Effects of Rapamycin on Body Weight and Random Blood Glucose of Diabetic Rats
The body weight and random blood glucose of the rats were examined after treatment with rapamycin for 0 w (before treatment), 4 w and 8 w. The mean body weights of the three diabetic groups (DC, DMSO, and RAPA group) were obviously lower than that of the control group, and the body weight in the RAPA group was lower than that in the DC and DMSO groups (Fig. 1A). Before treatment with rapamycin (0 w), at 4 w and at 8 w, the values of random blood glucose in the three diabetic groups were higher than those in the normal group, while there were no significant differences among the three diabetic groups (Fig. 1B).
Rapamycin Prevented Formation of Cataracts in Diabetic Rats
The lenses of rats in each group were observed and photographed under a slit lamp microscope at 0 w, 4 w, and 8 w (Fig. 1C, D). The lenses in the NC group remained transparent for 8 w. At 4 w, the lenses in the three diabetic groups showed various degrees of opacification. The degree of opacity of most lenses in the DC group (11/20) and the DMSO group (12/20) was grade 2, the vacuoles at the equator of the lenses had gradually expanded toward the center, and light cloud-like opacity appeared in the nuclear area of the lenses. In the RAPA group, the degree of opacity of most lenses was grade 1, and vacuoles only appeared around the equator of lenses. There was no significant difference in the degree of opacity between the DC group and the DMSO group (adjusted P > 0.05), while the degree of lens opacity in the RAPA group was significantly lower than that in the DC group and the DMSO group (all adjusted P < 0.05). At 8 w, lens opacity in the three diabetic groups continued to increase. The degree of opacity of most lenses in the DC group (14/20) and the DMSO group (13/20) was grade 3, and the vacuoles became denser and extended to the nuclear area. In the RAPA group, the degree of opacity of most lenses (13/20) was grade 2. The statistical analysis was consistent with that at 4 w. These results suggested that rapamycin could delay and prevent the development of cataracts in diabetic rats.
Histological changes in the lenses are shown in Fig. 1E and F. In the NC group, the LECs underneath the center anterior lens capsule were in a single layer, and at the equatorial region, they showed a physiological bow arrangement. The lens fibers in the superficial cortex had an orderly and tight arrangement. In the DC and DMSO groups, the lens epithelium was in multiple layers, and some LECs migrated into the superficial cortex. Lens fibers in the cortex showed edema and vacuole formation. After treatment with rapamycin (RAPA group), the lens epithelium was basically maintained in a single flat structure, and the edema of lens fibers in the cortex was reduced.
Rapamycin Activated Autophagy and Inhibited EMT in the Lens of Diabetic Rats
At 8 w, the proteins in the LECs of the rats in each group were extracted, and autophagic marker proteins were detected by Western blot assays (Fig. 2A-C). Compared with that of the NC group, the expression of LC3 Ⅱ/I in the LECs of the DC group was significantly reduced (P < 0.0001), while the expression of SQSTM1/p62 was increased (P < 0.0001). The expression levels of LC3 Ⅱ/I and SQSTM1/p62 were not significantly different between the DC and DMSO groups (P > 0.05), indicating that autophagic activity in the lens epithelium of diabetic rats was inhibited. After treatment with rapamycin, the expression of LC3 Ⅱ/I increased in the RAPA group (P < 0.0001), and the expression of SQSTM1/p62 decreased compared with that in both the DC group and the DMSO group (all P < 0.001), but the RAPA and NC groups showed significant differences in the expression of LC3Ⅱ/I and SQSTM1/p62 (all P < 0.01).
Autophagosomes were observed under a TEM to further detect the changes in autophagic activity in the LECs of rats (Fig. 2D, E). The number of autophagosomes in the DC group was significantly reduced compared with that in the NC group (P < 0.0001). However, the number of autophagosomes increased in the RAPA group. Collectively, this evidence suggested that rapamycin could partially rescue the reduced autophagic activity in the LECs of diabetic rats.
EMT marker proteins were detected by Western blot assays (Fig. 2F-H). The expression of E-cadherin was significantly downregulated (P < 0.001), and the expression of α-SMA was significantly upregulated (P < 0.001) in the DC group compared with the NC group. In addition, the expression of E-cadherin and α-SMA showed no differences between the DC and DMSO groups (P < 0.05), indicating that EMT occurred in the lens epithelium of diabetic rats. Compared with those of the DC and DMSO groups, the expression of E-cadherin increased (all P < 0.05) and the expression of α-SMA decreased (all P < 0.05) in the RAPA group, suggesting that rapamycin could not only elevate the autophagic activity but also partially prevent EMT in the lens epithelium of diabetic rats. Alterations in both E-cadherin and α-SMA were also detected by immunofluorescence staining in whole mount lens epithelium, and the results were consistent with those in the Western blot assays (Fig. 2I).
