Objective To study the effects of advanced glycation end (AGEs) products induced by bovine serum albumin (BSA) on the survival and the morphology of bovine retinal endothelial cells (BREC) and pericytes (BRP). Methods BSA with the final concentration of 50 mg/ml was incubated in PBS, containing 500 mmol/L D-glucose, for 12 weeks under 37℃. AGEs-BSA was purified by Sephacryl S-300 column chromatography and was confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of AGEs-BSA was determined by the method of commassie protein assay. In order to detect the toxic effects of AGEs-BSA on cultured BREC and BRP, groups of AGEs-BSA and BSA with different concentration and untreated control were set up. Phase contrast microscope was used to observe the effect of AGEs-BSA and BSA (with the concentration of 500mu;g/ml and actuation duration of 48 hours) on morphology of BREC and BRP. Results As the dosage of AGEs-BSA increased, the number of inhibited cells increased. When the concentration of AGEs-BSA was 500mu;g/ml, the inhibited BREC in AGEs-BSA group was (72.8plusmn;15.9)% of which in untreated control group, and the inhibited BRP was (64.8plusmn;9) % of which in untreated control group. AGEs-BSA with low concentration promoted the proliferation of endothelial cells, but there was no significant difference between AGEs-BSA and the control group (P=0.231). Inhibited proliferation and abnormal morphology were seen under the phase contrast microscope while the normal morphology of cells was found in BSA and control group. Conclusion AGEs-BSA with the high concentration may inhibit the growth of both BREC and BRP, which leads the loss of BRP and damage of vascular function. These results suggest that nonenzymatic glycosylation plays a major role in diabetic complications. (Chin J Ocul Fundus Dis, 2006, 22: 11-15)
Cell migration is defined as the directional movement of cells toward a specific chemical concentration gradient, which plays a crucial role in embryo development, wound healing and tumor metastasis. However, current research methods showed low flux and are only suitable for single-factor assessment, and it was difficult to comprehensively consider the effects of other parameters such as different concentration gradients on cell migration behavior. In this paper, a four-channel microfluidic chip was designed. Its characteristics were as follows: it relied on laminar flow and diffusion mechanisms to establish and maintain a concentration gradient; it was suitable for observation of cell migration in different concentration gradient environment under a single microscope field; four cell isolation zones (20 μm width) were integrated into the microfluidic device to calibrate the initial cell position, which ensured the accuracy of the experimental results. In particular, we used COMSOL Multiphysics software to simulate the structure of the chip, which demonstrated the necessity of designing S-shaped microchannel and horizontal pressure balance channel to maintain concentration gradient. Finally, neutrophils were incubated with advanced glycation end products (AGEs, 0, 0.2, 0.5, 1.0 μmol·L−1), which were closely related to diabetes mellitus and its complications. The migration behavior of incubated neutrophils was studied in the 100 nmol·L−1 of chemokine (N-formylmethionyl-leucyl-phenyl-alanine) concentration gradient. The results prove the reliability and practicability of the microfluidic chip.
