Fig 2: Proposed mechanism for how hypoxic preconditioning rejuvenates OM-MSCs against ICH. In the microenvironment of ICH, the basal autophagy in normoxia OM-MSCs is insufficient, which accelerates the cellular senescence of transplanted OM-MSCs. Compared with normoxia OM-MSCs, hypoxic preconditioning upregulates the expression of miR-326 in OM-MSCs. miR-326 could promote autophagy by targeting PTBP1/PI3K signaling pathway. The sufficient autophagy can maintain OM-MSC functional survival. As a result, hypoxic preconditioning delays OM-MSC senescence and augments the therapeutic efficacy of OM-MSCs in ICH.

Fig 3: NMDA induces autophagy and injury of RGCs. (a) The viability of RGCs treated with different concentrations (0, 50, 100 or 150 µmol/L) of NMDA was evaluated by MTT assay. (b) The expression of autophagy-related proteins (LC3-I, LC3-II, Beclin-1 and ATG5) in RGCs treated with different concentrations (0, 50, 100 or 150 µmol/L) of NMDA was examined by Western blotting. (c) The expression of PI3K/AKT signaling-associated proteins (pAKT, AKT, pPI3K and PI3K) in RGCs treated with different concentrations (0, 50, 100 or 150 µmol/L) of NMDA was tested by Western blotting. *p < 0.05, ** p < 0.01, ***p < 0.001

Fig 4: Effect of PD-1 blockade on the PD-1 downstream signaling molecules in PBL from BVDV-infected mice. (A) shown are the results of densitometric analyses of the levels of p-PI3K (1), p-Akt (2), p-mTOR (3), p-ERK (4) in bar graph format as well as the representative results (5) of Western blot analysis of PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, ERK, p-ERK, and ß-actin in CP BVDV-infected mice. (B) shown are the results of densitometric analyses of the levels of p-PI3K (1), p-Akt (2), p-mTOR (3), p-ERK (4) in bar graph format as well as the representative results (5) of Western blot analysis of PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, ERK, p-ERK, and ß-actin in NCP BVDV-infected mice. ***p < 0.001, **p < 0.01, *p < 0.05, NS, not significant. The animals were assigned into 7 experimental groups, including the mock-infected group, CP BVDV-infected group, CP BVDV+anti-PD-1 group, CP BVDV+mouse IgG group, NCP BVDV-infected group, NCP BVDV+anti-PD-1 group, and NCP BVDV+mouse IgG group. CP and NCP BVDV-infected mice were used as the controls. Data are presented as mean ± SD (n = 5 per group) and analyzed using one-way ANOVA.

Fig 5: Klotho affected the IGF-1/PI3K signaling pathway. (a) IGF-1R, p-IGF-1R, and IGF-1 protein levels were tested by western blot. (b) Immunofluorescence was used to detect p-IGF-1R levels. (c) Western blot of PI3K/p-PI3K, Akt/p-Akt, and mTOR/p-mTOR protein levels. *p < 0.05 compared with the NC group. #p < 0.05 compared with the HG group.