| Background and aimsIschemia-reperfusion (I/R) injury is the pathophysiological processes including initial damage due to cell ischemia and hypoxia, and the further damage induced by reperfusion, leading to cell death and organ dysfunction. Lung I/R injury is still one of the major complications of cardiothoracic surgeries such as heart bypass surgery and lung transplantation. In I/R progress, DNA damage is induced, abnormal folding of newly synthesized proteins accumulate, and apoptosis associated with endoplasmic reticulum stress is also facilitated. Recently two main protein degradation pathways containing the ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP) have indicated to be involved in eukaryotes, and UPS and ALP participate in the I/R pathophysiological process. In our study, the mechanisms, interrelations and regulations of UPS and ALP in the lung I/R injury are not fully known. The exploration of UPS and ALP role in lung I/R injury would benefit the new therapeutics of lung I/R injury, the lung function protection after cardiopulmonary surgery, successful lung transplantation and long-term survival of patients receiving the lung transplantation.Methods(1) Rat lung epithelial cells (CCL149) were cultured. CL1 was linked to pEGFP (Green fluorescent protein) for reconstruction of UPS reporting genes, while LC3 (Light chain 3) was linked to pAsRed2 (Red fluorescent protein, RFP) for ALP reporting vectors. The confused reporting vectors were transfected into CCL149 respectively, followed by G418 screen and monoclonal cell culture repeatedly to obtain pEGFP-Nl-CL1 CCL 149 and pAsRed2-N1-LC3 CCL 149 cells.(2) Oxygen glucose deprivation hypoxia/reoxygenation (OGD/R) method was applied to induce anoxia/reoxygenation (A/R) and to modify I/R in vitro. pEGFP-N1-CL1 CCL 149 were incubated with proteasome inhibitor (MG132) and activator (Adriamycin) respectively, while pEGFP-N1-CL1 CCL 149 without any interventions were treated as control.Similarly, pAsRed2-N1-LC3 CCL 149 were incubated with autophagy inhibitor (3 MA) and activator (Rapamycin respectively), while pAsRed2-Nl-LC3 CCL 149 without any interventions were treated as control. pEGFP-N1-CL1 CCL 149 and pAsRed2-N1-LC3 CCL 149 cells received four different degrees of A/R (0-h/0-h,2-h /2-h,4-h/4-h, and 6-h/6-h) respectively and were both divided into ten groups as follows:0-h/0-h,2-h/2-h,4-h/4-h,6-h/6-h,2-h/2-h i,4-h/4-h i,6-h/6-h i,2-h/2-h a, 4-h/4-h a,6-h/6-h a. I represented inhibitor and a represented activator.(3) Fluorescence intensity (using fluorescence microscope), ultrastructural changes (using transmission electron microscopy), apoptosis rate (using TUNEL assay), Caspase 3 protein expression (using western blot), cytokines containing IL-10, MCP-1 and ICAM-1 (using ELISA assay), and endoplasmic reticulum stress-related genes (BIP, XBP-1 and CHOP) expressions (using real-time PCR and western blot) of pEGFP-Nl-CL1 CCL 149 and pAsRed2-N1-LC3 CCL 149 cells in the above ten groups were observed respectively. Expressions of ubiquitinated proteins, GFP, UPS related signal molecules (NF-?B, I?B-?) were performed in ten groups of pEGFP-N1-CL1 CCL 149, while Red and ALP related signal molecule (Beclin 1) expressions were detected in ten groups of pAsRed2-N1-LC3 CCL 149 (using western blot).Results(1) Accompanied with increasing A/R time, fluorescent intensity of GFP in pEGFP-N1-CL1 CCL 149 decreased gradually (P<0.05). Compared with cells without any interventions at the respective A/R time (2-h/2-h,4-h/4-h,6-h/6-h), GFP intensity in cells incubated with MG132 obviously increased, while GFP intensity in cells incubated with doxorubicin obviously decreased(P<0.05). Accompanied with increasing A/R time, fluorescent intensity of Red in pAsRed2-N1-LC3 CCL 149 increased gradually (P<0.05). Compared with cells without any interventions at the respective A/R time (2-h/2-h,4-h/4-h,6-h/6-h), Red intensity in cells incubated with 3 MA obviously decreased, while RFP intensity in cells incubated with rapamycin obviously increased (except for 6-h/6-h;P<0.05).