BACKGROUND: Runx2 plays a central role in osteogenic differentiation, which is of important significance in new bone formation.OBJECTIVE: To observe the effect of Runx2 over-expression on osteogenic differentiation of human umbilical cordblood mesenchymal stem cells.METHODS: Runx2 was generated by RT-PCR and the recombinant adenovirus (pAd-Runx2) was constructed. The viraltiter was determined by air dilution method. After being transfected into human umbilical cord blood mesenchymal stemcells, the green fluorescence was observed under fluorescence microscope. Real-time fluorescent quantitative PCR andwestern blot were used to detect mRNA expression changes of osteogenesis related genes, Runx2, OCN, BMP-2, ALP,in human umbilical cord blood mesenchymal stem cells at 1, 3, 7 and 14 days after transfection.RESULTS AND CONCLUSION: The recombinant adenovirus was successfully constructed and the titer was1.7×1010 pfu/L. After infection by pAd-Runx2, human umbilical cord blood mesenchymal stem cells expressed greenfluorescence protein clearly under the fluorescence microscope. Cells in the transfected group differentiated intoosteoblast-like cells, and those in the control group stayed the same as pre-infected. The expression of Runx2, OCN,BMP-2 and ALP in the transfected group increased over time to some extent, but these changes were not detected in thecontrol group. These findings indicate that Runx2 over-expression can promote the osteogenic differentiation of humanumbilical cord blood mesenchymal stem cells.

ObjectiveTo investigate the effect of anisodamine on the expression of connexin 43 (Cx43) in swine ventricular myocardium after resuscitation from cardiac arrest.Methods The experiment was conducted on healthy pigs, and they were randomly divided into three groups, namely sham group, epinephrine group (control group) and anisodamine group (experimental group, animals were resuscitated combined with injection of 0.4 mg/kg of anisodamine), with 5 pigs in each group. Model of ventricular fibrillation was reproduced by alternating current challenge, and cardiopulmonary resuscitation (CPR) was performed 8 minutes after cardiac arrest. Left ventricular myocardium was harvested at 24 hours after restoration of spontaneous circulation. The expression and distribution of Cx43 were observed by immunofluorescence, Cx43 mRNA expression was assessed with reverse transcription-polymerase chain reaction (RT-PCR), and the protein expressions of Cx43 and phosphorylation of Cx43 (p-Cx43) were analyzed by Western Blot.Results The positive expression of Cx43 in ventricular muscle was distributed uniformly, mostly at the end-to-end linkage of myocardial cells, with a few side-to-side linkage in sham group. The positive expression of Cx43 in control group was significantly weaker than that in the sham group, and the signal intensity was significantly declined (4.35±2.10 vs. 10.02±3.66,P< 0.01). The positive expression of Cx43 at the end-to-end linkage and side-to-side linkage was irregular in experimental group, and the signal intensity was obviously higher than that in the control group (7.91±2.54 vs. 4.35±2.10,P< 0.05), but it was significantly weaker than that in the sham group (7.91±2.54 vs. 10.02±3.66,P< 0.05). For control group and experimental group, the Cx43 mRNA and protein expressions were significantly lower than those of the sham group [Cx43 mRNA (A value): 0.32±0.05, 0.32±0.03 vs. 0.48±0.07; Cx43 protein (A value): 0.43±0.03, 0.50±0.07 vs. 0.65±0.04, allP< 0.01], and there were no significant differences between experimental group and control group (allP> 0.05). The p-Cx43 protein expression of control group was significantly lower than that of the sham group (A value: 0.22±0.03 vs. 0.37±0.06,P< 0.01), and it was significantly higher in the experiment group than that in the control group (A value: 0.29±0.07 vs. 0.22±0.03, P< 0.01), but there was no significant difference with the sham group (P> 0.05). No significant difference in p-Cx43/Cx43 was found among sham, control, and experiment groups (0.57±0.09, 0.51±0.05, 0.58±0.06, all P> 0.05).ConclusionAnisodamine can improve the abnormal expression of Cx43 in ventricular muscle of pigs with cardiac arrest, which may be related to the protection effect of anisodamine on cardiac conduction.

