Liraglutide, a TFEB-Mediated Autophagy Agonist, Promotes the Viability of Random-Pattern Skin Flaps

Figure 5

3MA reverses the effect of LIR in random flaps. (a) LC3II expression in flap tissues was detected by immunofluorescence analysis, and autophagosomes are shown (green) (scale bar: 10 μm). (b) Histogram of LC3II-positive cell percentage. (c) Laser Doppler blood flow scanning (scale bar: 0.5 cm) on the 7th day after surgery. (d) Histogram showing the intensity of the blood flow signal. (e) The degree of flap necrosis in the LIR and LR+3MA groups on the 3rd and 7th days after surgery (scale bar: 0.5 cm). (f) Histogram of the survival area percentage of the flaps on day 7 after surgery. (g) Images of tissue oedema in each group on the 7th day after surgery (scale bar: 0.5 cm). (h) Histogram of the water content percentage of each tissue. (i) Haematoxylin and eosin (H&E) staining showing blood vessels in area II of skin flaps in each group (magnification: 200x; scale bar: 50 μm). (j) A histogram of the mean vascular density (MVD) in H&E images was quantitatively analysed. (k and o) Western blots of cadherin 5, VEGF, MMP9, SOD1, HO1, and eNOS in the LIR and LIR+3MA groups. (i and p) ImageJ was used to quantitatively analyse the optical density values of cadherin 5, VEGF, MMP9, SOD1, HO1, and eNOS in each group. (m) Immunohistochemistry analysis of CD34-positive blood vessels in flap area II of each group (magnification 200x; scale, 50 μm). (n) CD34-positive blood vessel density histograms. (q and r) Western blots were used to detect the autophagy indices for VPS34, Beclin-1, LC3II, P62, CTSD, and ATG-5 in each group and the pyroptosis indices ASC, caspase-1, GSDMD-N, IL-1 inhibitor, IL-18, and NLRP3. (s and t) ImageJ was used to quantitatively analyse the histogram of optical density values of autophagy and pyroptosis indicators in each group. The data are presented as the , for each group. ns stands for nonsignificance, and .

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