f zu{ } 19 í } 10 2015 03 05 Çf Chinese Journal of Tissue Engineering Research March 5, 2015 Vol.19, No.10 Îε«Œ Œ o Ù ŒNF-κBq 1 k 2 Ä m 3 i 1 k 1 ý 4 Æ 5 ( 1 Þ{ 030001 2 030001 3 t{ Ý t n 610041 4 Þ{ œ þ Þ 030001 5 Þ{ œ ÞICU 030001) s 1 Í Îε«Œ Œ²Ÿƒ» Œ o Œ ³² ºŠ Àr Îε«Œ äq Œ NF-κB q n 2 µé h ³Îε«Œ³² ŒÉ q Œv 10 ùã± ÙŒ o Í xîî⵫œ ŒÉ q Œv 10 ºñ ùœ Œ o ý ²u º ºÀ Œ z Îε«Œ Œ o NF-κB Œ q 2 gè ÎΠ«Œz Œ o NF-κB Œ q««ù rn t ~k ~ع yu«ùèr(2011-105) r ñ y Èr(12016905) Îε«Œ º o n º Í {º Íùˆ r rq Îε«Œ o ÙŒ o qùœ Í 36 ù Wistar Ùà Ég 3 œ Œ o Îε«Œ o ý 2 nr { Œ o œ Ùu Ùr { Îε«Œ o ý ÅŒ Îε«Œ 1 ml(1 10 9 L 1 ) ºw 2 ü mkr 1 ml 6 h ý 3 Ùž fq Œv 10 Œ q 2 ²Ÿ À ok q ~ Ù Œ Œ yp 24» Ég 3 d Ø mkr Œ o Ø Œ o Ùž Îε«Œ È Ø Œ o Ùž Îε«Œºü È p 37 ƒzé g 5%CO 2»~ 1 h n Œ n ºŠ À Œ NF-κB(P65) q À Ùg Í œ r Œ o Îε«Œ o ž Œ q 2 éî(p < 0.05) Îε«Œ o Œ q 2 ƒpœ o (P < 0.05) ü ž q Œv 10 i(p > 0.05) Œ o Îε«Œ o ¼ Œ «¼Çž Îε«Œ o og±œ o ñ Œ Í d r Œ o Îε«Œ È Œq NF-κB(P65) q éî(p < 0.05) Îε«Œ È ƒpœ o (P < 0.05) Ÿ Îε«Œ Œ o ùœ Œ NF-κB(P65) q ˆºÃ Œ Œ q 2 Ÿ² ² à f Œ Í n Œ Îε«Œ q Œv 10 à 1986 { 2014 Þ{ x À µ Ôd Ü Þ{ œ Þ ICU 030001 f É ú:r394.2 h u:b ú:2095-4344 (2015)10-01556-06 ÈÍ 2015-02-09 http://www.crter.org k Ä m i k ý Æ. Îε«Œ Œ o Ù Œ NF-κB q [J].f zu{ 2015 19(10):1556-1561. doi:10.3969/j.issn.2095-4344.2015.10.015 Bone marrow mesenchymal stem cells regulate nuclear factor kappab expression in alveolar macrophages of acute lung injury rats with sepsis Zhang Ji-feng 1, Zhang Zi-qi 2, Luo Xiao-tian 3, Hou Lin-yi 1, Jiang Qin 1, Lv Jie-ping 4, Zhang Wen-kai 5 ( 1 Shanxi Medical University, Taiyuan 030001, Shanxi Province, China; 2 Taiyuan Fifth High School, Taiyuan 030001, Shanxi Province, China; 3 Rehabilitation Medicine Center, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China; 4 Department of Anesthesiology, First Affiliated Hospital of Shanxi Medical University, Taiyuan 030001, Shanxi Province, China; 5 Department of Surgical ICU, Second Affiliated Hospital of Shanxi Medical University, Taiyuan 030001, Shanxi Province, China) Abstract BACKGROUND: Bone marrow mesenchymal stem cells have a therapeutic effect on acute lung injury, but the Zhang Ji-feng, Master, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China Corresponding author: Zhang Wen-kai, Master, Chief physician, Department of Surgical ICU, Second Affiliated Hospital of Shanxi Medical University, Taiyuan 030001, Shanxi Province, China Accepted: 2015-02-09 1556 P.O. Box 10002, Shenyang 110180
