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AIM: To investigate the relationship between vascular smooth muscle cell (VSMC) injury, organelle stress response and autophagic cell death (autophagy) and ferroptosis induced by the chemical hypoxia inducer cobalt chloride (CoCl2) through the bioinformatics analysis and in vitro cell experimentation. METHODS: The dataset GSE119226 of VSMC treated with cobalt chloride was acquired from the gene expression database (GEO). The R language was used to investigate the relationship between CoCl2 treatment and organelle stress response (Golgi stress, endoplasmic reticulum stress) and two forms of cell death (ferroptosis and autophagic cell death). With primary cultured rat VSMC (rVSMC) and CoCl2-induced anoxia model, the changes in cell viability were detected by CCK-8 method, and reactive oxygen species (ROS) levels were measured using DCFH-DA method. The expression levels of HIF-1α (a key molecule in hypoxia), Golgi stress markers GM130 and p115, endoplasmic reticulum stress markers GRP78 and CHOP, autophagy markers LC3-II / LC3-I and Beclin1, and ferroptosis markers GPx4 and xCT were detected by Western blot. The effect of inducing or inhibiting organelle stress and cell death on the CoCl2-induced cell damage was also observed. RESULTS: Differentially expressed genes analysis of GSE119226 dataset showed that CoCl2 treatment of VSMCs had significant effects on organelle function and stress response, autophagy and ferroptosis-related genes, in which endoplasmic reticulum stress, protein processing in endoplasmic reticulum, regulation of Golgi to plasma membrane protein transport, autophagy / autophagic cell death, and ferroptosis pathways were remarkably enriched. The results of in vitro experiment showed that compared with normal rVSMC, cell viability was significantly decreased after CoCl2 treatment, as well as HIF-1α protein expression and ROS levels in rVSMCs were increased. In rVSMC treated with Co-Cl2, the expression levels of Golgi structural proteins GM130 and p115 (reflecting the occurrence of Golgi stress) were decreased, while the markers GRP78 and CHOP (reflecting the occurrence of endoplasmic reticulum stress) were increased. At the same time, CoCl2 treatment also reduced the expression of autophagy markers LC3-II/LC3-I and Beclin1 (indicating the decrease levels of autophagy), while the expression of ferroptosis markers GPx4 and xCT were decreased (indicating the occurrence of ferroptosis). Compared with CoCl2 treatment group, induced Golgi stress, endoplasmic reticulum stress, or ferroptosis could further reduce cell viability, while inhibition of these processes could improve cell viability. On the other hand, increasing the level of autophagy can improve the cell viability. CONCLUSION: Hypoxia induced by cobalt chloride can lead to VSMC injury. Golgi stress, endoplasmic reticulum stress, ferroptosis, and the reduction of autophagy level play an important role in it. Inhibition of organelle stress response and ferroptosis, or increase of autophagy level can improve VSMC injury caused by cobalt chloride.
