLUSIONS PNS can promote the production of angiogenic factors and inhibit the production of pro-inflammatory factors， thereby promoting wound healing in rats after anal fistula surgery. The above effects are related to the activation of HIF-1α/VEGF/VEGFR2 signaling pathway.

Effect and mechanism of Astragalus polysaccharide on peritoneal fibrosis and angiogenesis in peritoneal dialysis rats / 中国药房

OBJECTIVE To investigate the effect and mechanism of Astragalus polysaccharide （APS） on peritoneal fibrosis and angiogenesis in rats with peritoneal dialysis （PD）. METHODS Rats were randomly divided into normal control group （Control group）， model group （PD group）， 70 mg/kg APS group （APS-L group）， 140 mg/kg APS group （APS-H group）， and 140 mg/kg APS+40 mg/kg hypoxia-inducible factor-1α （HIF-1α） agonist DMOG group （APS-H+DMOG group）， with 12 rats in each group. PD rat models were constructed in the last four groups of rats. Administration groups were given APS intragastrically and DMOG intraperitoneally. Control group and PD group were given constant volume of normal saline intragastrically， once a day， for 4 consecutive weeks. After the last medication， the peritoneal ultrafiltration （UF）， mass transfer of glucose （MTG）， the levels of serum creatinine （Scr） and blood urea nitrogen （BUN） were detected in rats； peritoneal histomorphology and peritoneal fibrosis （peritoneal thickness and proportion of collagen fiber deposition） were observed； the microvascular density and the expression levels of α-smooth muscle actin （α-SMA）， laminin （LN）， HIF-1α and vascular endothelial growth factor （VEGF） proteins were detected in peritoneal tissue of rats. RESULTS Compared with Control group， the mesothelium of rats in the PD group was loosely arranged and shed， inflammatory cells infiltrated， the peritoneal thickness and proportion of collagen fiber deposition were increased significantly （P＜0.05）. The levels of MTG， Scr and BUN in serum， microvascular density and the expressions of α-SMA， LN， HIF-1α and VEGF proteins were significantly increased， while the level of UF was significantly decreased （P＜ 0.05）； compared with PD group， the levels of above indexes were significantly reversed in APS-L and APS-H groups （P＜0.05）， and the improvement of APS-H group was better than APS-L group （P＜0.05）. Compared with APS-H group， the levels of above indexes in APS-H+DMOG group were all reversed （P＜0.05）. CONCLUSIONS APS inhibits peritoneal fibrosis and angioge-nesis in PD rats by inhibiting HIF-1α/VEGF signaling pathway.

Inhibition of Pannus Formation by Wutoutang in AIA Rats with Wind-cold-dampness Bi Syndrome Via Down-regulating HIF-1α/VEGFA/Ang Signaling Pathway / 中国实验方剂学杂志

ObjectiveThis study aims to investigate the inhibitory effect of Wutoutang on pannus formation in adjuvant-induced arthritis （AIA） rats with wind-cold-dampness Bi syndrome and its potential mechanism. MethodA total of 40 male SD specific pathogen-free （SPF） rats were selected and divided into blank group， wind-cold-dampness Bi syndrome group ［Complete Freund's Adjuvant （CFA）， 200 μg］， Wutoutang group （15 g·kg-1·d-1）， and indometacin group （10 mg·kg-1） according to random number table method. Except for the blank group， the other groups were given wind-cold-dampness stimulation before the CFA injection. After the rats were administered for 30 days， the basic conditions， onset time， arthritis index score， and foot swelling volume of AIA rats with wind-cold-dampness Bi syndrome were observed. Finally， peripheral arterial blood， ankle joint， and synovial tissue were taken. Enzyme-linked immunosorbent assay （ELISA） was used to detect serum hypoxia-inducible factor-1α （HIF-1α）， vascular endothelial growth factor A （VEGFA） protein content， and rheumatism， including anti-O （ASO）， C-reactive protein （CRP）， and rheumatoid factor （RF）. Hematoxylin-eosin （HE） staining revealed the changes in joint histomorphology. Immunohistochemistry was used to detect the expression of HIF-1α and VEGFA， two important proteins in the ankle pathway. Quantitative real-time polymerase chain reaction （Real-time PCR） was used to reveal mRNA levels of HIF-1α， VEGFA， angiopoietin-1 （Ang-1）， and angiopoietin-2 （Ang-2） in rat synovial tissue. ResultThe foot swelling volume and arthritis score of AIA rats with wind-cold-dampness Bi syndrome were substantially higher （P<0.01） compared with the blank group. Serum CRP， RF， and ASO levels were considerably elevated （P<0.01）. HE staining showed obvious hyperplasia of ankle synovium and synovial inflammation， angiogenesis and pannus formation， and aggravated bone destruction， indicating successful modeling. After the intervention of Wutoutang， the onset time was delayed （P<0.01）. Foot swelling volume and arthritis score were decreased （P<0.01）. Serum CRP， RF， and ASO levels were significantly decreased （P<0.01）. The inflammatory hyperplasia of synovial tissue， angiogenesis and pannus formation， and bone destruction were alleviated. The mRNA levels of HIF-1α， VEGFA， Ang-1， and Ang-2 in the synovial membrane were significantly decreased （P<0.05， P<0.01）. The expressions of HIF-1α and VEGFA in serum and ankle joints were decreased （P<0.01）. In the indomethacin group， the onset time of the disease was delayed （P<0.01）. Foot swelling volume and arthritis score were decreased （P<0.01）. Serum CRP， RF， and ASO levels were significantly decreased （P<0.01）. HIF-1α/VEGFA/Ang signaling pathway was activated， and pathological tissue injury was improved. ConclusionWutoutang can delay the onset time of AIA rats with wind-cold-dampness Bi syndrome， reduce foot swelling volume， arthritis score， rheumatic activity， and improve joint histopathology. It can inhibit pannus formation， and its mechanism may be related to down-regulating the expression of the HIF-1α/VEGFA/Ang pathway.

