Objective:To explore the effects and the possible mechanism of bone marrow mesenchymal stem cell (BMMSC) transplantation on apoptosis in rats cerebral cortex after cardiac arrest/cardiopulmonary resuscitation (CA/CPR).Methods:The BMMSC of 2 Sprague-Dawley (SD) rats aged 4-5weeks was extracted, and the 3rd passage was used in experimental study. Eighteen Sprague-Dawley (SD) rats were divided into sham group, model group (CA/CPR group) and intervention group (BMMSC group) according to random number table method, with 6 rats in each group. CPR was performed 6 minutes after asphyxia induced CA. In sham group, CA was not induced except performing general surgical procedure. At 1 hour after return of spontaneous circulation (ROSC), 0.5 mL phosphate buffered saline (PBS) was injected through tail vein in CA/CPR group. 2×10 9/L green fluorescence protein (GFP)-labeled BMMSC was injected through tail vein 1 hour after ROSC in BMMSC group. Neurological deficit score (NDS) were assessed in every group at 72 hours after CPR. Serum S100 calcium binding protein B (S100B) levels were assayed by enzyme linked immunosorbent assay (ELISA). Distribution of BMMSC in brain was observed under a fluorescent microscope. Apoptosis rate in cerebral cortex was assayed by TdT-mediated dUTP nick-end labeling (TUNEL). Western blotting was performed to measure the expression levels of active aspartic acid specific cysteine proteinase (caspase-8 and caspase-9) in cerebral cortex. Results:At 3 days after CPR, compared with sham group, the apoptosis of cerebral cortex cells was increased and brain damage was obvious, NDS score was decreased significantly (56.6±5.5 vs. 80.0±0.0, P < 0.05), and serum S100B was increased markedly (ng/L: 45.1±4.7 vs. 19.1±1.4, P < 0.05), apoptosis rate of cerebral cortex cells increased significantly [(52.9±11.8)% vs. (10.1±1.5)%, P < 0.05], the level of active caspase-8 expression in cerebral cortex was significantly higher (caspase-8/GAPDH: 0.689±0.047 vs. 0.330±0.108, P < 0.05), and there was no significant difference in active caspase-9 protein expression (caspase-9/GAPDH: 0.428±0.014 vs. 0.426±0.021, P > 0.05) in CA/CPR group. After BMMSC transplantation, GFP-labeled BMMSC were primarily detected in cerebral cortex, compared with CA/CPR group, the apoptosis of cerebral cortex cells and brain injury were significantly improved in BMMSC group, NDS score increased significantly (70.6±2.1 vs. 56.6±5.5, P < 0.05), serum S100B levels in BMMSC group were lower (ng/L: 32.0±3.2 vs. 45.1±4.7, P < 0.05), apoptosis rate of cerebral cortex cells decreased significantly [(31.1±3.4)% vs. (52.9±11.8)%, P < 0.05], and the active caspase-8 expression in cerebral cortex in BMMSC group was significantly decreased (caspase-8/GAPDH: 0.427±0.067 vs. 0.689±0.047, P < 0.05). The active caspase-9 expression in cerebral cortex in BMMSC group and CA/CPR group were not significantly different (caspase-9/GAPDH: 0.431±0.022 vs. 0.428±0.014, P > 0.05). Conclusion:BMMSC transplantation can alleviate rat brain damage after CA/CPR possibly by inhibiting the death receptor mediated apoptotic pathway to inhibit the apoptosis of brain cells.

