Purpose:To study the effect of early restrictive fluid resuscitation (EFR) on inflammatory and immune factors in patients with severe pelvic fracture (SPF).Methods:A total of 174 SPF patients in the Department of Orthopaedics,the First Affiliated Hospital of Chengdu Medical College from July 2015 to June 2018 were involved in this study and divided into EFR group (n =87) and control group (n =87) using the random number table method.Conventional fluid resuscitation (CFR) was performed in control group,and EFR was performed in EFR group.The incidences of acute respiratory distress syndrome (ARDS) and multiple organ dysfunction syndrome (MODS) during rescue,successful rescue rate,blood transfusion volume,fluid input,and resuscitation time were compared between the two groups.The parameters including prothrombin time (PT),hematocrit (HCT),platelet (PLT) and blood lactate (BL) at the 4th hour after fluid resuscitation were recorded.The levels of inflammatory factors (TNF-α,IL-6,CRP) and immune factors (CD3+,CD4+,CD8+,CD4+/CD8+) were compared between the two groups before treatment and 7 days after treatment.The revised acute physiologic and chronic health evaluation system and the sequential organ failure assessment scores were adopted for evaluation before treatment and 7 days after treatment.Results:The incidences of ARDS and MODS during rescue in EFR group were significantly lower than those in control group (p=0.015 and 0.010 respectively),and the successful rescue rate in EFR group was significantly higher than that in control group (p =0.011).The blood transfusion volume,fluid input,resuscitation time in EFR group were significantly lower than those in control group (p =0.016,0.002 and 0.001 respectively).At the 4th hour after fluid resuscitation,PTand BL in EFR group were significantly lower than those in control group (p =0.021 and 0.003 respectively),while HCT and PLT in EFR group were significantly higher than those in control group (p =0.016 and 0.021 respectively).On day 7 after treatment,TNF-α,IL-6,CRP and CD8+ in EFR group were significantly lower than those in control group (p =0.003,0.004,0.007 and 0.003 respectively),while CD3+,CD4+ and CD4+/CD8+ in EFR group were significantly higher than those in control group (p =0.004,0.000,0.007 respectively).On day 7 after treatment,the revised acute physiologic and chronic health evaluation (APACHE) system and the sequential organ failure assessment (SOFA) scores in EFR group were significantly lower than those in control group.Conclusion:EFR can effectively eliminate inflammatory factors,improve immune function,maintain the stability of blood components,reduce the incidences of ARDS and MODS,and elevate the successful rescue rate in patients with SPF.

BACKGROUNDIsocitrate lyase (ICL) was previously demonstrated to play a pivotal role in the intracellular metabolism of Mycobacterium tuberculosis (MTB). Presently several lines of evidence suggest that ICL from MTB (MTB-ICL) may play some roles in the interaction between MTB and host macrophage. However, there has been no research on the interaction between MTB-ICL and host macrophage.

METHODSMTB-icl and M. smegmatis (MS)-icl genes were amplified by polymerase chain reaction (PCR) and cloned into the E. coli-mycobacterium shuttle plasmid pUV15 to obtain recombinant shuttle plasmids pMTB-icl and pMS-icl. Following transformation into MS by electroporation, the expression of pMTB-icl and pMS-icl was verified by reverse transcriptase (RT)-PCR. The expression of recombinant plasmids derived from pUV15 when rMS was phagocytized by macrophage was also verified via fluorescence microscope. Ms 1 - 2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were used to infect RAW264.7 cells and the survival of intracellular MS was monitored by bacterial culture at 0, 24 and 48 hours after infection. The culture supernatants from macrophage infected by Ms 1 - 2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were collected and the interferon (IFN)-gamma and nitric oxide (NO) concentrations were measured by ELISA or by Griess assay, respectively. The apoptosis of macrophage was assayed by the in situ TUNEL technique.

