Fig 2: Effects of high glucose (5.6, 12.5, 25, and 50 mM) on autophagy, apoptosis, and senescence in chondrocytes.A Western blot results and quantification analyses of p62, and LC3 in chondrocytes under various glucose concentrations. B Western blot results and quantification analyses of p53, p21, and p16 treated with various glucose concentrations. C Representative micrographs of SA-β-gal staining representing the effects of glucose on senescence. Scale bar 100 μM. D Western blot results and quantification analyses of Bcl2, Bax, and cleaved caspase-3 treated with various glucose concentrations. E Representative micrographs of TUNEL staining representing the effects of glucose on apoptosis. F TUNEL-positive cell intensity under various glucose concentrations. Scale bar = 200 μM. Data are presented as means ± SD (n = 5). *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 3: TBMS induces AMPK-dependent autophagy. (A) Western blot analysis of AMPK and AMPK phosphorylation (T172) in HepG2 cells treated with the indicated concentrations of TBMS for 24 h. β-actin was used as the internal control. (B) HepG2 cells were treated with TBMS in the absence or presence of CC for 24 h. The mRNA expression of Beclin 1 and LC3-I was measured by RT-qPCR assay. (C) HepG2 cells were treated with TBMS in the absence or presence of CC for 24 h. The protein expression of LC3-I and LC3-II was measured by western blot analysis. β-actin was used as the internal control. (D) Densitometric analysis of LC3-II/LC3-I is shown in the histogram. The data represent the mean ± SD. *P<0.05, **P<0.01 vs. NC group. TBMS, tubeimoside I; AMPK, AMP-activated protein kinase; CC, compound C; LC3, microtubule-associated protein 1 light chain 3; RT-qPCR, reverse transcription-quantitative PCR.

Fig 4: Ginsenoside Rg2 increases the expression of autophagy-related proteins beclin 1, LC3 and ATG5. (A) HCM cells were incubated with 200 µM ginsenoside Rg2 for 12 h and exposed to 100 µg/ml TZM for another 24 h. The expression levels of beclin 1, LC3 and ATG5 were measured by western blotting. The relative levels of (B) beclin 1 and (C) ATG5 were normalized to β-actin and quantified using ImageJ software. (D) The expression of LC3 in cells was detected with immunofluorescence staining. Magnification, x200. (E) Quantification of LC3-positive cells. Data were representative of three separate experiments. **P<0.01 vs. the control group. TZM, trastuzumab; HCM, human cardiomyocyte; LC3, microtubule associated protein 1 light chain 3 α; ATG5, autophagy protein 5; Rg2, ginsenoside Rg2.

Fig 5: Effect of rhHMGB1 and Beclin1 siRNA. (A) Beclin1 protein levels detected by western blot following Beclin1 siRNA treatment in normal and OGD/R (8 h OGD followed by 24 h reperfusion) groups and quantitative analysis of Beclin1 was performed following Beclin1 siRNA treatment relative to GAPDH; (B) autophagy related proteins detected by western blot; (C) quantitative analysis of LC3II protein relative to LC3I; (D) quantitative analysis of Beclin1 protein relative to GAPDH; (E) quantitative analysis of P62 protein relative to GAPDH; (F) quantitative analysis of cytoplasmic HMGB1 protein relative to GAPDH. Data are presented as the mean ± standard deviation of 3 samples per group. *P<0.05 vs. normal control group, #P<0.05 vs. OGD/R group, &P<0.05 vs. IPOC group. N, normal control group; NCRNA, negative control siRNA; siRNA, small interfering RNA; OGD/R, oxygen and glucose deprivation and reperfusion; LC3, microtubule associated-protein 1A/1B-light chain 3; P62, sequestome 1; HMGB1, recombinant human high mobility group box 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IPOC (OGD/R plus 3 cycles of IPOC), ischemic post-conditioning; rhHMGB1, recombinant human HMGB1.