Objective:To explore the protective effects of bradykinin postconditioning on cardiopulmonary resuscitation (CPR) rats, and to assess the underlying mechanisms.Methods:Forty-eight adult male Sprague-Dawley (SD) rats were randomly divided into four groups according to random number table: Sham operation group, cardiac arrest (CA) group, bradykinin treatment (BK) group, and AMP-activated protein kinase (AMPK) inhibitor Compound C+ bradykinin treatment (CP+BK) group, finally, 8 rats in each group were taken for follow-up experiment. CA was induced by asphyxia. Rats in the Sham group received arteriovenous catheterization, endotracheal intubation, and mechanical ventilation, without CA. Compound C (250 μg/kg) was intraperitoneally injected in CP+BK group 30 minutes before CA, and the same volume of dimethyl sulfoxide (DMSO) was given in the remaining groups. Bradykinin (150 μg/kg) was intraperitoneally injected in BK group and CP+BK group 48 hours after restoration of spontaneous circulation (ROSC), and same volume of saline was given in the remaining groups. The neural function of rats in each group was evaluated with neurological deficit score (NDS) 72 hours after ROSC. Microtubule-associated protein light chain 3 (LC3) and p62 expressions were detected by immunohistochemistry, autophagosomes were observed by transmission electron microscopy, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method (TUNEL) assay was used to assess apoptosis.Results:Compared with the Sham group, the NDS was decreased (60.75±5.80 vs. 80.00±0.00, P < 0.01), the expression levels of LC3 and p62 elevated [LC3 ( A value): 1.04±0.64 vs. 0.40±0.14, p62 ( A value): 2.75±0.57 vs. 0.36±0.12, both P < 0.05], the number of autophagosomes and apoptotic cells increased in the CA group [(39.00±8.00)% vs. (3.87±1.90)%, P < 0.05]. Compared with the CA group, the NDS (67.75±6.32 vs. 60.75±5.80, P < 0.05), the expression of LC3 ( A value: 1.60±0.34 vs. 1.04±0.64, P < 0.05), and the number of autophagosomes increased in the BK group, while the expression of p62 and the rate of apoptotic cells reduced [p62 ( A value): 1.51±0.32 vs. 2.75±0.57, apoptotic cells rate: (23.03±1.91)% vs. (39.00±8.00)%, both P < 0.05]. Compared with the BK group, the NDS (59.00±8.19 vs. 67.75±6.32, P < 0.05), the expression of LC3 ( A value: 0.62±0.41 vs. 1.60±0.34, P < 0.05) and the number of autophagosomes declined in the CP+BK group, while the expression of p62 and the rate of apoptotic cells elevated [p62 ( A value): 3.50±0.47 vs. 1.51±0.32, apoptotic cells rate: (44.53±10.15)% vs. (23.03±1.91)%, both P < 0.05]. Conclusion:Bradykinin postconditioning played a neuroprotective role in CPR rats by activating autophagy and reducing apoptosis.

Objective To investigate the effects of bone marrow mesenchymal stem cells (BMSCs) transplantation on the expression of nerve growth factor (NGF) and Caspase-3 in rat hippocampus after cardiac arrest (CA). Methods Sprague-Dawley (SD) rats were randomly divided into 3 groups: sham group (n=6), CA group (n=6), and BMSCs group (n=6). CPR was performed on the groups after the induction of asphyxial cardiac arrest. Animals in the BMSCs group or the CA group were respectively injected with a dose of 1×106 BMSCs in 0.5 mL phosphate buffer solution (PBS) or 0.5 mL PBS alone via the vena caudalis 1 h after successful resuscitation. The neurological status after restoration of spontaneous circulation (ROSC) were assessed by modified neurological severity scores (mNSS); serum levels of S100B were assayed, and the expression of NGF and Caspase-3 in hippocampus was detected by immunohistochemistry. Results Compared with the CA group, mNSS and S100B levels were lower in the BMSCs group on the 7th day after ROSC [(0.9±0.3) vs (4.5±0.6), (90.12±4.62) pg/mL vs (182.30±2.58) pg/mL, both P<0.05] with higher expression of NGF and lower expression of Caspase-3 [(11.391±1.297) vs (7.744±1.334), (6.256±1.036) vs (8.506±1.742), both P< 0.05]. Conclusions BMSCs transplantation might improve rat's neurological functions after cardiac arrest, which may be related to up-regulation of NGF expression and down-regulation of Caspase-3 expression.

