Effects of microRNA-34a on regulating silent information regulator 1 and influence of the factor on myocardial damage of rats with severe burns at early stage / 中华烧伤杂志

Objective@#To explore the effects of microRNA-34a on regulating silent information regulator 1 (SIRT1) and influence of SIRT1 on myocardial damage of rats with severe burns at early stage.@*Methods@#(1) Twenty-four Sprague-Dawley (SD) rats were divided into sham injury (SI) group, simple burns (SB) group and SIRT1 agonist (SA) group according to the random number table (the same grouping method below), with 8 rats in each group. Rats in groups SB and SA were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burns) on the back, and rats in group SI were sham injuried on the back. Immediately after injury, rats in groups SI and SB were intraperitoneally injected with normal saline of 50 mL/kg, and rats in group SA were intraperitoneally injected with normal saline of 50 mL/kg and 1 mg/mL resveratrol of 50 mg/kg. At 6 h post injury, abdominal aortic blood was collected to make serum and myocardial tissue of rats was collected. (2) Myocardial cells of twelve neonatal SD rats were collected and divided into microRNA-34a mimic control (MMC) group, microRNA-34a mimic (MM) group, microRNA-34a inhibitor control (MIC) group, and microRNA-34a inhibitor (MI) group, which were respectively transfected with gene sequences of mimic control, mimic, inhibitor control, and inhibitor of microRNA-34a. The microRNA-34a expression level and protein expression level of SIRT1 in myocardial cells were respectively detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Another batch of myocardial cells were divided into microRNA-34a inhibitor control+ burn serum (MCB) group, microRNA-34a inhibitor+ burn serum (MB) group, and microRNA-34a inhibitor+ burn serum + EX527 (MBE) group. Myocardial cells in group MCB were transfected with gene sequence of inhibitor control, and myocardial cells in the later groups were transfected with gene sequence of inhibitor of microRNA-34a. After transfection of 48 h, myocardial cells in group MBE were cultured in Dulbecco′s modified Eagle′s medium (DMEM) solution for 6 hours, with serum in group SB of volume fraction of 10% and final amount-of-substance concentration of 1 mol/L, and myocardial cells in the other 2 groups were cultured in DMEM solution with serum from rats of group SB of volume fraction of 10%. The protein expression levels of myocardial cells of SIRT1, cleaved-caspase-3, and Bax were detected by Western blotting. (3) Myocardial tissue from (1) was collected to detect expression levels of microRNA-34a and mRNA of SIRT1 in groups SI and SB by real-time fluorescence quantitative RT-PCR. Morphology of myocardial tissue of rats in groups SI, SB, and SA was observed with biological image navigator. The mRNA expression levels of interleukin 1β (IL-1β) and tumor necrosis factor (TNF-α) of rats in groups SI, SB, and SA were detected by real-time fluorescence quantitative RT-PCR. The expression levels of cleaved-caspase-3, and Bax of myocardial tissue of rats in groups SI, SB, and SA were detected by Western blotting. Data were processed with one-way analysis of variance and least-significant difference test.@*Results@#(1) After transfection of 48 h, the expression level of microRNA-34a of myocardial cells in group MM was 4.67±0.92, significantly higher than 1.03±0.04 in group MMC (P<0.01); the protein expression level of SIRT1 of myocardial cells in group MM was 0.35±0.06, significantly lower than 1.12±0.11 in group MMC (P<0.01). After transfection of 48 h, the expression level of microRNA-34a of myocardial cells in group MI was 0.26±0.07, significantly lower than 1.33±0.07 in group MIC (P<0.01); the protein expression level of SIRT1 of myocardial cells in group MIC was 1.12±0.16, significantly lower than 1.74±0.34 in group MI (P<0.01). At 6 h after culture, compared with those in group MCB, the SIRT1 protein expression level of myocardial cells in group MB was significantly increased (P<0.05), while cleaved-caspase-3 and Bax protein expression levels of myocardial cells in group MB were significantly decreased (P<0.05). Compared with those in group MB, the SIRT1 protein expression level of myocardial cells in group MBE was with no significantly statistical difference (P>0.05), and cleaved-caspase-3 and Bax protein expression levels were significantly increased (P<0.05). (2) At 6 h post injury, compared with that in group SI, the microRNA-34a expression level of myocardial tissue in group SB was significantly increased (P<0.01), and the mRNA expression level of SIRT1 of myocardial tissue in group SB was significantly decreased (P<0.01). At 6 h post injury, myocardial cells in group SI arranged neatly with normal nucleus and no inflammatory cells infiltration; myocardial cells in group SB arranged disorderly, with no abnormal nucleus, and obvious inflammatory cells infiltration; myocardial cells in group SA arranged neatly, with normal nucleus and little inflammatory cells infiltration. At 6 h post injury, compared with those in group SB, the mRNA expression levels of IL-1β and TNF-α, and the protein expression levels of cleaved-caspase-3 and Bax of myocardial tissue in groups SI and SA were significantly decreased (P<0.01).@*Conclusions@#The microRNA-34a expression level of myocardial tissue of rats with severe burns at early stage increases, which decreases the expression level of SIRT1, and increases the expression levels of IL-1β, TNF-α, cleaved-caspase-3 and Bax, leading to obvious myocardial damage. Activation of SIRT1 can alleviate myocardial damage of rats with severe burns at early stage through decreasing expression levels of IL-1β, TNF-α, cleaved-caspase-3, and Bax.