RESULTSRT-PCR showed that both pMTB-icl and pMS-icl could be expressed in MS. Fluorescence microscopic observation showed that recombinant plasmids derived from pUV15 (pUV15-IG) could also be expressed in MS when MS were phagocytized by macrophage. Bacterial culture data demonstrated that rMS-pMTB-icl exhibited significantly increased intracellular survival in the murine macrophage cell line RAW264.7 compared with Ms 1 - 2c, rMS-pUV15 and rMS-pMS-icl. This increased intracellular survival was not accompanied by the upregulation of IFN-gamma and NO in host macrophage. But a lower apoptosis rate of macrophages infected with rMS-pMTB-icl was observed when compared with macrophages infected with other strains of MS.

CONCLUSIONSMTB-ICL could promote the intracellular survival of MS. Suppressing the apoptosis of host macrophage may be one of the important mechanisms involved in this increased intracellular survival.

Animals ; Apoptosis ; genetics ; physiology ; Cell Line ; In Situ Nick-End Labeling ; Interferon-gamma ; metabolism ; Isocitrate Lyase ; genetics ; metabolism ; Macrophages ; cytology ; metabolism ; microbiology ; Microbial Viability ; Microscopy, Fluorescence ; Mycobacterium smegmatis ; enzymology ; genetics ; growth & development ; Mycobacterium tuberculosis ; enzymology ; genetics ; Nitric Oxide ; metabolism ; Plasmids ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transformation, Genetic

BACKGROUNDCartilage injury has a very poor capacity for intrinsic regeneration. The cell-based treatment strategy for the cartilage repair using differentiated bone marrow mesenchymal stem cells (BMSCs) is, however, a promising approach to the chondral repair. This study was aimed to explore the chondrogenic potential of the goat BMSCs in the Transwell co-culture system and the poly-laetide-co-glycolide (PLGA) scaffolds.

METHODSThe BMSCs were isolated from the goat iliac crest while the chondrocytes were obtained from the goat's last costal cartilage. In the Transwell co-culture system, the BMSCs co-cultured with chondrocytes were designed as group A, whereas the goat's BMSCs induced with the chondrogenic medium were group B. Both groups A and B were the experimental groups, while group C that only contained BMSCs was the control group. In the PLGA scaffolds co-culture system, BMSCs were seeded into the PLGA scaffolds, which were suspended in the 24-well plate, and the control group was established by presence or absence of chondrocytes at the bottom of the 24-well plate. Toluidine blue staining, Alcian blue staining, collagen II immunofluoresence, collagen II immunochemical staining, collagen I, collagen II, COL2a Q-PCR and osteopontin Q-PCR were used to examine the chondrogenic conditions as well as the expressions of chondrogenic and osteogenic genes.

RESULTSCells isolated from the aspirates of the goat bone marrow proliferated rapidly and gained characteristics of stem cells in Passage 4. However, the differentiations of chondrocytes were not apparent in Passage 3. The results from Toluidine blue staining, collagen II immunofluoresence and PCR showed the transformation of BMSCs to chondrocytes in the Transwell co-culture system and PLGA scaffolds. Although the cartilage gene expressions were upgraded in both chondrogenesis group and co-culture system, the osteopontin gene expression, which represents osteogenic level, was also up-regulated.

CONCLUSIONSThe Transwell co-culture system and the PLGA scaffolds co-culture system can promote the chondrogenic differentiation of the goat's BMSCs, while up-regulated osteopontin gene expression in the Transwell co-culture system implies the osteogenic potential of BMSCs.

Animals ; Bone Marrow Cells ; physiology ; Cell Culture Techniques ; methods ; Chondrocytes ; physiology ; Chondrogenesis ; physiology ; Coculture Techniques ; Goats ; Mesenchymal Stromal Cells ; physiology ; Tissue Engineering ; methods ; Tissue Scaffolds

