Comprehensive insights into berberine's hypoglycemic mechanisms: A focus on ileocecal microbiome in db/db mice.

The efficacy of berberine in managing diabetes through modulation of gut microbiome has been established through fecal sample analyses. However, relying solely on fecal materials constrains our comprehension of berberine's effects on diverse gastrointestinal locations. This study specifically explores the ileocecal region, a segment characterized by higher microbial diversity than fecal samples. Berberine exhibits a robust hypoglycemic impact by significantly reducing glucose levels in blood and urine. Beyond glycemic control, berberine ameliorates various diabetes-related symptoms in serum, including increased insulin and leptin, but decreased NEFA and MDA. Notably, berberine demonstrates liver-protective functions by alleviating oxidative stress and enhancing hepatic glycogen abundance. These outcomes prompted a high-throughput sequencing analysis of the ileocecal microbiome, revealing an augmentation of beneficial bacterial genera (four genera in the Lachnospiraceae family, Erysipelatoclostridium, and Escherichia-Shigella), along with a reduction in harmful bacterial genera (Romboutsia). Additionally, we predicted the impact of the ileocecal microbiome on clinically relevant factors associated with diabetes. These findings elucidate the multi-pathway mechanisms of berberine in treating T2D, underscoring its potential as a natural anti-diabetic agent or functional food, particularly through the modulation of the gut microbiota.

Scutellarin suppresses the metastasis of triple-negative breast cancer via targeting TNFα/TNFR2-RUNX1-triggered G-CSF expression in endothelial cells.

Triple-negative breast cancer (TNBC) is heterogeneous and aggressive, with high vascularity and frequent metastasis. We have already found natural flavonoid scutellarin (SC) suppressed spontaneous TNBC metastasis via normalizing tumor vasculature in vivo. In this study, supernatant from tumor necrosis factorα (TNFα)-treated human mammary microvascular endothelial cell (HMMEC) promoted cell migration and pseudopod formation in TNBC cells, but these phenomena were disappeared in SC-co-treated HMMEC. TNFα enhanced the expression of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in both HMMEC and human umbilical vein endothelial cell (HUVEC). G-CSF promoted TNBC migration and invasion in vitro, while G-CSF neutralization antibody and SC both inhibited TNBC metastasis in Balb/c mice. SC had no inhibition on the G-CSF-induced TNBC cell migration, but reduced G-CSF content in TNBC tumor tissues and TNFα-stimulated endothelial cells (ECs). SC restricted the nuclear translocation of runt-related transcription factor 1 (RUNX1) in TNBC tumor vessels and TNFα-treated ECs. RUNX1 was found to directly bind to the promoter of G-CSF in TNBC tumor vessels and regulated G-CSF expression. TNF receptor 2 (TNFR2) was crucial for regulating the TNFα-induced RUNX1 activation and G-CSF expression. Notably, SC hindered the interaction between TNFα and TNFR2 via binding to TNFR2. This work demonstrated that SC reduced TNBC metastasis by targeting TNFα/TNFR2-initiated RUNX1 activation and subsequent G-CSF production in TNBC-associated ECs.

Isotoosendanin exerts inhibition on triple-negative breast cancer through abrogating TGF-ß-induced epithelial-mesenchymal transition via directly targeting TGFßR1.

As the most aggressive breast cancer, triple-negative breast cancer (TNBC) is still incurable and very prone to metastasis. The transform growth factor ß (TGF-ß)-induced epithelial-mesenchymal transition (EMT) is crucially involved in the growth and metastasis of TNBC. This study reported that a natural compound isotoosendanin (ITSN) reduced TNBC metastasis by inhibiting TGF-ß-induced EMT and the formation of invadopodia. ITSN can directly interact with TGF-ß receptor type-1 (TGFßR1) and abrogated the kinase activity of TGFßR1, thereby blocking the TGF-ß-initiated downstream signaling pathway. Moreover, the ITSN-provided inhibition on metastasis obviously disappeared in TGFßR1-overexpressed TNBC cells in vitro as well as in mice bearing TNBC cells overexpressed TGFßR1. Furthermore, Lys232 and Asp351 residues in the kinase domain of TGFßR1 were found to be crucial for the interaction of ITSN with TGFßR1. Additionally, ITSN also improved the inhibitory efficacy of programmed cell death 1 ligand 1 (PD-L1) antibody for TNBC in vivo via inhibiting the TGF-ß-mediated EMT in the tumor microenvironment. Our findings not only highlight the key role of TGFßR1 in TNBC metastasis, but also provide a leading compound targeting TGFßR1 for the treatment of TNBC metastasis. Moreover, this study also points out a potential strategy for TNBC treatment by using the combined application of anti-PD-L1 with a TGFßR1 inhibitor.