Non-coding RNAs ( ncRNA ), including ribosomal RNA( rRNA), transfer RNA( tRNA), MicroRNA ( miRNA), long noncoding RNA(lncRNA) and small nucleolar RNA(snoR-NA), are a class of RNA that have multiple functions and are not translated to proteins. MicroRNA and lncRNA are involved in the injury, remodeling and aging of blood vessels, and it is necessary to understand the regulatory roles of MicroRNA and lncRNA in these processes. It is reported that MicroRNA and lncRNA are not only participated in the regulation of oxidative response, inflammation, cell proliferation and migration, and phenotype transition, they are also involved in the regulation of gene expression by conducting different mechanisms, including transcriptional regulation, post-transcriptional modification and chromatin remodeling. These aspects of regulation by MicroRNA and lncRNA are related to cardiovascular diseases, such as ath-erosclerosis, hypertension, myocardial infarction, stroke, pul-monary hypertension and diabetes, and thus provide a new way for genetic diagnosis and therapy of cardiovascular diseases.

This study was aimed to determine effect of Bu-Shen Kang-Shuai (BSKS) Tablet on HO-1 mRNA and its associated oxidative stress levels among atherosclerotic rabbits. A total of 56 rabbits were randomly divided into the normal group (8 rabbits) and the experimental group (48 rabbits). Normal diet was given to the normal group. Atherosclerotic rabbits models were established in the experimental group. At the eighth week, rabblits in the experi-mental group were randomly divided into the model group, BSKS Tablet group and simvastatin group. Blood samples were collected before medication, 8-, 12-, 16-week after medication from rabbits of each group. Rabbits were sacri-ficed under aseptic conditions at the last blood collection. Expressions of aortic HO-1 mRNA and PPARα mRNA were measured by Q-PCR method. The level of MMP-9 was measured by immunohistochemical assay. Serum HbCO, COX-2 activity and cGMP level were measured by ELISA assay. The results showed that after the intervention of BSKS Tablet, serum HbCO level decreasd, cGMP was obviously increased. However, there was no obvious change on the COX-2 activity. The immunohistochemical assay showed that BSKS Tablet obviously reduced MMP-9 level of rabbits. There was only small amount of aortic HO-1 mRNA expression in the normal group. However, the expres-sion of aortic HO-1 mRNA in the atherosclerosis group was increased. After intervention of BSKS Tablet, the ex-pression of HO-1 mRNA was increased with statistical significance (P < 0.05). Simvastatin had similar antioxidant effect. It was concluded that the compound preparation of traditional Chinese medicine (TCM) BSKS Tablets had an important antioxidant effect in treatment of atherosclerosis. Its protective mechanism may be through the regulation of HO-1 mRNA gene expression and effects of HO-1/CO-cGMP pathway activities of related enzymes while peroxida-tion stability of atherosclerotic plaque.

Objective:To recognize the characteristics of necrotizing fasciitis patients complicated with sepsis and summarize the experience the treatment.Methods:A retrospective study was conducted. The clinical data of 57 patients with necrotizing fasciitis complicated with sepsis admitted to Guangdong Provincial People's Hospital from July 2009 to December 2019 was analyzed by collecting such factors as gender, age, complications, infection sites, pathogens, surgery information, treatment options and outcome. The patients were divided into debridement group ( n = 14) and control group ( n = 43) according to whether the debridement was completed within 48 hours of admission, and the mortality during hospitalization between the two groups was compared. A telephone follow-up had been done to record the long-term outcome of these patients. Results:Among 57 patients with necrotizing fasciitis complicated with sepsis, there were 43 males and 14 females with the average age of (57.9±12.1) years old. Most of the underlying diseases were diabetes mellitus (70.17%), other diseases included hypertension (8.77%), tumor chemotherapy (7.02%), liver disease (hepatitis, cirrhosis, 7.02%), coronary artery heart disease (3.51%), systemic lupus erythematosus (3.51%), etc. Most of the infection site was lower limbs (71.93%). There were 78 pathogens cultured in 57 patients, in which 52 were non-drug resistant bacteria (66.67%), and 26 were drug resistant bacteria (33.33%). There were 40 Gram positive (G +) bacteria (51.28%), 29 Gram negative (G -) bacteria (37.18%), 8 fungi (10.26%) and 1 mixed bacteria (1.28%). Finally, of 57 patients, 46 patients were cured, and 11 patients died with hospital mortality of 19.30%. Among 57 patients, the hospital mortality in the debridement group was significantly lower than that in the control group [0% (0/14) vs. 25.58% (11/43), P < 0.05]. Among the 46 cured patients, 11 had accepted amputations, accounting for 23.91%. In December 2020, 43 patients who were cured (3 patients were lost to follow-up) were followed up by telephone. Twenty-three patients were completely self-care, 9 patients were partly self-care, 8 patients were completely unable to take care of themselves, and 3 patients died. Conclusions:Necrotizing fasciitis with sepsis mostly occurs in people with weakened immunity, and has a high mortality and disability rate. Early identification and active surgical debridement may be the key to improve the treatment effect.