Background Cartilage injury has a very poor capacity for intrinsic regeneration. The cell-based treatment strategy for the cartilage repair using differentiated bone marrow mesenchymal stem cells (BMSCs) is, however, a promising approach to the chondral repair. This study was aimed to explore the chondrogenic potential of the goat BMSCs in the Transwell co-culture system and the poly-laetide-co-glycolide (PLGA) scaffolds.Methods The BMSCs were isolated from the goat iliac crest while the chondrocytes were obtained from the goat's last costal cartilage. In the Transwell co-culture system, the BMSCs co-cultured with chondrocytes were designed as group A,whereas the goat's BMSCs induced with the chondrogenic medium were group B. Both groups A and B were the experimental groups, while group C that only contained BMSCs was the control group. In the PLGA scaffolds co-culture system, BMSCs were seeded into the PLGA scaffolds, which were suspended in the 24-well plate, and the control group was established by presence or absence of chondrocytes at the bottom of the 24-well plate. Toluidine blue staining,Alcian blue staining, collagen Ⅱ immunofluoresence, collagen Ⅱ immunochemical staining, collagen Ⅰ, collagen Ⅱ, COL2a Q-PCR and osteopontin Q-PCR were used to examine the chondrogenic conditions as well as the expressions of chondrogenic and osteogenic genes.Results Cells isolated from the aspirates of the goat bone marrow proliferated rapidly and gained characteristics of stem cells in Passage 4. However, the differentiations of chondrocytes were not apparent in Passage 3. The results from Toluidine blue staining, collagen Ⅱ immunofluoresence and PCR showed the transformation of BMSCs to chondrocytes in the Transwell co-culture system and PLGA scaffolds. Although the cartilage gene expressions were upgraded in both chondrogenesis group and co-culture system, the osteopontin gene expression, which represents osteogenic level, was also up-regulated.Conclusions The Transwell co-culture system and the PLGA scaffolds co-culture system can promote the chondrogenic differentiation of the goat's BMSCs, while up-regulated osteopontin gene expression in the Transwell co-culture system implies the osteogenic potential of BMSCs.

AIM:To investigate the interaction of Ca 2+-sensing proteins , stromal interaction molecule 1 (STIM1) and calcium release-activated calcium channel protein 1 (Orai1), in Ca2+-sensing receptor (CaSR)-mediated extracellular Ca2+ influx and production of nitric oxide ( NO).METHODS: Human umbilical vein endothlial cells (HUVECs) were incubated with CaSR agonist spermine [activating store-operated calcium channels (SOC) and receptor-operated calcium channels ( ROC) ] alone or combined with CaSR negative allosteric modulator Calhex 231+ROC analogue TPA (activating ROC, blocking SOC), protein kinase C (PKC) inhibitor Ro 31-8220, or PKCα/β1 selective inhibitor Go 6976 (activate SOC, blocking ROC).The protein expression of STIM1 and Orai1 was determined by the method of immu-nofluorescence .The interaction between STIM 1 and Orai1 was examined by co-immunoprecipitation .The second to third passages of HUVECs were divided into STIM 1 and Orai1 short hairpin RNA group ( shSTIM1+shOrai1 group ) , vehicle-STIM1+vehicle-Orai1 group and control group , and then incubated with the 4 different treatments above .The intracellular Ca2+concentration ( [ Ca2+] I ) was detected using the fluorescent Ca 2+indicator Fura-2/AM.The production of NO was also determined by DAF-FM DA fluorescent probe .RESULTS:The protein expression of STIM 1 and Orai1 was located in the cytoplasm.Compared with control group , the localization of STIM1 and Orai1 in the cytoplasm was reduced after the HUVECs were incubated with Calhex 231+TPA, Ro 31-8220 or Go 6976, and the interaction of STIM1 and Orai1 was de-creased significantly .The [ Ca2+] I and the net NO fluorescence intensity in shSTIM 1+shOrai1 group were significantly re-duced after the 4 different treatments (P<0.05).CONCLUSION:STIM1 and Orai1 are components of SOC and ROC in store-and receptor-operated Ca 2+entry and NO generation .