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Objective To investigate the early resuscitation effect of hemoglobin-based oxygen carriers (HBOC) in rats with uncontrolled hemorrhagic shock.Methods 170 Sprague-Dawley (SD) rats were randomly divided into five groups:lactate Ringer solution (LR) control group,whole blood control group,and 0.5％,2.0％,5.0％ HBOC groups,with 34 rats in each group.The uncontrolled hemorrhagic shock model in SD rats was reproduced by cutting off the splenic artery branch,and induced mean arterial pressure (MAP) reducing to 40 mmHg (1 mmHg =0.133 kPa).The corresponding solution was infused after model reproduction in each group,maintaining MAP at 50 mmHg for 1 hour,then completely ligating and hemostasis,and maintaining MAP at 70 mmHg for 1 hour and 80 mmHg for 1 hour respectively,after maintaining MAP 80 mmHg,all were supplemented with LR to 2 times blood loss volume.The survival rate and blood loss rate were observed in 16 rats in each group,hemodynamics parameters including MAP,left ventricular systolic pressure (LVSP) and the maximum rate of left ventricular pressure rise (+dp/dtmax) were determined in another 10 rats,and cardiac output (CO) and tissue oxygen supply (DO2) were observed in the rest 8 rats.Results ① When resuscitation by LR alone,the blood loss rate of animals was as high as 60％ to 70％.Compared with the LR control group,whole blood recovery could significantly reduce the blood loss rate before hemostasis in uncontrolled hemorrhagic shock rats [(46.6 ± 4.5)％ vs.(62.3 ± 4.0)％,P ＜ 0.01];0.5％,2.0％,5.0％ HBOC could significantly decrease the blood loss rate,especially in 5.0％ HBOC group with significant difference as compared with that in the LR control group [(45.6±4.1)％ vs.(62.3±4.0)％,P ＜ 0.01].② When LR was used alone for resuscitation,the rats died quickly and survived for a short time.Only one rat survived for 12 hours,and no rat survived for more than 24 hours.Compared with the LR control group,whole blood resuscitation could improve the survival rate of uncontrolled hemorrhagic shock rats,and the survival time was significantly prolonged (hours:20.4± 4.6 vs.3.5 ± 1.1,P ＜ 0.01);0.5％,2.0％ and 5.0％ HBOC also significantly prolonged the survival time of rats.The 5.0％ HBOC group had the best effect,4 rats survived in 24 hours,and the survival time was significantly longer than that of the LR control group (hours:18.4 ± 4.0 vs.3.5 ± 1.1,P ＜ 0.01),and it was the same as the whole blood control group.③ Compared with pre-shock,CO,DO2 and hemodynamic parameters of uncontrolled hemorrhagic shock rats were significantly decreased,and the above parameters were gradually increased with the prolongation of rehydration time.Compared with the LR control group,whole blood resuscitation could significantly increase CO and DO2,and improve hemodynamics in rats with uncontrolled hemorrhagic shock at different time points.Three concentrations of HBOC could also increase CO,DO2 and other hemodynamic parameters of rats at 1 hour of maintaining MAP of 80 mmHg after hemostasis and 1 hour and 2 hours after resuscitation.The effect of 5.0％ HBOC group was more significant than that of the LR control group with statistically significant difference [CO (× 10-3,L/min):72.84±2.84 vs.63.11±2.38 at 1 hour of maintaining MAP of 80 mmHg,70.25±4.55 vs.59.88 ± 9.31 at 1 hour after resuscitation,71.51 ± 2.90 vs.53.24 ± 6.32 at 2 hours after resuscitation;DO2 (L· min-1 · m-2):271.9± 13.5 vs.159.1 ±25.4 at 1 hour of maintaining MAP of 80 mmHg,261.0± 15.0 vs.145.7±20.1 at 1 hour after resuscitation,249.6± 12.0 vs.107.4± 18.2 at 2 hours after resuscitation;MAP (mmHg):82.1±2.1 vs.74.0±2.8 at 1 hour of maintaining MAP of 80 mmHg,107.5±9.3 vs.64.0±5.7 at 1 hour after resuscitation,104.0±9.7 vs.73.0±4.2 at 2 hours after resuscitation;LVSP (mmHg):128.6±7.9 vs.103.8±0.8 at 1 hour of maintaining MAP of 80 mmHg,129.3±± 15.0 vs.99.4±0.0 at 1 hour after resuscitation,127.5± 11.3 vs.97.4±0.0 at 2 hours after resuscitation;+dp/dt max (mmHg/s):6 534.2±± 787.6 vs.5 074.0± 71.7 at 1 hour of maintaining MAP of 80 mmHg,5 961.5 ±± 545.4 vs.4 934.5 ± 510.2 at 1 hour after resuscitation,5 897.4± 350.5 vs.4 534.7 ±489.2 at 2 hours after resuscitation,all P ＜ 0.05].Conclusions HBOC infusion prolonged the survival time,increased survival rate,and improved hemodynamics,cardiac function and tissue oxygen supply in a dose-dependent manner in the early stage of uncontrolled hemorrhagic shock.The recovery effect of 5.0％ HBOC was similar to that of the whole blood.