Objective To investigate the potential protective effects of valproic acid (VPA) on gut barrier function after major burn injury in rats and its mechanism.Methods Forty male Sprague-Dawley (SD) rats were divided into sham + normal saline (NS),sham + VPA,scald + NS,and scald + VPA groups,with 10 rats in each group.Rat with 55％ total body surface area (TBSA) third-degree severe-bums model was reproduced by immersing into 80 ℃ water,and the rats in sham groups were given sham-bums by immersing into 37 ℃ water.The rats after severebums were immediately treated with 0.25 mL of 300 mg/kg VPA or NS by subcutaneous injection.Rats were sacrificed at 2 hours and 6 hours after injury,and abdominal aortic blood and ileal tissue were harvested.The levels of vascular endothelial growth factor (VEGF) were determined by enzyme-linked immunosorbent assay (ELISA).The intestinal permeability was evaluated by fluorescein isothiocyanate-dextran (FITC-dextran) determination.The histomorphological changes in gut barrier were evaluated by Chiu grading system.Levels of acetylated lysine at the ninth position of histone 3 protein (Ac-H3K9),hypoxia-inducible factor 1α (HIF-1α),zona occludens 1 (ZO-1) and myosin light chain kinase (MLCK) were determined by immunofluorescence staining and Western Blot.Results Compared with sham + NS group,rats in scald + NS group showed intestinal mucosal damage 2 hours after bum injury,as well as increased mucosal permeability,protein expression levels of HIF-1 α,VEGF,MLCK,and lowered levels of AC-H3K9 and ZO-1.These changes were much more prominent at 6 hours after injury.VPA treatment significantly attenuated the bum-induced intestinal damage.Compared with scald + NS group,the protective effects in scald + VPA group was not evident at 2 hours after injury;however,intestinal damage was much less severe at 6 hours after injury (Chiu score:2.03 ± 0.27 vs.3.12 ± 0.15),intestinal permeability was significantly decreased [FITC-dextran (μg/L):709 ± 76 vs.1138 ± 75],histone acetylation was enhanced [Ac-H3K9 (gray value):1.55 ± 0.12 vs.0.48±0.12],ZO-1 degradation was significantly inhibited (gray value:0.69 ± 0.12 vs.0.43 ± 0.16),the protein expression levels of VEGF and MLCK were significantly down-regulated [VEGF (ng/mg):51.7±3.7 vs.71.2±4.3,MLCK (gray value):1.98±0.20 vs.2.80±0.24],while the HIF-1 α protein expression levels were significantly reduced at both 2 hours and 6 hours after injury (gray value:2.50±0.39 vs.3.88±0.42 at 2 hours,1.83±0.42 vs.4.42±0.41 at 6 hours,all P ＜ 0.05).Conclusions Severe bum injury can induce histone deacetylation,ZO-1 degradation and intestinal barrier dysfunction.VPA can improve the levels of histone acetylation and ZO-1,and protect intestinal epithelial barrier function.These may probably be mediated through inhibiting HIF-1α and its downstream gene VEGF and MLCK.