Purpose:: To study the effect of early restrictive fluid resuscitation (EFR) on inflammatory and immune factors in patients with severe pelvic fracture (SPF ). Methods:: A total of 174 SPF patients in the Department of Orthopaedics, the First Affiliated Hospital of Chengdu Medical College from July 2015 to June 2018 were involved in this study and divided into EFR group (n = 87) and control group (n = 87) using the random number table method. Conventional fluid resuscitation (CFR) was performed in control group, and EFR was performed in EFR group. The incidences of acute respiratory distress syndrome (ARDS) and multiple organ dysfunction syndrome (MODS) during rescue, successful rescue rate, blood transfusion volume, fluid input, and resuscitation time were compared between the two groups. The parameters including prothrombin time (PT), hematocrit (HCT), platelet (PLT) and blood lactate (BL) at the 4th hour after fluid resuscitation were recorded. The levels of inflammatory factors (TNF-α, IL-6, CRP) and immune factors (CD3^＋, CD4^＋, CD8^＋, CD4＋/CD8＋) were compared between the two groups before treatment and 7 days after treatment. The revised acute physiologic and chronic health evaluation system and the sequential organ failure assessment scores were adopted for evaluation before treatment and 7 days after treatment. Results:: The incidences of ARDS and MODS during rescue in EFR group were significantly lower than those in control group (p=0.015 and 0.010 respectively), and the successful rescue rate in EFR group was significantly higher than that in control group (p = 0.011). The blood transfusion volume, fluid input, resuscitation time in EFR group were significantly lower than those in control group (p = 0.016, 0.002 and 0.001 respectively). At the 4th hour after fluid resuscitation, PT and BL in EFR group were significantly lower than those in control group (p = 0.021 and 0.003 respectively), while HCT and PLT in EFR group were significantly higher than those in control group (p = 0.016 and 0.021 respectively). On day 7 after treatment, TNF-α, IL-6, CRP and CD8^＋ in EFR group were significantly lower than those in control group (p = 0.003, 0.004, 0.007 and 0.003 respectively), while CD3^＋, CD4^＋ and CD4＋/CD8＋in EFR group were significantly higher than those in control group (p = 0.004, 0.000, 0.007 respectively). On day 7 after treatment, the revised acute physiologic and chronic health evaluation (APACHE) system and the sequential organ failure assessment (SOFA) scores in EFR group were significantly lower than those in control group. Conclusion: EFR can effectively eliminate inflammatory factors, improve immune function, maintain the stability of blood components, reduce the incidences of ARDS and MODS, and elevate the successful rescue rate in patients with SPF.

The present study was to investigate the role of the interaction between canonical transient receptor potential channel 1 (TRPC1) and calcium release-activated calcium modulator 1 (Orai1) in extracellular Ca-sensing receptor (CaR)-induced extracellular Ca influx and nitric oxide (NO) production. Human umbilical vein endothelial cells (HUVECs) were incubated with CaR agonist Spermine [activating store-operated calcium channels (SOC) and receptor-operated calcium channels (ROC)] alone or in combination with the following reagents: CaR negative allosteric modulator Calhex231 plus ROC analogue TPA (activating ROC and blocking SOC), Ro31-8220 (PKC inhibitor that activates SOC and blocks ROC) or Go6967 (PKCs and PKCµ inhibitor that activates SOC and blocks ROC). The protein expressions and co-localization of TRPC1 and Orai1 were determined using immunofluorescent staining. The interaction between TRPC1 and Orai1 was examined by co-immunoprecipitation. We silenced the expressions of their genes in the HUVECs by transfection of constructed TRPC1 and Orai1 shRNA plasmids. Intracellular Ca concentration ([Ca]) was detected using Ca indicator Fura-2/AM, and NO production was determined by DAF-FM staining. The results showed that TRPC1 and Orai1 protein expressions were co-located on the cell membrane of the HUVECs. Compared with Spermine+Ca group, Calhex231+ TPA+Spermine+Ca, Ro31-8220+Spermine+Ca and Go6976+Spermine+Ca groups exhibited down-regulated protein expressions of TRPC1 and Orai1 in cytoplasm and decreased co-localization on the cell membrane. Co-immunoprecipitation results showed that the interaction between TRPC1 and Orai1 was reduced by Calhex231 plus TPA, Ro31-8220 or Go6976 addition in the Spermine-stimulated HUVECs. Double knockdown of Trpc1 and Orai1 genes significantly decreased [Ca] level and NO production in all of the Spermine+Ca, Calhex231+TPA+Spermine+Ca, Ro31-8220+Spermine+Ca and Go6976+Spermine+Ca groups. These results suggest that TRPC1/Orai1 may form a complex that mediates Ca influx and No production via SOC and ROC activation.