mechanism is unclear. If the mechanism is understood, the majority of patients with acute lung injury can obtain a benefit. OBJECTIVE: To explore the possible mechanism underlying bone marrow mesenchymal stem cells in the treatment of acute lung injury with sepsis in rats. METHODS: (1) Thirty-six adult Wistar rats were randomly divided into three groups, sham operation group (sham group), sepsis group and bone marrow mesenchymal stem cells group (cell treatment group). In the sepsis and cell treatment groups, animal models of sepsis with acute lung injury were established by cecal ligation and puncture, while in the sham group, the cecum was not ligated and punctured. Then, 1 ml normal saline was injected via the femoral vein in the sepsis and sham groups, and 1 ml bone marrow mesenchymal stem cell suspension (1 10 9 /L) was injected into the cell treatment group. After 6 hours, interleukin 10 and macrophage inflammatory protein-2 levels in serum were measured in the three groups. Lung tissues were taken for pathological observation using hematoxylin-eosin staining. (2) Rat alveolar macrophages were obtained by bronchoalveolar lavage, seeded into 24-well culture plates, and divided into three groups: control group (group A), sepsis model group (group B) and intervention group of bone marrow mesenchymal stem cells (group C). Normal saline, septic plasma, and co-intervention of septic plasma and mesenchymal stem cells were used in the groups A, B, C, respectively. Then, cells in the three groups were cultured in a 5% CO 2 incubator at 37 for 1 hour. After that, alveolar macrophages were taken to detect whether nuclear factor-κb (P65) protein entered into the nucleus using laser scanning confocal microscopy. RESULTS AND CONCLUSION: (1) The results of animal experiments showed that compared with the sham group, the macrophage inflammatory protein-2 levels in the sepsis group and cell treatment group were significantly increased (P < 0.05), but the macrophage inflammatory protein-2 level in the cell treatment group was significantly lower than that in the sepsis group (P < 0.05); there were no significant differences in serum interleukin 10 levels among the three groups (P > 0.05); inflammatory cell infiltration, interstitial pulmonary edema and pulmonary hemorrhage existed in the sepsis and cell treatment groups, but these symptoms were significantly reduced in the cell treatment group compared with the sepsis group. (2) Results from cell experiments showed that compared with the group A, in group B and group C, the number