Hypoglycemic mechanisms of Polygonatum sibiricum polysaccharide in db/db mice via regulation of glycolysis/gluconeogenesis pathway and alteration of gut microbiota.

Polygonatum rhizoma polysaccharide (PP) is a main ingredient of Polygonatum rhizoma , which is both food and traditional herbal medicine. In this study, we aimed to investigate the hypoglycemic effect of PP and the underlying mechanisms in db/db mice. Our finding showed that PP significantly ameliorates diabetic symptoms by reducing glucose levels in blood and urine and increasing insulin and leptin abundance in the serum. Histopathological examination revealed that PP improved the pathological state and increased hepatic glycogen storage in liver. Additionally, RT-qPCR results indicated that PP significantly down-regulated the expression of phosphoenolpyruvate carboxykinase 1. Furthermore, 16s rRNA sequencing results demonstrated that PP intervention resulted in an increase in beneficial bacteria genus and a reduction in harmful genus. Redundancy analysis revealed the correlation between intestinal flora and clinical factors. Taken together, these results suggest that PP has a significant hypoglycemic effect on type 2 diabetes (T2D) through up-regulating serum insulin and leptin, as well as hepatic glycogen storage, and down-regulating hepatic phosphoenolpyruvate carboxykinase 1 expression, as well as modulating gut microbiota composition. In conclusion, this study investigated the mechanisms of PP in the treatment of diabetes in db/db mice. To the best of our knowledge, this is the first study to explore the positive and negative correlations between gut microbiota and clinical factors, such as oxidative stress injury in liver and glucose related indicators in the blood.

Scutellarin suppresses triple-negative breast cancer metastasis by inhibiting TNFα-induced vascular endothelial barrier breakdown.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with high vascularity and frequent metastasis. Tumor-associated abnormal vasculature was reported to accelerate TNBC metastasis. Scutellarin (SC) is a natural flavonoid with a cardiovascular protective function. In this study, SC reduced TNBC metastasis and alleviated tumor-associated vascular endothelial barrier injury in vivo. SC rescued the tumor necrosis factor-α (TNFα)-induced diminishment of endothelial junctional proteins and dysfunction of the endothelial barrier in vitro. SC reduced the increased transendothelial migration of TNBC cells through a monolayer composed of TNFα-stimulated human mammary microvascular endothelial cells (HMMECs) or human umbilical vein endothelial cells (HUVECs). TNFα induced the nuclear translocation of enhancer of zeste homolog-2 (EZH2), and its chemical inhibitor GSK126 blocked TNFα-induced endothelial barrier disruption and subsequent TNBC transendothelial migration. TNF receptor 2 (TNFR2) is the main receptor by which TNFα regulates endothelial barrier breakdown. Extracellular signal-regulated protein kinase (ERK)1/2 was found to be downstream of TNFα/TNFR2 and upstream of EZH2. Additionally, SC abrogated the TNFR2-ERK1/2-EZH2 signaling axis both in vivo and in vitro. Our results suggest that SC reduced TNBC metastasis by suppressing TNFα-initiated vascular endothelial barrier breakdown through rescuing the reduced expression of junctional proteins by regulating the TNFR2-ERK1/2-EZH2 signaling pathway.

Toosendanin and isotoosendanin suppress triple-negative breast cancer growth via inducing necrosis, apoptosis and autophagy.