Objective To investigate the effects of transplantation with different amount of bone marrow mesenchymal stem cells(BMSCs)on osteoporosis in ovariectomized rats.Methods The rat model of osteoporosis was prepared by ovariectom(OVX)and verified after 3 months.A total of 60 female Sprague-Dawley rats were randomly divided into 6 groups(n=10 each).The rats with shamed operation served as a sham-injured control group(sham control group).The 5 ovariectom (OVX) groups with osteoporosis were study groups as follows:(1)the negative therapy group(simple OVX group);(2)the positive therapy group(OVX+ estrogen,E2 group) by intramuscular injection;others were treated with transplantation of BMSCs by tail vein injection in low dose(LS group),middle dose (MS group)and high dose(HS group).At 8,12 and 16 weeks after intervention,body mineral density (BMD)were detected by dual-energy x-ray absorptiometry scans.After 16 weeks of intervention,rat shinbone was obtained and stained by hematoxylin-eosin(HE) staining.Results Compared with the sham control group,simple OVX group showed a reduced total body BMD and the decreased proportion of trabecular bone to bone marrow cavity area (P ＜0.05).The total body BMD and the proportion of trabecular bone to bone marrow cavity area were higher in each BMSCs transplantation groups than in simple OVX group at 8,12,16 weeks after intervention(P ＜0.05),which showed a increased trend in the total body BMD and the proportion with the increased dose of transplantation BMSCs(P＜0.05).Rats in the HS group had highest BMD and best proportion of trabecular bone to bone marrow cavity area among three doses of transplantation BMSCs.Conclusions BMSCs transplantation can significantly improve osteoporosis of ovariectomized rats with an increased total body BMD and higher proportion of trabecular bone to bone marrow cavity area,and better and longer outcomes can be achieved.

Objective To explore the effects of bone marrow mesenchymal stem cells (MSCs) transplantation on receptor-interacting protein kinase 1 (RIP1) and RIP3 in rat brain after cardiac arrest (CA).Methods Sprague Dawley (SD) rats were randomly (random number) divided into sham group (n=8),CA group (n=8) and MSCs group (n=8).Animals were subjected to asphyxial cardiac arrest and followed by cardiopulmonary resuscitation (CPR).In MSCs group or CA group,animals received intravenous injection of 1 × 106 MSCs in 0.5 mL phosphate buffer solution (PBS) or 0.5 mL PBS alone at 1 h after successful resuscitation.Neurological deficit scores (NDS) were assessed at 3 d after CPR.Donor MSCs in brain were detected under a fluorescent microscope.HE staining of brain tissue was performed to observe necrotic neurons.Western blot analysis was performed to measure the levels of RIP1 and RIP3 in brain.Multiple comparisons were made by analysis of variance or Kruskal-Wallis H test.Results At 3 d after CPR,MSCs group demonstrated higher NDS than CA group [72.5(71.5,73.2) vs.63.0(62.5,64.1),Z=3.376,P=0.001].DAPI-labeled MSCs were primarily observed in the cerebral cortex.The percentage of necrotic neurons in MSCs group was significantly lower than that in CA group [(29.6±5.9)％ vs.(57.2±6.4)％,t=8.922,P＜0.01].The levels of RIP1 and RIP3 expression in brain in MSCs group were significantly lower than those in CA group [RIP1:0.227(0.193,0.243) vs.0.599(0.535,0.629),Z=3.151,P=0.001;RIP3:0.217(0.203,0.274) vs.0.543(0.533,0.555),Z=3.361,P=0.001].Conclusion MSCs transplantation improves neurological function after CPR from CA in rats likely associated with inhibiting necroptosis.

Objective To investigate the improvement of ischemic hypoxic injury of brain after the transplantation of bone marrow mesenchymal stem cells (BMSCs).Methods Rats were randomly (random number) divided into sham operation group,cardiac arrest group and BMSCs treatment group (n =10 in each group).The model of cardiac arrest was induced by asphyxia.One hour after restoration of spontaneous circulation (ROSC),green fluorescent protein labeled BMSCs were transplanted via tail vein injection.At 3 and 7 days after transplantation,frozen sections of hippocampus was stained with hematoxylin-eosin (HE).The rest of brain tissue was weighed by an electronic balance.Brain water content (％) was calculated as (wet weight-dry weight) / wet weight × 100％.Results ①BMSCs were observed in hippocampus at 3 and 7 days after transplantation under fluorescent microscopy.②Compared with sham operation group and BMSCs treatment group,brain water content in cardiac arrest group was higher (all P ＜ 0.05).HE staining results showed that BMSCs transplantation could lessen hypoxia ischemia damage on brain.Conclusions BMSCs reduced the neurons damage induced by cardiac arrest and promoted neurological function recovery.

