Adipose-derived Mesenchymal Stem Cells Can Promote Functional Recovery In Rats With Ischemic Stroke Through Unfold Protein Reaction Regulation And Inflammation Suppression

Backgroud:Cerebral ischemia is very common in neurolgoy department,whose characteristics are high morbility,high mortality and high recurrence.It has become the leading cause of death in our country.Intravenous thrombolysis and machinery thrombectomy are the only two therapies approved by FDA in acute ischemic stroke.Because of their short therapy time window,only few people could benefit from the treatment.Hence,a new method for ischemic stroke is urgently needed.In recent years,scientists pay more attention to stem cells,especially mesenchymal stem cells.Accumulated evidence has demonstrated its safety and effectiveness,which contribute to their application in clinical trials.Mesenchymal stem cells can be derived from bone marrow,adipose tissue,umbilical blood,etc.Although bone marrow mesenchymal stem cells(BMSCs)are the most popular cells,adipose derived mesenchymal stem cells(ADMSCs)are proved easier to be obtained and cultured in comparion with BMSCs.In addition,some evidence also suggested ADMSCs have the similar biology features to BMSCs.Even in the treatment of other diseases,ADMSCs were proved to lead to better prognosis than BMSCs.Preclinical studies also demonstrated ADMSCs can reduce infarction volume and promote functional recovery in ischemic stroke.So,ADMSCs probably become the optimal candidate cells for clinical application.Although the benefit can be get from ADMSCs,the mechanism of the action is unclear and complex.Understanding the mechanism could contribute to the translation from preclinic to clinic.The present evidence has showed that ADMSCs play an important role in anti-inflammation,anti-apoptosis and angiogenesis,neurogenesis after ischemic stroke.Especially in the acute phase,anti-inflammation and anti-apoptosis play the key role.However,there is no study about the relationship between ADMSCs and blood-brain barrier(BBB)in transient MCAO model.We have already required that inflammation have a close connection to blood-brain barrier,and unfold protein reaction can contribute greatly to apoptosis and inflammation.So,in present study,ADMSCs were applied in transient MCAO rat and probable mechanism may be futher explored.Aim:(1)check the surface markers on ADMSCs and the capabilities to differentiate into osteoblast and lipoblast;(2)check the influence on BBB permeability in ADMSCs treatment;(3)check if ADMSCs therapy can reduce the infarction volume and promote the functional recovery;(4)check the influence of ADMSCs on the microglia activation and inflammatory cytokines release;(5)check the apoptosis and proteinexpression in UPR pathway.Method:(1)250g-300 g SD rats were prepared to make transient MCAO model;(2)ADMSCs were prepared from epididymis adipose tissue and cultured up to passage 3.flow cytometry test were applied to check the surface markers on ADMSCs before transplantation.Besides,ADMSCs were cultured in differentiation culture until day21.Oil red staining and alizarin red staining were applied to check the differentiation capabilities of ADMSCs to osteoblast and lipoblast;(3)The rats were divided into 3groups,sham group,MCAO group and ADMSCs group.The same volume of PBS or ADMSCs were intravenously transplanted to rats on time point 0,12 h and 24 h after MCAO model;(4)check the m NSS score in every rat on D1,D3,D7 and D14;(5)TTC staining were applied to check the infarction volume;(6)Evan blue staining were applied on D1,D7 and D14 to check the BBB permeability;(7)The rats were sacrificed on D14,BBB tight connection protein ZO-1,Claudin-5 and activated microglia marker OX-42 were checked through immunofluorescent staining.Immunofluorescent intense were compared in all groups;(8)PT-PCR test to check cytokines TNF-?,IL-1?,IL-6 release and compare the expression in every group;(8)TUNEL staining were applied to check the percentage of apoptosis;(9)westernblot were applied to check the protein expression in UPR pathway,including GRP78,PERK,p-PERK,e IF2?,p-e IF2?,ATF4,CHOP,Bax,Bcl-2,XBP-1.Results:(1)ADMSCs significantly expressed CD44,CD90,CD29,the surface marker of mesenchymal stem cells,but not CD34,CD45,the surface marker of hematopoietic stem cell;(2)Oil red staining and alizarin red staining showed ADMSCs can differentiated into osteoblast and lipoblast;(3)TTC staining showed ADMSCs can significantly reduce the infarction volume in MCAO model(P<0.01)(4)m NSS score decreased in ADMSCs group,which began on D7 up to D14(P<0.01);(5)Evan blue staining showed BBB disruption became the worst on D1,and it became better when the times went on.After ADMSCs therapy,BBB permeability were reduced on D7;(6)PT-PCR test showed the release of TNF-?,IL-1?,IL-6 was higher in MCAO group in comparison with sham group(P<0.01).The differences were also significant between MCAO group and ADMSCs group((P<0.01);(7)immunofluorescent staining also found ADMSCs significant increased the immunofluorescent intense of ZO-1,Claudin-5(P < 0.01).And the intenst fo OX-42 were reduced(P <0.01);(8)TUNEL staining showed neuron apoptosis percentage were decreased in infarction boundary in ADMSCs group(P<0.01);(9)In westernblot test,ADMSCs increased the expression of GRP78 and Bcl-2,but reduce the expression of p-PERK,p-e IF2?,ATF4,CHOP,Bax,XBP-1.Conclusions:(1)ADMSCs are easily obtained and cultured.They can differentiate into osteoblast and lipoblast.No immunological rejection was showed.They maybe the most optimal candidate cells for clinical application.(2)repeated intravenously transplantation of ADMSCs can reduce infarction volume and promote neurology functional recovery in MCAO rats.(3)earlier transplantation of ADMSCs can alleviate the BBB disruption,which is parallel to the functional recovery.(4)ADMSCs can suppress the inflammation response,which lead to further BBB recovery.(5)Transient MCAO can trigger UPR which contribute to further apoptosis.ADMSCs could suppress the apoptosis by UPR regulation,finally promote functional recovery.