Toosendanin (TSN) and isotoosendanin (ITSN) are two natural triterpenoids isolated from Fructus Meliae Toosendan or Cortex Meliae. This study aims to observe the inhibition of TSN and ITSN on the growth of triple-negative breast cancer (TNBC) and the preliminary engaged mechanism. Cell viability assay showed that both TSN and ITSN had obvious cytotoxicity in a variety of tumor cells, and they had the best inhibitory effect on TNBC cells including MDA-MB-231, BT549 and 4T1. Propidium iodide (PI) staining results showed the increased number of necrotic MDA-MB-231 and 4T1 cells induced by TSN (20 nM) and ITSN (2.5 µM). Annexin V-FITC and PI double-staining results showed that TSN (20 nM) and ITSN (2.5 µM) induced cell apoptosis in both MDA-MB-231 and 4T1 cells. Moreover, TSN (20 nM) and ITSN (2.5 µM) induced the cleavage of pro-caspase-3 and pro-caspase-9, and decreased the expression of anti-apoptotic Bcl-xL in both MDA-MB-231 and 4T1 cells. Results from scanning electron microscope observation and detecting the expression of microtubule-associated protein 1 light chain 3B (LC3B) and Beclin 1 evidenced that TSN (20 nM) and ITSN (2.5 µM) induced autophagy in both MDA-MB-231 and 4T1 cells. TSN and ITSN decreased 4T1 xenograft tumor growth without inflicting toxicity on vital organs in mice. Collectively, this study shows that natural compound TSN and ITSN suppress TNBC growth via inducing necrosis, apoptosis and autophagy. TSN and ITSN could be promising drugs for TNBC treatment.

Scutellarein alleviates the dysfunction of inner blood-retinal-barrier initiated by hyperglycemia-stimulated microglia cells.

AIM: To investigate the alleviation of scutellarein (SN) against inner blood-retinal-barrier (iBRB) dysfunction in microglia cells stimulated by hyperglycemia and to elucidate the engaged mechanism. METHODS: Microglia BV2 cells were stimulated by using 25 mmol/L D-glucose. The same concentration of mannitol (25 mmol/L) was applied as an isotonic contrast. Real-time PCR, Western-blot assay and immunofluorescence staining assay was performed. The dysfunction of iBRB in vitro was detected by using transendothelial electrical resistance (TEER) assay. Additionally, the leakage of fluorescein isothiocyanate (FITC)-conjugated dextran (70 kDa) was detected. RESULTS: SN abrogated microglia BV2 cells activation and reduced the phosphorylated activation of extracellular signal-regulated protein kinase (ERK)1/2. SN also decreased the transcriptional activation of nuclear factor κB (NFκB) and the elevated expression of tumor necrosis factor α (TNFα), interleukin (IL)-6 and IL-1ß in BV2 cells treated with D-glucose (25 mmol/L). SN attenuated iBRB dysfunction in human retinal endothelial cells (HRECs) or choroid-retinal endothelial RF/6A cells when those cells were treated with TNFα, IL-1ß or IL-6, or co-cultured with microglia cells stimulated by D-glucose. Moreover, SN restored the decreased protein expression of tight junctions (TJs) in TNFα-treated HRECs and RF/6A cells. CONCLUSION: SN not only alleviate iBRB dysfunction via directly inhibiting retinal endothelial injury caused by TNFα, IL-1ß or IL-6, but also reduce the release of TNFα, IL-1ß and IL-6 from microglia cells by abrogating hyperglycemia-mediated the activation of microglia cells.

Erianin alleviates diabetic retinopathy by reducing retinal inflammation initiated by microglial cells via inhibiting hyperglycemia-mediated ERK1/2-NF-κB signaling pathway.

Blood-retinal barrier (BRB) breakdown is a typical event in the early stage of diabetic retinopathy (DR). This study aims to elucidate the protection of erianin, a natural compound isolated from Dendrobium chrysotoxum Lindl, against DR development. Erianin alleviated BRB breakdown and rescued the reduced claudin1 and occludin expression in retinas from streptozotocin-induced diabetic mice. Erianin reduced microglial activation, ERK1/2 phosphorylation, NF-κB transcriptional activation, and the elevated TNF-α expression both in vitro and in vivo. ERK1/2 inhibitor U0126 abrogated NF-κB activation in d-glucose-treated BV2 cells. Erianin reduced cellular glucose uptake, and molecular docking analysis indicated the potential interaction of erianin with glucose transporter (GLUT)1. GLUT1 inhibitor (STF31) reduced the activation of the ERK1/2-NF-κB signaling pathway. Coculture with d-glucose-stimulated microglial BV2 cells and with TNF-α stimulation both induced inner BRB and outer BRB damage in human retinal endothelial cells and APRE19 cells, but erianin improved all these damages. In summary, erianin attenuated BRB breakdown during DR development by inhibiting microglia-triggered retinal inflammation via reducing cellular glucose uptake and abrogating the subsequent activation of the downstream ERK1/2-NF-κB pathway. Moreover, erianin also alleviated BRB damage induced by TNF-α released from the activated microglia.-Zhang, T., Ouyang, H., Mei, X., Lu, B., Yu, Z., Chen, K., Wang, Z., Ji, L. Erianin alleviates diabetic retinopathy by reducing retinal inflammation initiated by microglial cells via inhibiting hyperglycemia-mediated ERK1/2-NF-κB signaling pathway.

