Published online Dec 7, 2015. doi: 10.3748/wjg.v21.i45.12822
Peer-review started: April 18, 2014
First decision: June 19, 2015
Revised: July 2, 2015
Accepted: September 2, 2015
Article in press: September 2, 2015
Published online: December 7, 2015
Processing time: 233 Days and 23.2 Hours
AIM: To investigate whether heat shock pretreatment (HSP) improves mesenchymal stem cell (MSC) repair via autophagy following hepatic ischemia-reperfusion injury (HIRI).
METHODS: Apoptosis of MSCs was induced by 250 mM hydrogen peroxide (H2O2) for 6 h. HSP was carried out using a 42 °C water bath for 1, 2 or 3 h. Apoptosis of MSCs was analyzed by flow cytometry, and Western blot was used to detect Bcl-2, Bax and cytochrome C expression. Autophagy of MSCs was analyzed by flow cytometry and transmission electron microscopy, and the expression of beclin I and LC3-II was detected by Western blot. MSCs were labeled in vivo with the fluorescent dye, CM-Dil, and subsequently transplanted into the portal veins of rats that had undergone HIRI. Liver levels of proliferating cell nuclear antigen (PCNA) were quantified by fluorescent microscopy. Serum aminotransferase activity and the extent of HIRI were also assessed at each time point.
RESULTS: HSP for 2 h reduced apoptosis of MSCs induced by H2O2 as seen by a decrease in apoptotic rate, a decrease in Bax and cytochrome C expression and an increase in Bcl-2 expression (P < 0.001). In addition, HSP for 2 h induced autophagy of MSCs exposed to H2O2 as shown by an increase in acidic vesicular organelle-positive cells, beclin 1 and LC3-II expression, and autophagosome formation (P < 0.05). Treatment with 3-methyladenine attenuated HSP-induced autophagy and abolished the protective effects of HSP on the apoptosis of MSCs. Rapamycin failed to have additional effects on either autophagy or apoptosis compared with HSP alone. The phosphorylation of p38MAPK was significantly elevated and the phosphorylation of mTOR was downregulated in heat shock pretreated MSCs. Treatment with the p38MAPK inhibitor, SB203580, reduced HSP-induced autophagy in MSCs. In vivo studies showed that the transplantation of HSP-MSCs resulted in lower serum aminotransferase levels, lower Suzuki scores, improved histopathology and an increase in PCNA-positive cells (P < 0.05).
CONCLUSION: HSP effectively induces autophagy following exposure to H2O2via the p38MAPK/mTOR pathway, which leads to enhanced MSC survival and improved MSC repair following HIRI in rats.
Core tip: We investigated the interaction between autophagy and apoptosis in mesenchymal stem cells (MSCs) exposed to H2O2. We found that heat shock pretreatment (HSP)-induced autophagy served as a protective mechanism. HSP for 2 h improved the therapeutic potential of MSCs in the treatment of ischemia-reperfusion (I/R) injury in rats and enhanced autophagy via the p38MAPK/mTOR pathway, which is involved in the protective effects of HSP on H2O2-induced MSC apoptosis. Systemic administration led to an increase in HSP-MSCs homing to I/R liver cells compared with MSCs, resulting in a significant improvement in liver function, accelerated mitogenic response and alleviation of histopathological damage.