Objective To explore the influence and mechanism of mechanistic target of rapamycin kinase (mTOR)/ receptor of advanced glycation end products (RAGE) pathway mediated-ferritinophagy on high glucose consumption promoting invasion and migration of colorectal cancer (CRC). Methods① Patients and tissue samples. Clinical data and tissues were collected from CRC patients underwent surgery and completed the dietary questionnaire in the Second Affiliated Hospital of Harbin Medical University between October 2022 and October 2023. Real-time quantitative reverse transcription PCR (qRT-PCR) was used to analyzed the expression of nuclear receptor coactivator 4 (NCOA4) and ferritin in CRC and para-carcinoma tissues.② Cell culture and treatment. The HT29 and HCT116 cells were treated by RPMI1640 medium containing 0, 35, 70, 105, 140 mmol/L glucose, and cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) activity analysis were performed to confirm 105 mmol/L glucose was the optimal concentration in the current study. Then the HT29 and HCT116 cells were randomly divided into: control group, glucose group; control group, glucose group, si-RAGE group, and glucose+si-RAGE group; control group, glucose group, rapamycin group, and glucose+rapamycin group. Untreated HT29 and HCT116 cells were considered as control group. The cells in glucose group were treated with 105 mmol/L glucose for 48 h. The CRC cells in si-RAGE group were transfected with si-RAGE for 6 h. The CRC cells in rapamycin group were treated with 10 nmol/L rapamycin for 48 h. The CRC cells in glucose+si-RAGE group were treated with 105 mmol/L glucose for 48 h combination transfected with si-RAGE for 6 h. The CRC cells in glucose+rapamycin group were treated with 105 mmol/L glucose for 48 h combination treated with 10 nmol/L rapamycin for 48 h. Then electron microscopy and western blot, wound healing assay and transwell assay were exhibited, respectively.③ Azoxymethane (AOM)-induced CRC rat model. The effects of glucose consumption on malignant behavior and ferritinophagy mediated by mTOR/RAGE pathway were evaluated in AOM-induced CRC rat models. A total of 16 rats were randomly divided into control group and glucose group, the colorectal tumor number was record and HE staining of colorectal tumor tissues was further performed. The expression of RAGE, mTOR, NCOA4, and ferritin in colorectal tissues of rats from each group was detected by qRT-PCR. Results① More lymphatic node metastasis and TNM Ⅲ/Ⅳ stages was observed in CRC patients from high glucose consumption group (P=0.004, P=0.004). Moreover, we confirmed that NCOA4 expression was significantly decreased (P<0.001) while ferritin was significantly increased (P<0.001) in CRC tissues especially in the CRC tissues from patients with positive lymph nodes metastasis. Additionally, high glucose consumption of CRC patients was negatively correlated with ferritinophagy flux. ② High glucose treatment significantly decreased autophagosomes in HT29 and HCT116 cells while si-RAGE transfection increased autophagic vacuoles compared to the control group. When compared with the glucose group, autophagosomes were increased in the glucose+si-RAGE group. Moreover, compared to the control group, the expression of RAGE, p-mTOR, and ferritin was increased (P<0.001) while the expression of NCOA4 was decreased (P<0.001) in glucose group, but the expression of RAGE, p-mTOR and ferritin was decreased (P<0.001) while the expression of NCOA4 was increased (P<0.001) in si-RAGE group; when compared with the glucose group, the expression of RAGE, p-mTOR and ferritin was downregulated (P<0.001) while the expression of NCOA4 was upregulated (P<0.001) in HT29 and HCT116 cells from the glucose+siRAGE group. Compared to the control group, the HT29 and HCT116 cells in the glucose group performed enhanced wound scratch healing and migration, invasion viability (P<0.05); but the HT29 and HCT116 cells in the si-RAGE group performed impaired wound scratch healing and migration, invasion viability (P<0.05). When compared with the glucose group, the HT29 and HCT116 cells in the glucose+si-RAGE group performed impaired wound scratch healing and migration, invasion viability (P<0.05).③ Rapamycin treatment significantly inhibited the expression of RAGE, p-mTOR and ferritin (P<0.05) but induced the expression of NCOA4 (P<0.05) compared to the control group. When compared with the glucose group, the expression of RAGE, p-mTOR and ferritin was downregulated (P<0.05) while the expression of NCOA4 was upregulated (P<0.05) in HT29 and HCT116 cells from the glucose+rapamycin group. Additionally, compared to the control group, rapamycin treatment performed inhibited effect on wound scratch healing and migration, invasion viability in the HT29 and HCT116 cells (P<0.05); while the HT29 and HCT116 cells in the glucose+rapamycin group performed impaired wound scratch healing and migration, invasion viability (P<0.05) when compared with the glucose group.④ In the AOM induced CRC rat model, we found the more colorectal tumors, aggravated cellular pleomorphism and upregulate expression of RAGE, p-mTOR, ferritin (P<0.05) while downregulated expression of NCOA4 (P<0.05) in the control group than those of the glucose group. ConclusionHigh glucose consumption promote invasion and migration in CRC through suppressing ferritinophagy via activating the mTOR/RAGE pathway.