TRPV1 mediates astrocyte activation and interleukin-1ß release induced by hypoxic ischemia (HI).

BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) is a serious birth complication with high incidence in both advanced and developing countries. Children surviving from HIE often have severe long-term sequela including cerebral palsy, epilepsy, and cognitive disabilities. The severity of HIE in infants is tightly associated with increased IL-1ß expression and astrocyte activation which was regulated by transient receptor potential vanilloid 1 (TRPV1), a non-selective cation channel in the TRP family. METHODS: Neonatal hypoxic ischemia (HI) and oxygen-glucose deprivation (OGD) were used to simulate HIE in vivo and in vitro. Primarily cultured astrocytes were used for investigating the expression of glial fibrillary acidic protein (GFAP), IL-1ß, Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), and activation of the nucleotide-binding, oligomerization domain (NOD)-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome by using Western blot, q-PCR, and immunofluorescence. Brain atrophy, infarct size, and neurobehavioral disorders were evaluated by Nissl staining, 2,3,5-triphenyltetrazolium chloride monohydrate (TTC) staining and neurobehavioral tests (geotaxis reflex, cliff aversion reaction, and grip test) individually. RESULTS: Astrocytes were overactivated after neonatal HI and OGD challenge. The number of activated astrocytes, the expression level of IL-1ß, brain atrophy, and shrinking infarct size were all downregulated in TRPV1 KO mice. TRPV1 deficiency in astrocytes attenuated the expression of GFAP and IL-1ß by reducing phosphorylation of JAK2 and STAT3. Meanwhile, IL-1ß release was significantly reduced in TRPV1 deficiency astrocytes by inhibiting activation of NLRP3 inflammasome. Additionally, neonatal HI-induced neurobehavioral disorders were significantly improved in the TRPV1 KO mice. CONCLUSIONS: TRPV1 promotes activation of astrocytes and release of astrocyte-derived IL-1ß mainly via JAK2-STAT3 signaling and activation of the NLRP3 inflammasome. Our findings provide mechanistic insights into TRPV1-mediated brain damage and neurobehavioral disorders caused by neonatal HI and potentially identify astrocytic TRPV1 as a novel therapeutic target for treating HIE in the subacute stages (24 h).

Natural flavonoid galangin alleviates microglia-trigged blood-retinal barrier dysfunction during the development of diabetic retinopathy.

Hyperglycemia-induced blood-retinal barrier (BRB) breakdown is an early and typical event of diabetic retinopathy (DR). Although chronic inflammation plays an important role in DR development, the concrete mechanism remains unclear. This study aims to investigate the role of microglia cells-triggered inflammatory response in hyperglycemia-induced BRB breakdown and the amelioration of galangin, a natural flavonoid. Galangin alleviated BRB breakdown in streptozotocin-induced diabetic mice. D-glucose (25 mM)-stimulated microglia BV2 cells induced BRB damage in vitro, but galangin reversed this injury. Galangin decreased the activation of microglia cells, ROS formation, the phosphorylation of extracellular-signal-regulated protein kinase (ERK)1/2, the transcriptional activation of nuclear factor κB (NFκB) and early growth response (Egr1) protein, and the elevated expression of tumor necrosis factor (TNF)-α both in vitro and in vivo. ERK1/2 inhibitor U0126 reduced ROS formation, the activation of NFκB and Egr1, and the elevated TNFα expression in D-glucose-stimulated BV2 cells. N-acetylcysteine, a well-known antioxidant, abrogated D-glucose-induced NFκB and Egr1 activation in BV2 cells. Galangin also reversed the decreased expression of claudin1 and occludin, and the increased BRB injury and ROS formation in TNFα-treated human retinal endothelial cells (HRECs) and ARPE19 cells. Galangin induced the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) in both HRECs and ARPE19 cells. Moreover, the galangin-provided attenuation on BRB breakdown was diminished in Nrf2 knockout diabetic mice. In conclusion, galangin alleviated DR by attenuating BRB damage via inhibiting microglia-triggered inflammation and further reversing TNFα-induced BRB dysfunction by abrogating oxidative stress injury via activating Nrf2.

Scutellarin alleviates blood-retina-barrier oxidative stress injury initiated by activated microglia cells during the development of diabetic retinopathy.

