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. 2020 Dec 18;8:592833. doi: 10.3389/fbioe.2020.592833

FIGURE 4.

FIGURE 4

Insulin relieved MGO-induced HIF-1α impairment in wound healing cells. (A) Insulin promoted MGO-induced cell viability impairment in a concentration-dependent manner. HUVECs and fibroblasts were pretreated with 0 (control group) and 800 μM MGO for 30 min. Thirty minutes after MGO treatment, 0, 10– 6, 10– 7, and 10– 8 M insulin were added to the culture medium. After 24 h of incubation, cells were subjected to CCK-8 analysis. The viability of each group was compared with that of the control group and presented as the percentage of the control group. Data are shown as mean ± SD. **/##p < 0.01, n = 3. (B) Insulin promoted MGO-induced HUVEC angiogenesis impairment. HUVECs were set into three groups: control group (Ctrl) treated without MGO and insulin; MGO group (MGO) treated with 800 μM MGO; insulin group (INS) treated with 800 μM MGO and 10– 6 M insulin. The treatment was described previously. Twelve hours later, the tube formation status in both groups was photographed. (C–E) Insulin alleviated MGO-induced HIF-1α accumulation impairment of HUVECs and fibroblasts. HUVECs and fibroblasts were set as Ctrl, MGO, and INS groups and treated as described above. Twenty-four hours under the hypoxia state (1% oxygen concentration), cells were harvested, and HIF-1α levels were analyzed. Photoshop was used in the quantification of immunoblots. Data are shown as mean ± SD. **/##p < 0.01, n = 3. *Means Ctrl vs. MGO; #means MGO vs. INS. (F,G). Insulin promoted HIF-1α translocation into HUVECs and fibroblasts. In Ctrl, MGO, and INS groups and after 24 h under the hypoxia state, cells were harvested and fixed with 4% paraformaldehyde. The cells were then subjected to immunofluorescence examination to analyze the HIF-1α accumulation and translocation to the nucleus. (H,I) Insulin promoted translation complex formation of HIF-1α and CBP in HUVECs and fibroblasts. Cell lysis of Ctrl, MGO, and INS was subjected to co-IP examination to analyze the HIF-1α and CBP complex formation. (J–M) Insulin improved MGO-induced HIF-1α pathway impairment in HUVECs and fibroblasts. Cell lysis of Ctrl, MGO, and INS was subjected to western blot to examine the expression of HIF-1α target genes VEGF-A and GLUT-1 (HUVECs) and collagen I and collagen III (fibroblasts). Photoshop was used in the quantification of immunoblots. Data are shown as mean ± SD. **/##p < 0.01, n = 5. *Means Ctrl vs. MGO; #means MGO vs. INS.