Correlation Between Expression of HIF-1α and Prognosis of Arsenic-containing Chinese Herbal Compound in Treatment of Myelodysplastic Syndrome / 中国实验方剂学杂志

ObjectiveMyelodysplastic syndromes （MDS） is a group of clonal hematopoietic stem cell disorders，and this study aims to investigate the expression of hypoxia-inducible factor-1α（HIF-1α） in the bone marrow cells of patients with MDS and its correlation with the clinical features of MDS，the therapeutic efficacy of arsenic-containing Chineseherbal compound，and the survival prognosis. MethodAccording to the inclusion and exclusion criteria，27 MDS patients treated with arsenic-containing Chinese herbal compound in the Department of Hematology，Xiyuan Hospital，China Academy of Chinese Medical Sciences from January 2022 to September 2022 were included，and their bone marrow samples were collected by myelotomy. HIF-1α expression level in bone marrow cells was detected by real-time polymerase chain reaction （PCR） to analyze its correlation with clinical features，and logistic and Cox regression was used to analyze the risk factors affecting the efficacy and prognostic survival of MDS patients. ResultThe HIF-1α mRNA expression level was lower in bone marrow cells of MDS patients than in healthy subjects. HIF-1α was positively correlated with the degree of myelodysplasia（r=0.384，P<0.05） and bone marrow granulocytic system%（G%）（r=0.560，P<0.01）. Logistic regression showed that HIF-1α was a risk factor for the prognosis in the follow-up of the efficacy of treatment（P<0.05）and Cox regression showed that HIF-1α was an independent factor affecting the survival prognosis of MDS patients ［odds ratio（OR）=398.968，95% confidence interval（CI）（1.281，116 858.743），P<0.05］. ConclusionThe level of HIF-1α expression in bone marrow cells of MDS patients was closely related to the degree of clinical myelodysplasia and G%，and HIF-1α was a risk factor for the efficacy for and survival prognosis of MDS patients.

Comparison of HIF-1α and survivin levels in patients with diabetes and retinopathy of varying severity

ABSTRACT Purpose: This study measured serum hypoxia--inducible factor-1 (HIF-1α) and survivin levels in patients with diabetes and investigated their association with the severity of retinopathy. Methods: This study included 88 patients with type 2 diabetes mellitus who underwent routine eye examinations. Three groups were created. Group 1 consisted of patients without diabetic retinopathy. Group 2 included patients with non-proliferative diabetic retinopathy. Group 3 included patients with proliferative diabetic retinopathy. To measure serum HIF-1α and survivin levels, venous blood samples were collected from patients. Results: The mean HIF-1α levels in groups 1, 2, and 3 were 17.30 ± 2.19, 17.79 ± 2.34, and 14.19 ± 2.94 pg/ml, respectively. Significant differences were detected between groups 1 and 3 (p=0.01) and between groups 2 and 3 (p=0.01). The mean survivin levels in groups 1, 2, and 3 were 42.65 ± 5.37, 54.92 ± 5.55, and 37.46 ± 8.09 pg/ml, respectively. A significant difference was only detected between groups 2 and 3 (p=0.002). Conclusion: The present study revealed that serum HIF-1α and survivin levels are increased in patients with non-proliferative diabetic retinopathy compared to those in patients without diabetic retinopathy.

Anlotinib suppressed tumor cell proliferation and migration in hypopharyngeal carcinoma

Abstract Objective The purpose of this study is to study the in-vitro effects of multitarget inhibitor anlotinib on hypopharyngeal cancer cell proliferation and cell migration, and the underlying mechanism, which will provide new drug choices for hypopharyngeal cancer treatment. Methods The Hypopharyngeal cancer Fadu cells were treated with anlotinib at a concentration of 0, 5, and 10 μmoL/L, respectively. Cell counting kit-8 and the colony-forming assay were used to detect the inhibition of cell proliferation. Wound-healing assay and transwell assay were used to detect the migration and invasion ability of cells. Flow cytometry was used to detect the effects of anlotinib on cell cycle and apoptosis. RT-qPCR and Western blot were used to measure gene expression levels. Results CCK-8 and colony-forming assay showed that anlotinib could significantly inhibit cell proliferative activity. Wound-healing assay and transwell assay showed that anlotinib could inhibit cell migration and scratch. These results showed that anlotinib has obvious antitumor activity. Flow cell cycle experiment showed that anlotinib could promote Fadu cell apoptosis and block the G2/M phase for inhibiting cell proliferation. In addition, anlotinib decreased the expression of HIF-1α. Conclusions Anlotinib has an excellent suppressing effect on the proliferation, migration, and invasion of hypopharyngeal cancer Fadu cells in-vitro. Moreover, it can play an anti-tumor role through blocking cell cycle G2/M and promoting apoptosis, which may be related to the decrease of HIF-1a expression. Our study would provide a potential treatment method for patients with hypopharyngeal cancer. Level of evidence: Level 3.