Therefore, we found that autophagy was suppressed but EMT was activated in the lens epithelium of diabetic rats. EMT could be effectively prevented by improving the autophagic activity with rapamycin, resulting in delayed diabetic cataracts.
Rapamycin Activated Autophagy by Inhibiting the mTOR/ULK1 Signaling Pathway in High Glucose-Induced HLE-B3 Cells
We conducted further investigations in vitro to explore whether activation of autophagy could regulate the EMT of LECs under high glucose conditions. HLE-B3 cells were stimulated with high glucose and treated with rapamycin. The expression of LC3 II/I was decreased (P < 0.001) and that of SQSTM1/p62 was increased (P < 0.0001) after stimulation with high glucose compared with those in the NC cells. After treatment with rapamycin, the expression of LC3 II/I was increased (P < 0.001) and that of SQSTM1/p62 was decreased (P < 0.0001) compared with those in high glucose conditions (Fig. 3A-C), but these parameters still differed from those in the NC group. Under a TEM, autophagosomes in HLE-B3 cells in the HG group were significantly reduced compared with those in the NC group (P < 0.0001). However, the number of autophagosomes increased in the RAPA group (Fig. 3G, H). Altered protein expression in the signaling pathway regulating autophagic activity in HLE-B3 cells was also observed by Western blot assays. The expression levels of p-AKT, p-mTOR, and p-ULK1 were all upregulated (all P < 0.0001) in the HLE-B3 cells under high glucose conditions (in both the HG and DMSO groups) compared with those in the NC cells, but the expression of AKT, mTOR, and ULK1 was not significantly changed (P > 0.05). After treatment with rapamycin, the expression levels of both p-AKT and AKT (P > 0.05), mTOR, and ULK1 were not significantly changed (P > 0.05), while the expression levels of p-mTOR and p-ULK1 were significantly decreased (P < 0.0001) compared with those under high glucose conditions, and they were not significantly different from those in the NC group (Fig. 3A, D-F). This evidence suggested that rapamycin could activate autophagy by inhibiting the mTOR/ULK1 signaling pathway.
In summary, autophagy was inhibited, whereas EMT was stimulated in the HLE-B3 cells under high glucose conditions. Rapamycin enhanced autophagic activity through the mTOR/ULK1 signaling pathway and could reduce EMT in HLE-B3 cells.
Rapamycin Inhibited EMT of HLE-B3 Cells Induced by High Glucose through the Notch Signaling Pathway.
HLE-B3 cells were stimulated with high glucose and treated with rapamycin. The expression of E-cadherin was downregulated (P < 0.001), and α-SMA expression was significantly upregulated (P < 0.0001) in the high glucose group (HG group) compared with those in normal cultured cells (NC group). After treatment with rapamycin (RAPA group), the expression of E-cadherin was increased (P < 0.05) and α-SMA expression was decreased (P < 0.05) significantly compared with those in the high glucose groups (HG and DMSO groups), but there were still significant differences between the RAPA group and NC group (all P < 0.05) (Fig. 4A-C).
Cell migration was determined by transwell and scratch wound assays. Under high glucose conditions, cell migration was significantly increased compared with that of normal cultured cells (all P < 0.0001). After treatment with rapamycin, it was inhibited compared with that in the high glucose- and DMSO-treated cells, suggesting that rapamycin could partially restrain the EMT of HLE-B3 cells induced by high glucose (Fig. 4D-G).
The Notch signaling pathway, Jagged1/Notch1/NICD/Snail, is a classic signaling pathway regulating EMT and is activated by TGFβ2 during EMT of LECs (Han et al. 2019; Chen et al. 2017). Western blot assays (Fig. 4H-L) showed that in the HG group and the DMSO group, the expression levels of Jagged1, Notch1, NICD and Snail were higher than those in the NC group (all P < 0.001). Compared with that of the HG group and the DMSO group, the expression of Jagged1 showed no significant changes after treatment with rapamycin (P > 0.05), but the expression levels of Notch1, NICD and Snail were significantly decreased (all P < 0.001), although they were still higher than those in the NC group (all P < 0.05), indicating that activation of autophagy could partially block the Notch signaling pathway stimulated by high glucose.
Immunofluorescence staining assays showed the colocalization of Notch1 and LC3 (Fig. 5A) and of Notch1 and SQSTM1/p62 (Fig. 5B) in the cytoplasm of HLE-B3 cells. Co-IP showed that Notch1 could bind with LC3 and SQSTM1/p62 (Fig. 5C), and LC3 could bind with SQSTM1/p62 (Fig. 5D). These results suggested that autophagy might mediate the selective degradation of Notch1 through its binding with LC3 and SQSTM1/p62, thus negatively regulating the Notch signaling pathway.