Effects of activating silent information regulator 1 on early kidney damage in rats with severe burn / 中华烧伤杂志

Objective@#To investigate the effects of activating silent information regulator 1 (SIRT1) on the early kidney damage in rats with severe burn.@*Methods@#Thirty healthy male SD rats were divided into sham injury group (SI), pure burn group (PB), and SIRT1 activator group (SA) according to the random number table, with 10 rats in each group. Rats in groups PB and SA were inflicted with 30% total body surface area full-thickness scald (hereinafter referred to as burn) on the back. Immediately after injury, rats in group PB were intraperitoneally injected with normal saline in the dosage of 50 mL/kg, and those in group SA with 1 mg/mL (final mass concentration) resveratrol in the dosage of 50 mL/kg. Rats in group SI were sham injured and intraperitoneally injected with normal saline in the dosage of 50 mL/kg immediately after injury. Kidney tissue and abdominal aorta blood of rats in the three groups were collected at 24 hours after injury. The morphology of kidney tissue was observed after HE staining. The serum content of creatinine and urea nitrogen was determined with enzyme-linked immunosorbent assay. Protein expressions of SIRT1, Bax, and Bcl-2 in kidney tissue were determined with Western blotting. mRNA expressions of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-10 in kidney tissue were determined with real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were processed with one-way analysis of variance and LSD-t test.@*Results@#(1) In rats of group SI, structures of kidney tubules and glomeruli were intact. In rats of group PB, structures of kidney tubules were not clear with casts in them, and glomeruli showed pyknosis. In rats of group SA, structures of kidney tubules were relatively intact, and the pyknosis of glomeruli were slighter as compared with that of group PB with fewer glomeruli showing pyknosis. (2) The serum content of creatinine and urea nitrogen in rats of group PB was (67±14) μmol/L and (22.0±4.4) mmol/L, respectively, which was significantly higher than that of group SI [(28±7) μmol/L and (5.5±1.2) mmol/L respectively, with t values respectively 6.07 and 11.53, P values below 0.01]. The serum content of creatinine and urea nitrogen in rats of group SA was (39±9) μmol/L and (14.1±1.7) mmol/L, respectively, significantly lower than that of group PB (with t values respectively 4.09 and 4.17, P values below 0.01). (3) Compared with those of group SI, protein expressions of SIRT1 and Bcl-2 in kidney tissue of rats in group PB were significantly decreased (with t values respectively 16.32 and 19.58, P values below 0.01), while the protein expression of Bax was significantly increased (t=5.98, P<0.01). Compared with those of group PB, protein expressions of SIRT1 and Bcl-2 in kidney tissue of rats in group SA were significantly increased (with t values respectively 6.94 and 5.37, P values below 0.01), while the protein expression of Bax was significantly decreased (t=3.44, P<0.01). (4) mRNA expressions of TNF-α, IL-1β, and IL-10 in kidney tissue of rats in group PB were 17.0±4.0, 2.27±0.59, and 2.5±0.9, respectively, significantly higher than those of group SI (1.0, 1.00, and 1.0, respectively, with t values from 3.27 to 8.93, P<0.05 or P<0.01). mRNA expressions of TNF-α and IL-1β in kidney tissue of rats in group SA were 6.8±1.2 and 1.18±0.26, respectively, significantly lower than those of group PB (with t values respectively 4.59 and 4.32, P values below 0.01). mRNA expression of IL-10 in kidney tissue of rats in group SA was 5.0±1.0, significantly higher than that of group PB (t=5.51, P<0.01).@*Conclusions@#Activating SIRT1 on early stage of severe burn in rats can decrease levels of creatinine and urea nitrogen, thus improving the kidney function. It can down-regulate the protein expression of Bax and up-regulate the protein expression of Bcl-2, thus reducing the apoptosis in kidney tissue. Meanwhile, it can inhibit expressions of TNF-α and IL-1β and promote the expression of IL-10, thus alleviating the inflammatory response in kidney.