of nuclear factor-κb (P65) proteins into the nucleus was significantly higher (P < 0.05), but it was lower in the group C than the group B (P < 0.05). These findings indicate that bone marrow mesenchymal stem cells in acute lung injury with sepsis can regulate nuclear factor-κb (P65) protein of alveolar macrophages into the nucleus, reduce expression of macrophage inflammatory protein-2, and thereby play a protective role in the lung via reducing neutrophil infiltration. Temporarily, this study cannot explain whether bone marrow mesenchymal stem cells have an effect on interleukin 10. Subject headings: Bone Marrow; Mesenchymal Stem Cell Transplantation; Sepsis; Lung Injury; NF-kappa B; Macrophage Inflammatory Proteins Funding: the Scientific Research Funded Project of Shanxi Scholarship Council, No. 2011-105; the Science and Technology Project of Taiyuan City, No. 12016905 Zhang JF, Zhang ZQ, Luo XY, Hou LY, Jiang Q, Lv JP, Zhang WK. Bone marrow mesenchymal stem cells regulate nuclear factor kappab expression in alveolar macrophages of acute lung injury rats with sepsis. Zhongguo Zuzhi Gongcheng Yanjiu. 2015;19(10):1556-1561. 0 Introduction Œ o ICU¼ í o o ä ö g {² ü o i Î r g oqä [1-2] Œ ¼ Š Ä y v«ö Œ ¼Ÿ² Ÿ Ž qºà [3-4] ²u{Ÿ ÎΠµ«œùx³²¼m o ä Ãr }،ñ µ oö y º q q o [5] g Í nr { Í Ù ³² ÅŒ Îε«Œ ý6 h z ok öž ä ù ƒ» Œ(alevolar macrophages AM) o Πε«Œ È oýr Œ± NF-κB(P65) q À ü Îε«Œ Œ o Ù fq nöùœ Í 1 Materials and methods à dùgƒ¼ Í Œ ƒ Í Âö Íp2013 9 Ž2014 2 ç y ¹º Í ÍÙgöÉ Ä Wistar Ù39ù ƒ«150175 g ºf36ùà Ég3 œ Œ o Îε«Œ o 12ù ù 3ùnp ÄÎε«Œ Œ x Í Ù n çy mk Í Ùgf g Ï Ù Œ q2 q Œv 10 ELISA Ïr(äs mgy ¹ú) 40 g/l Š n fž Œ q RPMI 1640» ö DAPIŒ FITC ˆ p ( Åmg z ¹ú) NF-κB P65 (Bioworld¹ ú) Í Îε«Œ ö¹ Ä 3 ÚWistar Ù 3ù ƒ«(150±15) g È ƒzé g75%j 3 min ÉyÓ Î ŒÎ 5ú¾ ÿƒzé g10% fž qƒ DMEM» v ÎŒÎe ö À ÎÎ Œ 4 ml yp6 cm Œ ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 1557
Ÿ 1 ü Ùž q Œv 10 Œ q 2 (x _ ±s n=12 ng/l) Table 1 Detection of serum interleukin 10 and macrophage inflammatory protein-2 Ì q Œv 10 Œ q 2 œ 45.94±2.79 60.39±4.01 Œ o 47.12±3.07 494.19±8.20 a Îε«Œ o 46.96±3.61 435.44±8.11 ab Ÿ œ ± a P < 0.05 Œ o ± b P < 0.05 Ÿ 2 ü Ù NF-κB(P65) q À (x _ ±s n=12) Table 2 Nuclear factor-κb (P65) protein into the nucleus in the lung tissue Ì NF-κB d 0.20±0.00 0.74±0.04 a Œ o Îε«Œ È 0.27±0.02 ab Ÿ d ± a P < 0.05 Œ o ± b P < 0.05 A B 1 Îε«Œ öÿå Ÿ² Figure 1 Morphology of bone marrow mesenchymal stem cells and the surface markers f A xîî⵫œ Ê ² 95% ü( 100) B x ŒyÉ CD34  (0.92%) CD44  (98.09%) 2 Wistar