Objective: To explore the effects of allogeneic bone marrow mesenchymal stem cells (BMSCs) on polarization of peritoneal macrophages isolated from rats with sepsis induced by endotoxin/lipopolysaccharide (LPS). Methods: (1) BMSCs were isolated, cultured and purified from 5 SD rats with whole bone marrow adherent method. The third passage of cells were collected for morphologic observation, detection of expressions of stem cell surface markers CD29, CD44, CD45, and CD90 with flow cytometer, and identification of osteogenic and adipogenic differentiation. (2) Another 45 SD rats were divided into sham injury group (SI, n=5), LPS control group (LC, n=20), and BMSCs-treated group (BT, n=20) according to the random number table. Rats in groups LC and BT were injected with LPS (5 mg/kg) via tail vein to induce sepsis; rats in group SI were injected with the same amount of normal saline to simulate the damage. At post injury hour (PIH) 1, rats in group BT were given 1 mL BMSCs (2×10⁶/mL) via tail vein injection; rats in another two groups were injected with equal volume of phosphate buffer saline. Five rats in group SI at PIH 24 and in groups LC and BT at PIH 6, 12, 24, and 48 were sacrificed to harvest lung tissue for pathological observation with HE staining. In addition, rats in group SI at PIH 24 and in groups LC and BT at PIH 24 and 48 were simultaneously performed with intraperitoneal injection of low-glucose DMEM. Then peritoneal fluid was harvested to culture peritoneal macrophages. Flow cytometer was used to assess the positive expression of cell makers of macrophages including CD68 (making gate), CD11c, and CD206 in group SI at PIH 24 and in groups LC and BT at PIH 24 and 48. Data were processed with one-way analysis of variance and LSD test. Results: (1) The third passage of cells showed uniform fiber-like shape similar to fibroblasts. These cells showed positive expressions of CD29, CD44, CD90 and weak positive expression of CD45. They were able to differentiate into osteoblasts and adipocytes. These cells were identified as BMSCs. (2) At PIH 24, the structure of pulmonary alveoli of rats in group SI was clear and complete with no congestion or inflammatory cell infiltration. At PIH 6, the structure of pulmonary alveoli of rats in groups LC and BT was clear with a small amount of inflammatory cell infiltration, slight congestion and pulmonary interstitial thickening. At PIH 12, the inflammatory responses in lung tissue of rats in group LC were more severe than those in group BT with a large amount of inflammatory cell infiltration, serious congestion, and obvious pulmonary interstitial thickening. The pathological results of rats in group BT at PIH 12 was consistent with the results at PIH 6. At PIH 24, the pathological results of rats in groups LC and BT were similar to the results at PIH 12. At PIH 48, the structure of pulmonary alveoli tissue of rats in group LC was still severely disrupted, with a large number of inflammatory cell infiltration and congestion in lung tissue, but pulmonary interstitial thickening was slightly alleviated than before. The condition of rats in group BT nearly recovered to that in group SI. (3) At PIH 24, the positive expression rate of CD11c in peritoneal macrophages of rats in group LC [(83±10)%] was close to that in group BT [(87±7)%, P>0.05], and they were both significantly higher than the rate in group SI [(55±12)%, with P values below 0.01]. The positive expression rate of CD11c in peritoneal macrophages of rats in group LC [(59±11)%] at PIH 48 was close to that in group SI at PIH 24 (P>0.05), and they were both significantly higher than the rate in group BT [(20±11)%] at PIH 48 (with P values below 0.01). At PIH 24, the positive expression percentages of CD206 in peritoneal macrophages of rats were similar among the three groups (with P values above 0.05). The positive expression percentage of CD206 in peritoneal macrophages of rats in group SI at PIH 24 was close to that in group BT at PIH 48 (P>0.05), and they were both significantly lower than the percentage in group LC at PIH 48 (with P values below 0.01). Conclusions BMSCs can reduce the pathological inflammatory responses in the lung of rats with sepsis and inhibit peritoneal macrophages from polarizing into M1 phenotype, whereas they can not promote macrophages to polarize into M2 phenotype.

OBJECTIVETo investigate the effect of bone marrow mesenchymal stem cells (BMSCs) on secretion of inflammatory cytokines in macrophages stimulated by lipopolysacharide (LPS).

METHODSRat BMSCs and macrophages were isolated, cultured, and identified. The BMSCs and macrophages, cultured alone or in co-culture, were treated with LPS or PBS or without treatment and tested for interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) concentrations in the supernatants at 1, 3, 5, 7, 12, and 24 h after the treatment using enzyme-linked immunosorbent assay.

RESULTSExposure to LPS caused significantly increased IL-10 and TNF-α concentrations in the supernatant of cultured macrophages but not in BMSC culture. Macrophages co-cultured with BMSCs showed significantly lowered IL-10 and TNF-α secretions in response to LPS exposure as compared with the macrophages cultured alone.

CONCLUSIONBMSCs can reduce LPS-induced secretion of inflammatory cytokines by the macrophages to ameliorate inflammatory reactions.

