The Role Of Akt/Nrf2 Pathway In Promoting The Pro-angiogenic Function Of BM-MSCs Induced By CBDL Rat Serum

Research background and objective:Hepatopulmonary syndrome(HPS)is defined by arterial hypoxemia and an abnormal age-corrected alveolar–arterial oxygen gradient caused by the development of intrapulmonary vascular dilatation(IPVD)in the setting of liver disease and/or portal hypertension.The prevalence of HPS in cirrhotic patients is assessed to range from 5 to 32%.The occurrence of HPS significantly increases the mortality of patients,and liver transplantation is considered the only efficient therapy.Common bile duct ligation(CBDL)rats are the only accepted animal model of HPS.Pulmonary angiogenesis plays a vital role in CBDL animals as experimental HPS develops.However,the mechanisms behind this process remain to be further investigated.Mesenchymal stem cells(MSCs)have been considered a regulator of vascular growth because of their potential for transdifferentiation into endothelial cells(ECs)or their paracrine effect.Over the years,there has been much focus on the therapeutic application of MSCs in different types of disease,especially trauma or ischaemia disease.However,there are relatively few studies on MSCs involved in the pathologic changes of angiogenesis-dependent illnesses.The mobilization of bone marrow MSCs(BM-MSCs)can occur in response to a variety of stimuli,including chronic hypoxia,inflammation and injury.The liver and remote organs can be damaged by an increase in bilirubin,endotoxins and inflammatory mediators in the early stages of CBDL.Meanwhile,hypoxia is accompanied by the whole late phase of CBDL,which indicates that BM-MSCs may be mobilized in the rat experimental model of HPS.The stromal cell-derived factor 1/C-X-C chemokine receptor type 4(SDF-1/CXCR4)axis,an important chemokine/chemokine receptor pair in BM-MSCs,not only plays a key role in the metastasis and infiltration of malignant tumours but is also critical in the directional migration of BM-MSCs.Taken together,BM-MSCs may play a role in the pulmonary angiogenesis of HPS.Nf-E2 related factor 2(Nrf2)is a transcription factor that can activate various antioxidant responsive element(ARE)-dependent genes in response to oxidative stress,and our previous study found that phospho-Akt and ERK1 levels are increased in lung tissues 3 weeks after CBDL,which has been shown to activate Nrf2.Nrf2 has been confirmed to control stem cell survival under oxidative damage,and the activity of Nrf2 declines during replicative senescence and ageing.Moreover,it also has been found that Nrf2 activation promotes the expression of CXCR4 in haematopoietic stem cells.Therefore,in this study,we assessed the number of MSCs in the peripheral blood of CBDL rats and explored the effects of CBDL rat serum on the pro-angiogenic functions of BM-MSCs,including their directional migration,proliferation and paracrine ability.Methods(four parts):1.Establishment of HPS rat model by using improved CBDL method and the culture of primary BM-MSCsTo build the HPS rat model,male Sprague-Dawley rats(200–250 g)were subjected to CBDL operation using an improved method compared to our previous study.The sham animals underwent common bile duct exposure without ligation.Lung tissues and the peripheral blood were collected for further analysis.Primary BM-MSCs were isolated from the femurs and tibias of Sprague-Dawley rats and cultured by using MSCs complete medium.The BM-MSCs after 3-5 passages were used for further experiments in vitro.2.CFU-F assay of PB-MSCs and BM-MSCs and flow cytometry identification.All the isolated BM cells were plated on a 25 cm2 flask at a density of 8×104/cm2 cells and cultured in complete culture medium,whereas for PB-MSCs,a cell density of 4×105/cm2 cells per 25 cm2 flask was used.The culture medium was replaced 2 days later,and adherent colonies(>50 cells)derived from the CFU-Fs were counted on days 8.The surface markers were detected using a LSR II cytometer(BD Biosciences)and the Flow Jo software was used for analysis.3.Determination of the pro-angiogenic function in BM-MSCs treated with CBDL rats serumCultured BM-MSCs were treated with CBDL rat serum for 12 h,24 h and 48 h,repectively.The protein levels of proliferating cell nuclear antigen(PCNA)and CXCR4 were detected by Western blotting and VEGF secretion of BM-MSCs was determined by ELISA assay.Moreover,the proliferation and migration ability was determined by MTT assay and transwell assay.4.The relationship between activation of Akt/Nrf2 signaling pathway induced by CBDL rat serum and enhancement of pro-angiogenic function in BM-MSCsCultured BM-MSCs were transfected with Nrf2 si RNA or treated with Akt or Erk inhibitor,and then stimulated with Sham and CBDL rat serum.The expression of p-Akt,p-Erk,Nrf2,PCNA and CXCR4 were detected by Western blotting and VEGF secretion of BM-MSCs was determined by ELISA assay.The transwell and MTT assay were performed to further examine the proliferation and migration function of BM-MSCs.Results:1.BM-MSCs mobilization is increased in the CBDL rat.The mean value of CFU-Fs was 10.2±3.6 per 106 cells in the PB of CBDL rats compared with 1.4±0.8 per 106 cells in the PB of sham rats.However,there was no significant difference between the CFU-F frequency in the BM of CBDL rats and in the BM of sham rats,with values of 35.2±7.4 and 37.8±5.8 CFU-Fs per 106 cells,respectively.2.Characterization and identification of BM-MSCs and PB-MSCs.A large number of cluster cells randomly distributed on the bottom of the plates after 24 h incubation.The primary cells showed fibroblast-like morphology on day 5.They reached an 80% fusion rate,and cell colonies were found on days 7-8.As determined by flow cytometry,these cells were marked positive for CD29 and CD44 but negative for CD34 and CD45.These results demonstrated that the characteristics of BM-MSCs and PB-MSCs are similar.3.The expression of CXCR4,PCNA in BM-MSCs and its paracrine activity of VEGF are promoted by CBDL rat serum.The western blot results showed that the protein levels of CXCR4 and PCNA,which are an indicator of directional migration and proliferation,respectively,were significantly increased in a time dependent manner.Consistently,the MTT assay showed that the proliferation ability of MSCs was improved by CBDL rat serum.We also found that the VEGF level in the HPS group was 185% compared with the control group after 48 h treatment,and the amount of VEGF in the HPS medium without BM-MSCs is similar with that of the control medium,which is significantly lower than in the BM-MSCs conditioned medium4.The Akt/Nrf2 signalling pathway mediated the enhanced proliferation,migration and paracrine effect of BM-MSCs induced by CBDL rat serumWe found that the protein level of Nrf2 and Nrf2 nuclear translocation in BM-MSCs induced by CBDL serum is obviously reduced by the Akt inhibitor LY294002,whereas the ERK inhibitor PD98059 caused no effect.We also found that the promoted proliferation,migration and paracrine function of BM-MSCs were attenuated by blocking the Akt/Nrf2 pathway,instead of the ERK/Nrf2 pathway.Conclusions:These findings indicated that the number of MSCs increased in the peripheral blood of CBDL rats,and the Akt/Nrf2 pathway plays a vital role in promoting the pro-angiogenic functions of BM-MSCs,which could be a potent contributor to pulmonary angiogenesis in HPS.