Crosstalk between EMT and the Autophagy Signaling Pathways
To investigate whether there is an interaction between the mTOR/ULK1 signaling pathway, which regulates autophagy, and the Jagged1/Notch1/NICD/Snail signaling pathway, which mediates EMT, we employed not only rapamycin but also DAPT, a γ-secretase inhibitor that can block the final step of Notch cleavage and activation and then block the Notch signaling pathway in vivo and in vitro (Park et al. 2015; Jiao et al. 2014), to evaluate the crosstalk between autophagy and the EMT signaling pathways in HLE-B3 cells.
Western blot assays showed that there were no significant differences in the expression of E-cadherin and α-SMA between the DAPT and RAPA groups, although the levels were changed significantly compared with those in the high glucose groups (HG and DMSO groups). However, after combined treatment with rapamycin and DAPT (R + D group), the alterations in EMT marker proteins were more significant than those in the single treatment RAPA and DAPT groups (Fig. 6A-C). In addition, transwell and scratch wound assays demonstrated that either rapamycin or DAPT could effectively inhibit cell migration induced by high glucose, and the combination of rapamycin and DAPT could prevent cell migration much more significantly than either of them alone (Fig. 6D-G).
The protein expression of LC3 and SQSTM1/p62 showed that either DAPT or rapamycin significantly improved the inhibitory activity of autophagy in HLE-B3 cells stimulated by high glucose, and the combined treatment of rapamycin and DAPT showed a stronger enhancement than any of them alone (Fig. 6H-J). Among the proteins that regulate autophagy, p-mTOR, mTOR, p-ULK1 and ULK1 were assessed. Rapamycin effectively inhibited the expression of p-mTOR and p-ULK1, while DAPT had no obvious effects on the expression of p-mTOR but significantly reduced the expression of p-ULK1 compared with those of the HLE-B3 cells under high glucose conditions (HG and DMSO groups). After treatment with the combination of rapamycin and DAPT, the expression of p-mTOR was not decreased more than that after rapamycin treatment, but the expression of p-ULK1 showed a greater reduction (Fig. 6H, K, L).
Among the key proteins of the Notch signaling pathway, Jagged1, Notch1, NICD and Snail all showed upregulated expression in the HLE-B3 cells under high glucose conditions. As expected, DAPT inhibited the expression of NICD and Snail, while rapamycin significantly inhibited the expression of Notch1, NICD and Snail. After treatment with the combination of rapamycin with DAPT, the expression of NICD and Snail was reduced more than that in each treatment group, but Notch1 expression was not reduced more than that in the RAPA group (Fig. 6M-Q).
Thus, activation of autophagy with rapamycin inhibited EMT through the Notch1/NICD/Snail signaling pathway, while inhibition of EMT with DAPT inhibited the NICD/Snail signaling pathway and simultaneously activated autophagy through downregulation of p-ULK1 expression. Therefore, the crosstalk between EMT and the autophagy signaling pathways was mediated by NICD to p-ULK1.
Enhanced Interaction of NICD and ULK1 in HLE-B3 Cells Stimulated by High Glucose.
We found that DAPT inhibited the release of NICD and then prevented the phosphorylation of ULK1 stimulated by high glucose, thereby activating autophagy. Immunofluorescence staining assays showed that NICD and ULK1 colocalized in the cytoplasm of HLE-B3 cells, and this colocalization increased significantly after stimulation with high glucose. Moreover, NICD showed increased expression and entered the nucleus (Fig. 7A). Co-IP assays proved that there was an interaction between NICD and ULK1 in HLE-B3 cells stimulated by high glucose (Fig. 7B, C).
To further confirm that the phosphorylation of ULK1 is regulated by NICD, we overexpressed NICD by transfection with pEGFP-C3-NICD in HLE-B3 cells (Fig. 7D, E), which resulted in upregulation of p-ULK1 and SQSTM1/p62 expression and downregulation of LC3II/I expression (Fig. 7F-I), indicating inhibition of autophagy.
Thus, the Notch signaling pathway was not activated in normally cultured HLE-B3 cells, as the amount of activated NICD in the cytoplasm was very low, so its effects on ULK1 were weak, and autophagy could proceed normally. Under high glucose conditions, the Notch signaling pathway was activated, and NICD increased substantially in the cytoplasm. The combination of NICD and ULK1 increased the phosphorylation of ULK1 and inhibited the activity of ULK1, thereby blocking the initiation of autophagy and inhibiting this process.