The breakdown of blood-retinal barrier (BRB) is an early and typical event during the development of diabetic retinopathy (DR). Scutellarin (SC) is a natural flavonoid. This study aims to investigate the protection of SC from BRB damage via focusing on inhibiting microglia-initiated inflammation and subsequent oxidative stress injury. SC attenuated BRB breakdown and the reduced expression of claudin-1 and claudin-19 in STZ-induced diabetic mice. SC reduced microglia cells activation both in vivo and in vitro. The results of transendothelial/transepithelial electrical resistance (TEER/TER) and fluorescein isothiocyanate (FITC)-conjugated dextran cell permeability assay showed that SC attenuated BRB damage induced by d-glucose (25 mM)-stimulated microglia BV2 cells. SC suppressed nuclear factor κB (NFκB) activation and tumor necrosis factor (TNF)-α expression induced by d-glucose (25 mM) in BV2 cells. SC decreased the phosphorylation of extracellular regulated protein kinase (ERK)1/2 both in vivo and in vitro. MEK1/2 inhibitor U0126 reduced the d-glucose-induced NFκB nuclear accumulation and TNFα expression in BV2 cells. Next, SC improved the decreased expression of claudin-1 and claudin-19, the increased BRB damage and cellular reactive oxygen species (ROS) formation, and enhanced nuclear accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2) in TNFα-treated human retinal endothelial cells (HRECs) and APRE19 cells. Moreover, the SC-provided alleviation on BRB breakdown in STZ-induced diabetic mice was diminished in Nrf2 knock-out mice. In conclusion, SC alleviates BRB breakdown via abrogating retinal inflammatory responses and subsequent oxidative stress injury initiated by microglia cells that is activated by hyperglycemia during DR development.

Ursodeoxycholic acid ameliorates diabetic retinopathy via reducing retinal inflammation and reversing the breakdown of blood-retinal barrier.

Ursodeoxycholic acid (UDCA) is the hydrolysis of tauroursodeoxycholic acid, which is the main ingredient from bear gall that has functions including clearing heat and detoxification, and improving eyesight. However, whether UDCA has improving effects on diabetic retinopathy (DR) is not known. This study aims to observe the amelioration of UDCA on DR and its engaged mechanisms. The results of Evans blue permeation assay showed that UDCA (15, 30 mg/kg) reversed the breakdown of blood-retinal barrier (BRB) and the decreased expression of claudin-1 and claudin-19 in STZ-induced diabetic mice. UDCA reversed the reduced thickness of both inner nuclear layer (INL) and outer nuclear layer (ONL) in STZ-induced diabetic mice. UDCA reduced retinal ionized calcium-binding adapter molecule 1 (Iba1) expression in STZ-induced diabetic mice. UDCA reduced the expression of phosphorylated the inhibitor of nuclear factor κB kinase (IKK) and the nuclear translocation of p65 subunit of nuclear factor κB (NFκB) in retinas from STZ-induced diabetic mice. UDCA also reduced retinal expression of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), intercellular cell adhesion molecule-1 (ICAM-1), inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF) in STZ-induced diabetic mice. In conclusion, UDCA attenuates BRB breakdown during DR development via inhibiting retinal inflammation and reversing the reduced expression of tight junctions (TJs) including claudin-1 and claudin-19.

Chlorogenic acid attenuates diabetic retinopathy by reducing VEGF expression and inhibiting VEGF-mediated retinal neoangiogenesis.

Diabetic retinopathy (DR) is one of the most common and serious complications of diabetes mellitus (DM). This study aims to investigate the amelioration of chlorogenic acid (CGA) on proliferative DR (PDR) via focusing on inhibiting retinal neoangiogenesis. CGA reduced the increased cell proliferation, migration and tube formation induced by vascular endothelial growth factor (VEGF) in human retinal endothelial cells (HREC) and choroid-retinal endothelial RF/6A cells. CGA abrogated VEGF-induced the phosphorylation of VEGFR2 and its downstream mitogen-activated extracellular regulated kinase (MEK1/2), extracellular regulated protein kinase (ERK1/2) and p38 kinase. CGA reduced high glucose (HG)-induced the activation of microglia BV-2 cells. CGA also reduced HG-induced the increased VEGF expression and hypoxia-inducible factor 1-alpha (HIF-1α) translocation into nucleus in BV2 cells. Retinal immunofluorescence staining with cluster of differentiation 31 (CD31) and retinal histopathological observation both demonstrated that CGA (1, 10mg/kg) decreased the increased retinal vessels in streptozotocin (STZ)-induced hyperglycemic mice. CGA reduced the elevated serum VEGF level and microglia activation in STZ-induced hyperglycemic mice. In conclusion, CGA inhibits retinal neoangiogenesis during the process of DR by abrogating HG-induced HIF-1α-mediated paracrine VEGF expression in microglia cells and inhibiting VEGF-induced angiogenesis in retinal endothelial cells.