Objective To investigate the changes of diffusion tensor imaging (DTI) in the cerebral white matter of rats with ischemia-reperfusion injury.Methods Adult SD rats were randomly divided into two groups,sham operation group and model group.The model of ischemia reperfusion injury was made by the Koizumi suture method to occlude the middle cerebral artery.Application of Zea-Longa score was carried out to determine the establishment of modeling,and the modified neurological severity score (mNSS) was used to evaluate the neurological deficit of rats.The Rotarod test instrument was used to observe the motor function of rats by using Rotarod fatigue balance signs,and the DTI sequence scanning observation of brain white matter nerve fiber damage was determined by using Brook 7.0T small animal magnetic resonance imaging system.Track Vis software was used to analyze the distribution of cerebral white matter nerve fibers,and the relative number of nerve fibers in the areas of ROI (region of interest,ROI),sensorimotor areas and striatum were calculated.Results The results showed varying degrees of neurological impairment in rats 2 h after cerebral ischemia reperfusion injury,and the Zea-Longa score and the mNSS score were gradually reduced at the 1 d,7 d and 14 d after ischemia reperfusion injury.The time of rats retaining on the rotating rod was shortened at the 7 d and 14 d after ischemia reperfusion injury.At the ischemic lateral,nerve fibers decreased significantly,and the number of sensory nerve fiber connections in the sensorimotor areas to striatum was reduced.Nissl staining showed that the cytoplasm of neurons in the sensorimotor cortex and striatum of the ischemic lateral were disappeared and the Nissl bodies were decreased.Conclusions The nerve fibers of sensory motor cortex connecting to striatum were damaged by ischemia reperfusion injury.

Objective: Numerous studies have shown that bone marrow-derived mesenchymal stem cells (MSCs) enhance neurological recovery after cerebral ischemia. However, the mechanisms are still not clear. The present study aimed to investigate the beneficial effects of MSCs on global cerebral ischemia induced by cardiac arrest (CA) and the underlying mechanisms. Methods: Rats subjected to asphyxial CA were injected intravenously with MSCs (5×106 ) at 2 hours after resuscitation. Whole brain histopathologic damage scores (HDS) were assessed by histopathology at 3 and 7 days after resuscitation. The distribution of donor MSCs in the brain was evaluated. The expression of tumor necrosis factor-α-induced protein 6 (TSG-6) and pro-inflammatory cytokines in cerebral cortex was assayed. After intravenous infusion of TSG-6 siRNA-MSCs, HDS and pro-inflammatory cytokines were reevaluated at 7 days after resuscitation. Results: Intravenously administered MSCs significantly reduced whole brain HDS after global cerebral ischemia. Immunofluorescence microscopy revealed that donor MSCs were primarily found in cerebral cortex and expressed TSG-6. MSCs treatment significantly increased the expression of TSG-6 and reduced the expression of pro-inflammatory cytokines in cerebral cortex. In addition, intravenous infusion of TSG-6 siRNA-MSCs failed to attenuate brain inflammation. Conclusion: Systemically administered MSCs reduced inflammatory damage to brain in rats with global cerebral ischemia via secretion of TSG-6.
Heart Arrest ; Mesenchymal Stromal Cells