Ù Œ( 200) Figure 2 Wistar rat alveolar macrophages ( 200) ê Œ f Œ ²s» 3 h ýù Í Ê q«œ Œ«f»r 37 ƒzé g5%co 2 ñx» 48 hë ôã «Œ xý Œ85% üý nÿedtaqœ q ä~x }3xÎε«Œ np Í n 1ù Îε«Œ Ê ŒyÉ CD34 (0.92%) CD44 (98.09%) Ùg Í õd h[6]ÿr { 36ù Wistar Ù 10% ü 0.3 g/kgø ƒzé g75%j Ù µq µ f É 3 cmé ø µé År Œ o Îε«Œ o Ù r ² ² É 1 cméø ˆ ¼ g Ç º q q 1 mlmkr œ Ù { Îε«Œ o ýíî ÅŒ Πε«Œ 1 ml(1 10 9 L 1 ) Í p ý6 h ÙŒ ž20 np ž Œ q2 q Œv 10 e d Š o  Ïr l Ù Òy Œ 40 g/l Šn Í okéf À ok ä Œ Í Ä Wistar Ù3ù ƒ«150 200 g È ƒzé g75%j 2 min Ù Åȵ Ó Èµq ö y ~ sy ÓÓ T ø 20 ml ¾ Í ~ ~ ÈÁPBS 6 ml Ù µ ± 5 min pèáy ~f ö ² 3 Ä1015 ml ~ y ô ù Ä Œ Ø ÿƒzé g10% fž q1640» yp24 ¼È ² qrjf 37 ƒzé g5%co 2»~f 3 h ôã «Œ PBS 3 Ò Œ95%g Œ( 2) Œ q ä20 min x Œg ŒÃ Ég3 d Œ o ÎΠµ«œ È ü» ¼ÈØ0.8 ml PBS d Ø mkr 0.2 ml Œ o Ø Œ o Ùž 0.2 ml Îε«Œ È Ø Œ o Ùž 0.4 ml Îε«Œ 0.8 mlºü È 37 ƒzé g5%co 2»~ 1 h 40 g/l Šn 15 min n ºŠ À(CLSM) Œ NF-κB(P65) q À n h[7] º Š À NF-κBq ä²ÿ ü NF-κBq ä é É 1558 P.O. Box 10002, Shenyang 110180
A B C 3 d Œ NF-κB(P65) q ºŠ À ( 600) Figure 3 Detection of nuclear factor-κb (P65) protein into the nucleus in the control group under laser scanning confocal microscope ( 600) f A ù NF-κB(P65) B ù Œ DAPI C gœ Œ ü A B C 4 Œ o Œ NF-κB(P65) q ºŠ À ( 600) Figure 4 Detection of nuclear factor-κb (P65) protein into the nucleus in the sepsis model group under laser scanning confocal microscope ( 600) f A ù NF-κB(P65) B ù Œ DAPI C gœ Œ ü A B C 5 Îε«Œ È Œ NF-κB(P65) q ºŠ À ( 600) Figure 5 Detection of nuclear factor-κb (P65) protein into the nucleus in the bone marrow mesenchymal stem cells group under laser scanning confocal microscope ( 600) f A ù NF-κB(P65)µÉ B ù Œ DAPI C gœ Œ ü A B C D E 6 œ Œ o öîî⵫œ o Ù ok ä Figure 6 Pathological changes of lung tissue slices in the sham group, sepsis group and bone marrow mesenchymal stem cells group f A gœ ù Ù ö «( 100) B C gœ o ù ü }q Âà ñ «µž Œ (B 100 C 400) D E gîî⵫œ o ù ± ü Âñ ñ «± µž Œ (D 100 E 400) ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 1559
È Í q Œffn0.01 mol/l PBS 30 min 10 min/ ˆž Á1 h Ø p65 Ù ƒ(1 800) 4 ² 0.05 mol/l TBST 30 min 10 min/ Ø FITC ˆ p (1 50) 37 1 h 0.05 mol/l TBST 30 min 10 min/ Ø DAPIŒ 30 min 0.05 mol/l TBST 30 min 10 min/ Ø Ï np ºŠ À nfv10-asw 3.0 Viewer±z É Œ ç Œ ç g ž q Œv 10 Œ q2 ok ä Œ NF-κB(P65) q À É nspss 19.0±zÉ xx _ ±s Ÿx  ± nlsd-t Í nöœ Í P < 0.05g i 2 Results 2.1 ð õøùgƒ¼ ÍWistar Ù 36ù ² É f² Œ 2.2 Ï 10c ~ 2 vÿ1 ùxrç Œ o Îε«Œ o ž q Œv 10 œ r i(p > 0.05) œ r Œ o Œ q2 éî(p < 0.05) Îε«Œ o Œ q2 š±œ éî(p < 0.05) ƒpœ o (P < 0.05) 2.3 NF-κB(P65)~ ã d r Œ o NF-κB éî(p < 0.05) Πε«Œ È NF-κB š± d éî(p < 0.05) ƒpœ o (P < 0.05) Ÿ2 35 2.4 { ì ÆgΫœ Ù ö «( 6A) Œ o ü }q Âà ñ «µž Œ ( 6B C) Îε«Œ o ü Âñ ñ «± µž Œ ( 6D E) x ÅŒ ÎΠµ«œ ùã± ÙŒ o 3 Discussion Œ o Œ ¹ oö öo ÍqºÀ [8]  } [9] u{ xœ o o ŒÉ q Œ p α q Œv 6 Œ q 2}ù f Œöž~¼q Œ Í Ùö µ o n Simpson} [10-11] u{ x ÙŒ o f q Œ Œ q2öî² äg Ÿ² Î 1560 ² äf Œ²zÍ f Œ p «qg Œ n² zí qþ Œö ùé y v«œ q2 p α} Œ Œ o Œ NF-κB fü} [12] u{ x¼ NF-κB Ùq Œv 8} o qÿ ² q Œv 8}Ø f Œ ä o n Kooguchi} [13-14] öj à Œq ff Œ ö ä qÿ² à NF-κB f± q â 5fqêƒ Rel(cRel) RelA(p65 NF-κB3) RelB p50(nf-κb1) p52(nf-κb2) ºfRelA öüp65 NF-κBõ Œ ov«q± â Œ q2 p α º oö f Í n [15] ² qu{ x o ŒqNF-κB ú³ ŸÂ ýùx㱜 q Ù q oö o oo oq i [16] Œ q2g nê ŒÉ Œ Œ g qå Œf Œ Œ o äq Œ Œ q 2} äf Œ ¼ŠÄ ² Ø oö Œ q2 qömö ²z f n [8] Îε«Œº o ä Ãr ¼m }ñ oö qøœ ÃsÎε«Œ uu{ Åq² º³² o ¼m ö ä ØŒ oœ o qu{k Í [17] ³¼ oœ Œ o Ù } Íf Gupta} [18] öjîî⵫œ o Ù Œ q2 p α ƒ ž f q Œv 10 é Π٠ñ éo Ùqm i q Œv 10 ƒ f q Ø } [19] u{q Œv 10 ä ù ÍNF-κB ä Gupta} [18] Nemeth} [20] u{öjîî⵫œ Œ fù ŒNF-κB ˆºq Œv 10É Ø v ñ oö Íf r { Œ o ýˆnîî µ«Œ o ž fq Œv 10 ² Gupta Nemeth}u{ rü ºñ ù Œ Œ o ý ²u º rðîî⵫œ Œ o Ù f NF-κBö Œ q2q ³± È q Ð u{f oÿœ ož ýq Œ rm ä Œ q2 ùx äf Œ ¼q²z Œ o ºÀ Œ i [3] Í Ÿ œ r Œ o ž P.O. Box 10002, Shenyang 110180
Œ q2 éî Îε«Œ o ž Œ q2 š±œ éî ƒpœ o ²Ÿ Îε«Œùxƒ j žf Œ q2 v à f Œ ¼ q²z gu{îî⵫œ ƒ jžf Œ q2 ̓» Œ ³² ºŠ À Œ¼ömq ä x d r Œ o ŒNF-κB(P65) q éî Îε«Œ È Œ NF-κB(P65) q š± d éî ƒpœ o Nemeth} [20] öjîî⵫œ Œ fù Œ v ñ o ö u{ r ²Ÿ Îε«Œ Œ o nqùœ Í ³² ŒNF-κB ˆºÃ Œ q2 v Í f Œ Œ o Í q n u{q ok kg ² or q rð u{ xîî⵫œ o q Œ q n [21-22] ºvÉ Í v Í Çq g º Í y q Îε«Œ Œ o ùx ŒNF-κB(P65) q ˆº Œ Œ q2ÿ²ã ² à f Œ Í n Œ Îε«Œ q Œ v 10 «h  Üc à cúî à c c³ó cé ÜcúÎ ò À à d à n Ü Ü d ÌrÀ{ À À í q d k «n t 2009 wethical issues in animal experimentationx üì íd «(myeloperoxidase MPO) m«î Þî «ê ³ ~ y «û i xû ± Ý ½ «ð n ß n d u k À í À d 4 õ h References [1] Bersten AD, Edibam C, Hunt T, et al. Incidence and mortality of acute lung injury and the acute respiratory distress syndrome in three Australian States. Am J Respir Crit Care Med. 2002;165(4):443-448. [2] Li G, Malinchoc M, Cartin-Ceba R, et al. Eight-year trend of acute respiratory distress syndrome: a population-based study in Olmsted County, Minnesota. Am J Respir Crit Care Med. 2011;183(1):59-66. [3] Æ,ý, Â,}. Œ Œ o fq n[j].f g g, 2013,13(7):876-878. [4] Downey GP, Dong Q, Kruger J, et al. Regulation of neutrophil activation in acute lung injury. Chest. 