Scutellaria barbata attenuates diabetic retinopathy by preventing retinal inflammation and the decreased expression of tight junction protein.

AIM: To observe the attenuation of ethanol extract of Herba Scutellaria barbata (SE) against diabetic retinopathy (DR) and its engaged mechanism. METHODS: C57BL/6J mice were intraperitoneally injected with streptozotocin (STZ, 55 mg/kg) for 5 consecutive days to induce diabetes. The diabetic mice were orally given with SE (100, 200 mg/kg) for 1mo at 1mo after STZ injection. Blood-retinal barrier (BRB) breakdown was detected by using Evans blue permeation assay. Real-time polymerase chain reaction (RT-PCR), Western blot and immunofluorescence staining were used to detect mRNA and protein expression. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum contents of tumor necrosis factor-α (TNF-α) and interleukin (IL)-1ß. RESULTS: SE (100, 200 mg/kg) reversed the breakdown of BRB in STZ-induced diabetic mice. The decreased expression of retinal claudin-1 and claudin-19, which are both tight junction (TJ) proteins, was reversed by SE. SE decreased the increased serum contents and retinal mRNA expression of TNF-α and IL-1ß. SE also decreased the increased retinal expression of intercellular cell adhesion molecule-1 (ICAM-1). SE reduced the increased phosphorylation of nuclear factor kappa B (NFκB) p65 and its subsequent nuclear translocation in retinas from STZ-induced diabetic mice. Results of Western blot and retinal immunofluorescence staining of ionized calcium-binding adapter molecule 1 (Iba1) demonstrated that SE abrogated the activation of microglia cells in STZ-induced diabetic mice. CONCLUSION: SE attenuates the development of DR by inhibiting retinal inflammation and restoring the decreased expression of TJ proteins including claudin-1 and claudin-19.

Lonicerae Japonicae Flos attenuates diabetic retinopathy by inhibiting retinal angiogenesis.

ETHNOPHARMACOLOGICAL RELEVANCE: Lonicerae Japonicae Flos (Jin-Yin-Hua) is a well-known traditional Chinese medicine used for clearing away heat and toxic material. AIM OF THE STUDY: This study aims to observe the attenuation of aqueous extract of Lonicerae Japonicae Flos (FL) against streptozotocin (STZ)-induced diabetic retinopathy (DR) and its engaged mechanism. MATERIALS AND METHODS: STZ-induced proliferative DR (PDR) for 5 month in C57BL/6 mice was used in this study. Retinal vessels were observed by immunofluorescence staining with cluster of differentiation 31 (CD31) and histopathological evaluation. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum vascular endothelial growth factor (VEGF) content. Cell proliferation was detected by 3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide (MTT) assay in choroid-retinal endothelial RF/6A cells. VEGF-induced tube formation in RF/6A cells was observed. The contents of chlorogenic acid (CGA), caffeic acid (CA), and luteolin in FL were detected by high-performance liquid chromatography (HPLC). RESULTS: Histopathological evaluation demonstrated that retinal vessels were increased in STZ-induced PDR mice, whereas FL decreased such increase. The results of CD31 staining also showed that FL decreased the increased number of retinal vessels in STZ-induced PDR mice. In addition, FL reduced the increased serum VEGF content in STZ-induced PDR mice. FL reduced VEGF-induced RF/6A cell proliferation in the concentration-dependent manner, but had no obvious effect on RF/6A cell viability without VEGF stimulation. VEGF-induced tube formation in RF/6A cells was inhibited by different concentrations of FL. CGA, CA and luteolin all inhibited VEGF-induced tube formation in RF/6A cells, and the lowest effective concentration of CGA and CA was both 0.625µM, but of luteolin was 5µM. Furthermore, the results of HPLC demonstrated that the amount of CGA was the highest in FL. CONCLUSIONS: FL ameliorates STZ-induced PDR by inhibiting retinal angiogenesis. Phenolic acid CGA is the main compound contributing to the inhibition of FL on retinal angiogenesis.