OBJECTIVE:To establish the quality standard of Flemingia philippinensis. METHODS:The properties and micro-scopic characteristics were observed;TLC was adopted for qualitative identification of genistin and genistein;the moisture,total ash and extract were detected;HPLC was adopted for contents determination of genistin and genistein:the column was Thermo BE-TASIL C18 with mobile phase of Acetonitrile-0.5% glacial acetic acid(gradient elution)at a flow rate of 1.0 ml/min,the detection wavelength was 261 nm,column temperature was 25℃,and the injection volume was 10μl. RESULTS:The properties and micro-scopic characteristics of F. philippinensis and F. macrophylla showed strong specificity. TLC spots of genistin and genistein we- re clear and well separated,with no interference in negative control. The moisture was 3.69%-8.37%,total ash was 1.72%-6.74%,and extract was 5.89%-19.65%. The linear range was 0.012 9-2.588 μg for genistin(r=0.999 9)and 0.004 6-0.923 2 μg for genistein (r=0.999 9);RSDs of precision,stability and reproducibility tests were lower than 3.0%;recoveries were 99.63%-101.87%(RSD=0.82%,n=6)and 97.19%-100.34%(RSD=1.23%,n=6). CONCLUSIONS:The method is simple,accurate and specif-ic,and can be used for the quality control of F. philippinensis.

Objective To explore the protective effect and possible mechanism of bone marrow mesenchymal stem cells (BMSCs) on pulmonary fibrosis induced by paraquat (PQ) in rats.Methods A total of 54 male SD rats were randomly (random number) divided into 3 groups (n =18 in each).Group A (normal control group),group B [PQ + phosphate buffer solution (PBS)],group C (PQ + BMSCs group).Rats in group B and C were induced to get pulmonary fibrosis by intragastric administration of PQ in a dosage of 120 mg/kg.In addition,BMSCs was given to rats in group C by injection in a dose of 1 × 106/mL via the vena caudalis,whereas an equal volume of PBS (1 mL) was given to rats in group B by injection instead.The survival rats in each groups were sacrificed separately at 7 days,14 days and 28 days after administration of PQ.The lower lobe of left lung were taken to observe histopathological changes with HE staining and Masson staining,and the pulmonary fibrosis was scored by using the Szapiel classification of alveolitis.At the same time,the lower lobe of right lung was harvested to detect the hydroxyproline (HYP),TGF-β1 and HGF in lung tissue by using immunohistochemistry.Results The pathological changes in lung tissue of rats were inflammatory change in alveoli space filled with massive amount of exudate obviously in group B at 7 days after exposure to PQ,but in group C,the inflammatory changes were much milder than those in group B.The exudation and edema in lunge alveoli were mitigated in group B at 14 days after exposure to PQ,but pulmonary interstitial fibers were increased.The degree of pulmonary interstitial fibrosis in group C was milder than that in group B.In group B,there were obvious pathological changes including destroyed alveoli septum,enlarged alveoli,thickened alveoli septum and the deposition of collagen fibers,disarranged alveolar structure at 28 days after exposure to PQ.The deposition off collagen fiber was slighter with well observed basic alveolar structure in group C than that in group B.The levels of HYP in lung tissue at different intervals in B group were escalating with time after exposure to PQ,and reached a peak at 28 day,which were significantly higher than those in group A (P ＜ 0.01).The levels of HYP in lung tissue at different intervals in C group were increasing with time after exposure to PQ,and reached a peak at 28 day,which were significantly lower than those in group B (P ＜ 0.01).The levels of TGF-β1 from different intervals in B group were increased greatly with wider distribution over large portion of lung structure than those in group A,and reached a peak at 7 days,then declined with time,which were significantly higher than those in group A (P ＜ 0.05).The levels of TGF-β1 at different intervals in group C were significantly lower than those in group B (P ＜ 0.05).The levels of HGF at different intervals in B group increased with time,but there was no significant difference among those at different intervals (P ＞0.05).The levels of HGF at different intervals in group B were little bit higher than those in group A,but there was no significant difference between them (P ＞ 0.05).The levels of HGF at different intervals in group C increased with time with significant differences among different intervals (P ＜ 0.05).The levels of HGF at 3 intervals in group C were significantly different from those of group A and group B group (P ＜0.05).Conclusions BMSCs can alleviate pulmonary fibrosis induced by PQ,and increase the survival rate,which may be attributed to decreasing the level of TGF-β1 and increasing the level of HGF.