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Objective To observe the protective effects of calcium-sensing receptor (CaSR) inhibitor Calhex231 on traumatic hemorrhagic shock rats. Methods 144 SD rats were divided into six groups by random number table method: normal group, shock group, lactated Ringer's solution (LR) group, LR + Calhex231 0.1 mg/kg group, LR + Calhex231 1 mg/kg group, and LR + Calhex231 5 mg/kg group, with 16 rats in each group for survival observation and 8 rats for hemodynamics test. 64 SD rats were divided into four groups: normal group, shock group, lactated Ringer's solution (LR) group, LR + Calhex231 1 mg/kg group, with 8 rats in each group for detecting organ blood flow and superior mesenteric artery vascular reactivity and the other 8 rats for mesenteric artery vascular reactivity. After the establishment of traumatic hemorrhagic shock model, the shock group did not receive resuscitation, and the LR group was resuscitated with LR equal to two times of the blood loss volume. The three LR + Calhex231 groups with different dosages were firstly given LR of equal volume to that of blood loss, and then the Calhex231 was dissolved into LR (equal to the blood loss volume) to resuscitate. The wound was ligated and sutured immediately after resuscitation. The effect of Calhex231 on animal's 24-hour survival since the beginning of resuscitation was observed. The hemodynamics including the mean arterial blood pressure (MAP), left intraventricular systolic pressure (LVSP), maximal rising, and declining rate of left intraventricular pressure (±dp/dtmax) were observed before shock, at the end of shock, 1 hour after resuscitation, and 2 hours after resuscitation. The effects of Calhex231 on vital organ blood flow and vascular reactivity were observed 2 hours after resuscitation. Results All the shock rats died within 9 hours after the shock model was established. The survival outcomes of LR group rats were slightly improved compared with the shock group rats(P ＜0.05). The survival time and 24 hour survival rate of LR + Calhex231 1 mg/kg group and LR + Calhex231 5 mg/kg group rats were significantly increased compared with the shock group rats (P ＜0.05). The hemodynamic indexes of LR + Calhex231 groups were higher than those of the LR group. The best effect was observed in LR + Calhex231 1 mg/kg group rats (P ＜ 0.01). The MAP, LVSP and ± dp/dtmax were restored to normal level (64.9％, 82.4％, 89.8％, and 77.8％, respectively). Meanwhile, the blood flow in liver and kidney of LR + Calhex231 1 mg/kg group rats were increased from 57.2％ and 41％ to 108.7％ and 95.1％, respectively. The vascular reactivity including superior mesenteric artery and mesenteric artery of LR + Calhex231 1 mg/kg group rats were also increased (P ＜0.01). Conclusions In rats with hemorrhagic shock, the calcium sensitive receptor inhibitor Calhex231 can improve the vascular reactivity, the hemodynamics, and the blood flow of important organs. It plays a role in protecting the cardiovascular function and reducing the mortality after traumatic hemorrhagic shock.
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Objective To investigate the effects of calcium sensing receptor (CaSR)on vasorelaxation/vasoconstriction of superior mes-enteric artery (SMA)in rats and its relationship to endothelium.Methods With endothelium-intact and endothelium-denuded SMA rings of rats,the effects of CaSR-specific allosteric modulator cinacalcet on the SMA rings pre-contracted with norepinephrine (NE),and vascular contractile response /relaxation reactivity were observed.Results Cinacalcet had no effects on resting tension of SMA rings with or without endothelium.Cinacalcet caused a significant relaxation in the endothelium-intact SMA rings pre-contraction with NE in a dose-dependent manner.Endothelium denudation abolished cinacalcet-induced vasorelaxation.Pretreatment with cinacalcet for 30 minutes decreased the con-tractile response of endothelium-intact SMA rings to NE,but had no significant influence on relaxation reactivity.In the endothelium-denuded SMA rings,cinacalcet did not affect both vasoconstriction and vasorelaxation.Conclusion CaSR plays an important role in the regulation of the vascular reactivity,and this effect is endothelium-dependent.