(2) Accompanied with increasing A/R time, Caspase3 expression in pEGFP-N1-CL1 CCL 149 and pAsRed2-N1-LC3 CCL 149 both increased gradually (P<0.05). The apoptosis rate of pEGFP-N1-CL1 CCL 149 and pAsRed2-N1-LC3 CCL 149 obviously increased at A/R of 4-h/4-h and 6-h/6-h compared to that at 0-h/O-h (P<0.05). Secretion of IL-10?MCP-1?ICAM-1 increased and expressions of BIP, XBP-1 and CHOP upregulated in the two transfected cells accompanied with A/R time gradually (P<0.05). Compared with cells without any interventions at the respective A/R time (2-h/2-h, 4-h/4-h,6-h/6-h), Caspase 3 expression, apoptosis rate, secretion of IL-10, MCP-1 and ICAM-1 in cells incubated with MG132 or 3 MA obviously decreased, while expressions of BIP, XBP-1 and CHOP obviously increased (P<0.05). Adriamycin or rapamycin showed the opposite effect.(3) Accompanied with increasing A/R time, GFP expression in pEGFP-N1-CL1 CCL 149 decreased gradually, in accordance with GFP intensity change, and ubiquitinated proteins accumulated (P<0.05). NF-?B expression increased, while I?B-? increased at 4-h/4-h but decreased at 6-h/6-h. Compared with cells without any interventions at the respective A/R time (2-h/2-h,4-h/4-h,6-h/6-h), GFP, ubiquitinated proteins and I?B-? expressions in pEGFP-N1-CL1 CCL 149 incubated with MG13 obviously increased, while NF-?B expression decreased (P<0.05). Adriamycin showed the opposite change of GFP and ubiquitinated proteins expression at A/R of 2-h/2-h,4-h/4-h and 6-h/6-h. However, I?B-? expression significantly decreased and NF-?B expression increased only at A/R of 6-h/6-h in cells incubated with adriamycin (P<0.05).(4) Accompanied with increasing A/R time, RFP expression in pAsRed2-N1-LC3 CCL 149 increased gradually, in accordance with change of Red intensity, and Beclin 1 expression increased (P<0.05). Compared with cells without any interventions at the respective A/R time (2-h/2-h,4-h/4-h and 6-h/6-h), Red and Beclin 1 expressions in cells incubated with 3MA obviously inhibited. Rapamycin showed the opposite change of RFP and Beclin 1 expressions.(5) Results of transmission electron microscope suggested the increasing presence of autophagic vacuoles and autophagosomes in pEGFP-N1-CL1 CCL 149 and pAsRed2-N1-LC3 CCL 149 cells under the A/R. Intervention of 3 MA inhibited autophagosome appearance in pAsRed2-N1-LC3 CCL 149 cells, while rapamycin promoted presence of autophagosomes, autolysosomes, and lysosomal bodies.Conclusion(1) pEGFP-N1-CL1 ? pAsRed2-Nl-LC3 sensitively reflected the time-effect relationship of UPS or ALP and A/R stress in the CCL 149 cells, and the effect of proteasome and autophagy inhibitors (or activators) on UPS and ALP activity respectively, indicating the transfected cell model as a reliable tool for subsequent research.(2) UPS and ALP were activated under A/R environment, involving NF-?B signaling pathway (NF-?B and I-?B) in UPS function, and Beclin 1 signal pathway in ALP function. (3) Activation of UPS and ALP promoted IL-10, MCP-1 and ICAM-1 secretions in CCL 149 cells, indicating inhibition of UPS or ALP may play a role in anti-flammation. (4) Our results indicated the association among UPS, ALP and apoptosis. Activation of ALP and UPS promoted apoptosis, while UPS and ALP inhibition significantly reduced apoptosis. (5) Anoxia/reoxygenation promoted expressions of endoplasmic reticulum stress genes (BIP, XBP-1, CHOP), indicating the common regulatory targets between UPS and ALP and some same effects. A/R resulted in ER stress, however UPS and ALP activation accelerated the degradation of abnormally accumulated proteins. Regulation of ER stress might benefit cell protection under A/R in cells?... |
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