Animals ; Cells, Cultured ; Coculture Techniques ; Enzyme-Linked Immunosorbent Assay ; Inflammation ; Interleukin-10 ; secretion ; Lipopolysaccharides ; Macrophages ; secretion ; Mesenchymal Stromal Cells ; cytology ; Rats ; Tumor Necrosis Factor-alpha ; secretion

To investigate the effect of venous super drainage applying in free flaps. Methods From June, 2017 to December, 2018, 7 cases who had severe soft tissue injuries were treated with free flap. Cause of injury: 1 electric injuries, 2 tumor-related wounds, 1 deep burns, 2 mechanical trauma, and 1 necrotizing fasciitis. All patients were underwent free flap transplantation. There were 5 cases of anterolateral thigh artery perforator flap, 1 case of superficial circumflex iliac artery perforator flap, and 1 case of first dorsal metatarsal artery perforator flap.The application of venous super-drainage technology was carried out according to needs and specific conditions. Two sets of venous passages were prepared in both recipient and donor site to form a double set of venous reflux super-drainage mode. Blood supply, swelling, exudation, secondary exploration and survival rate of the flap were observed after operation, and regularly followed-up. Results All 7 flaps survived. Venous super drainage technique was ap-plied in 7 cases. No arteriovenous crisis occurred after the operation. The flaps had good blood circulation, slight swelling, less exudation, rapid edema regression and no secondary surgical exploration. Followed-up for 2-18 (average 10.5) months, there was no infection recurred. Flaps survived well, and the donor sites healed well without sensory loss.The flexion and extension function of joint was normal. Conclusion The technique of venous super-drainage prepares 2 sets of venous systems for the free flap in the treatment of vascular pedicle in the free flap repair operation, which is conducive to reducing the venous crisis after flap surgery, reducing flap edema, reducing exudation, reducing secondary surgical exploration and improving the survival rate of the flap.

OBJECTIVETo analyze the development of liver damage and reactivation of hepatitis B virus (HBV) during the treatment of extremely severe burn injury in HBsAg positive patients, in order to provide reference for prevention and treatment of liver damage in patients with HBV infection after extremely severe burn.

METHODSMedical records of 54 HBsAg positive patients after extremely severe burn injury admitted from January 2004 to December 2014 were retrospectively analyzed. Development of liver damage and HBV reactivation of these patients during the treatment were analyzed according to the classification of their gender, results of hepatitis B e antigen (HBeAg) and HBV DNA examinations on admission, and development of sepsis in the process of treatment. Data were processed with chi-square test.

RESULTS(1) The incidence of liver damage in the process of treatment of these patients was 85.2% (46/54). Among all the patients, the proportion of liver damage was 35/38 in male, which was significantly higher than that in female (11/16, χ² = 4.867, P<0.05). Liver damage was found in all of 26 patients who were HBeAg positive on admission, 34 patients who were HBV DNA positive on admission, and 36 patients who developed sepsis in the process of treatment; the proportions were significantly higher than those in patients who were HBeAg negative on admission (20/28), patients who were HBV DNA negative on admission (12/20), and patients who did not develop sepsis in the process of treatment (10/18), with χ² values respectively 11.801, 18.384, and 20.574, P values below 0.01. (2) The incidence of HBV reactivation in these patients was 29.6% (16/54). Among all the patients, the proportion of HBV reactivation was 13/38 in male and 3/16 in female, with no statistically significant difference between them (χ² = 0.656, P>0.05). The proportions of HBV reactivation in patients who were HBeAg positive on admission, patients who were HBV DNA positive on admission, and patients who developed sepsis in the process of treatment were respectively 13/26, 16/34, and 15/36, and they were significantly higher than those in patients who were HBeAg negative on admission (3/28), patients who were HBV DNA negative on admission (0/20), and patients who did not develop sepsis in the process of treatment (1/18), with χ² values respectively 9.979, 18.615, and 5.873, P<0.05 or P<0.01.

CONCLUSIONSPatients who are HBsAg positive, HBeAg positive, HBV DNA positive on admission, and develop sepsis in the process of treatment of extremely severe burn injury are more likely to develop liver damage and HBV reactivation. It is necessary to dynamically monitor the changes in HBV DNA and liver function, in order to identity the reactivation of virus.

Alanine Transaminase ; blood ; Burns ; complications ; drug therapy ; Chemical and Drug Induced Liver Injury ; DNA, Viral ; Female ; Hepatitis Antibodies ; blood ; Hepatitis B ; drug therapy ; epidemiology ; virology ; Hepatitis B Surface Antigens ; blood ; immunology ; Hepatitis B virus ; drug effects ; immunology ; isolation & purification ; Hepatitis B, Chronic ; blood ; pathology ; virology ; Humans ; Incidence ; Liver ; pathology ; Male ; Retrospective Studies