1999;116(1 Suppl): 46S-54S. [5] Müller I, Lymperi S, Dazzi F. Mesenchymal stem cell therapy for degenerative inflammatory disorders. Curr Opin Organ [6] Transplant. 2008;13(6):639-644. Ä,î, Å}. Ù x ý Slit2 örobo4qÿ²[j].fé ç, 2014,23(3):261-266. [7] Davin MS.Confocal scanning optical microscopy and its applications for biological specimens. J Cell Science. 1989; 94(2):175-182. [8] é, m. v o «r / {² ü [J].fé ç, 2006,15(11):965-966. [9] Â,, i,}. ä¾ Œ o Ù q n[j].f ç ê, 2013,11(16):403-404. [10] Simpson R, Alon R, Kobzik L, et al. Neutrophil and nonneutrophil-mediated injury in intestinal ischemiareperfusion. Ann Surg. 1993;218(4):444-253. [11] Xiao F, Eppihimer MJ, Young JA, et al. Lung neutrophil retention and injury after intestinal ischemia/reperfusion. Microcirculation. 1997;4(3):359-367. [12] fü,ëå,. {² ü öofq Œ v«[j].féí o ç, 2003,15(3):186-189. [13] Kooguchi K, Hashimoto S, Kobayashi A, et al. Role of alveolar macrophages in initiation and regulation of inflammation in Pseudomonas aeruginosa pneumonia. Infect Immun. 1998; 66(7):3164-3169. [14] Maus UA, Koay MA, Delbeck T, et al. Role of resident alveolar macrophages in leukocyte traffic into the alveolar air space of intact mice. Am J Physiol Lung Cell Mol Physiol. 2002;282(6): L1245-1252. [15] µ ±,, Ú,}. -κβ ä Œ o ö ofq n[j].f í o ç,2000,12(6):334-337. [16] Hu X, Qian S, Xu F, et al. Incidence, management and mortality of acute hypoxemic respiratory failure and acute respiratory distress syndrome from a prospective study of Chinese paediatric intensive care network. Acta Paediatr. 2010;99(5):715-721. [17] s,. q o² [J].fé ç, 2012,21(3):235-238. [18] Gupta N, Su X, Popov B, et al. Intrapulmonary delivery of bone marrow-derived mesenchymal stem cells improves survival and attenuates endotoxin-induced acute lung injury in mice. J Immunol. 2007;179(3):1855-1863. [19] Ø,,«,}.qv 10 ä ¼ Œ «B äö p q [J].f í o ç,2001,13(5):284-286. [20] Németh K, Leelahavanichkul A, Yuen PS, et al. Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)-dependent reprogramming of host macrophages to increase their interleukin-10 production. Nat Med. 2009;15(1):42-49. [21] Tai WL, Dong ZX, Zhang DD, et al. Therapeutic effect of intravenous bone marrow-derived mesenchymal stem cell transplantation on early-stage LPS-induced acute lung injury in mice. Nan Fang Yi Ke Da Xue Xue Bao. 2012;32(3):283-290. [22] Rojas M, Xu J, Woods CR, et al. Bone marrow-derived mesenchymal stem cells in repair of the injured lung. Am J Respir Cell Mol Biol. 2005;33(2):145-152. ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 1561