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Objective To investigate platelet-derived growth factor ( PDGF ) protection on blood flow and mitochondrial function of hemorrhagic shock rats. Methods Ninety-six SD rats were randomly divided into six groups including shock group, lactated ringer's solution (LR) resuscitation group,PDGF treatment groups(1,3. 5,7,15μg/kg). Laster-Doppler and oxygen concentration determination method were applied to observe the protective effect of PDGF treatment on animal survival,blood flow and mitochondrial function in liver and kidney. Re-sults As compared with LR resuscitation group,PDGF treatment increased animal survival rate and also improved blood fiow of liver and kindy,mitochondrial respiration control ration(RCR),of which the group with 3. 5μg/kg had the best result. Conclusion This finding sug-gests that PDGF may be a potential agent to treat acute critical such as hemorrhagic shock.
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Objective To investigate the beneficial effects of cyclosporin A ( CsA) on traumatic hemorrhagic shock in rats. Methods The traumatic hemorrhagic shock model was adopted in 144 SD rats which were divided into 6 groups: sham-operated group,shock control group,lactated Ringer's solution ( LR) group,CsA 1 mg/kg,5 mg/kg and 10 mg/kg group. The effects of three doses of CsA on the animal survival time and 24 h survival rate were observed,and the effects of CsA on hemodynamic parameters,including mean arterial blood pressure ( MAP) ,left intraventricular systolic pressure ( LVSP) ,left ventricular end-diastolic pressure ( LVEDP) ,maximal change rate of left intraven-tricular pressure ( ± dp/dtmax ) and heart rate ( HR) were also observed. Results CsA at the concentration of 5 mg/kg and 10 mg/kg can significantly increase the survival time and 24 h survival rate of shock rats,the survival rate was increased to 56. 3% from 25% of LR group. After shock,the hemodynamic parameters were significantly decreased including MAP,LVSP and ± dp/dtmax ,LR infusion only improved the hemodynamics to some extent,which were significantly lower than those in sham-operated group. CsA (5 mg/kg and 10 mg/kg) can signifi-cantly improve the hemodynamics of shock rats including LVSP and ± dp/dtmax ,which were increased at 2 h after resuscitation as compared to LR group,and return to about normal levels. 1 mg/kg of CsA also restored the hemodynamic parameters, but there were no significant differences between CsA 1 mg/kg group and LR group. Conclusion CsA has good beneficial effect on traumatic hemorrhagic shock,and 5 mg/kg and 10 mg/kg of CsA showed a better effect.
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Objective To investigate the effects of arginine vasopressin (AVP) combined with norepinephrine (NE) in treatment of uncontrolled hemorrhagic shock (UHS) in rats.Methods UHS models were produced in rats and divided into three groups according to the random number table,which were resuscitated with LR equivalent to 1/2 (17.5 ml/kg) of shed blood,LR equivalent to 1/4 (8.75 mL/kg) of shed blood and without LR respectively.Each group was subdivided into six groups:AVP1 (0.04 U/kg) group,AVP2 (0.4 U/kg) group,NE (3 μg/kg) group,AVP1 + NE group,AVP2 + NE group and LR control group,with 10 rats per group.Effects of single AVP or NE infusion or combined infusion respectively grouped with different doses of LR on survival time and hemodynamics of UHS rats were observed.Results Compared with AVP,NE and AVP + NE groups without LR or with LR equivalent to 1/2 of shed blood respectively,AVP2 + NE group with LR equivalent to 1/4 of shed blood provided better main artery pressure (MAP),prolonged survival time and enhanced 4-hour survival rate in treatment of UHS rats.Moreover,survival time and 24-hour survival rate were increased significantly and hemodynamic parameters like MAP,left intraventricular systolic pressure (LVSP) and maximal change rate of left intraventricular pressure (± dp/dt max) were improved after hemostasis.Conclusion AVP (0.04 U/kg) + NE (3 μg/kg) infusion with LR equivalent to 1/4 of shed blood prior to hematosis can win the time for definitive treatment and improve the treatment outcome.