Operational options and outcomes evaluation in cicatricial alopecia / 中国医师杂志

Objective To summarize the operational experience and explore the best surgery strategy in cicatricial alopecia.Methods Clinical date of 80 cases of cicatricial alopecia during January 2012 to December 2016 were analyzed retrospectively.The operation methods and related adverse events were recorded.Patients were followed-up on the postoperative 1 week,1 month,3 months,6 months and 1-2 years.The outcomes were evaluated by a 4-levels questionnaire:very satisfied,satisfied,not satisfied,and no effect.Results Forty cases were operated with expanded skin flap + Follicular unit extraction (FUE) transplantation,10 cases with scar resection + FUE repair,and 30 cases only with FUE.Twenty cases were completed treatment with single-stage surgical operation,and 60 cases with two-stage surgical operation.A percentage (70％) of patients was very satisfied and 30％ were satisfied after one-stage surgical operation.A percentage (85％) of patients was very satisfied and 15％ were satisfied after two-stage surgical operation.Conclusions The cicatricial alopecia needs comprehensive surgical treatment.FUE is a best additional operation technology.The effect of combined treatment is better than single therapy method in large area cicatricial alopecia.

Effects of mouse adipose-derived stem cell conditioned medium on the apoptosis of keratinocytes induced by thermal injury in vitro / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the effects of mouse adipose-derived stem cell conditioned medium (ADSC-CM) on apoptosis of keratinocytes (human epithelial cell line HaCaT) induced by thermal injury in vitro.</p><p><b>METHODS</b>(1) Adipose-derived stem cells (ADSCs) from inguinal adipose tissue of 5 healthy BALB/c mice were isolated, cultured, and purified by collagenase digestion in vitro. The 3rd passage of cells were collected for morphologic observation, detection of expressions of surface markers CD31, CD34, CD45, CD90, and CD105 with flow cytometer, and identification of adipogenic and osteogenic differentiation. (2) HaCaT cells were incubated in water at 51.5 °C for 35 seconds to reproduce thermal injury model, and then the apoptosis rate was detected immediately after injury by flow cytometer. (3) Thermally injured HaCaT cells were divided into routine culture group (RC, cultured with DMEM containing 10% FBS), serum-free group (cultured with serum-free DMEM), 50%ADSC-CM group (cultured with DMEM containing 50%ADSC-CM), and 100%ADSC-CM group (cultured with 100%ADSC-CM) according to the random number table. After 24 hours, apoptosis of HaCaT cells was observed by acridine orange-ethidium bromide (AO-EB) staining; apoptotic rate was determined by flow cytometer; the mRNA and protein levels of Bcl-2 and caspase-3 were respectively determined by real-time fluorescent quantitative RT-PCR technique and Western blotting (protein level was denoted as gray value); the cell cycles were determined by flow cytometer. All above experiments were repeated for 3 times. Data were processed with one-way analysis of variance and LSD- t test.</p><p><b>RESULTS</b>(1) The 3rd passage of cells proliferated well showing fusiform shape similar to fibroblasts. The positive expression rates of CD31, CD34, and CD45 were less than 10.0%, while those of CD90 and CD105 were above 90.0%. The cells could differentiate into adipocytes and osteoblasts. They were identified as ADSCs. (2) Immediately after injury, apoptotic rate of HaCaT cell was (9.8 ± 0.4)%. (3) The number of apoptotic cells was significantly higher in serum-free group than in the other three groups with AO-EB staining. The apoptotic rate of serum-free group [(8.1 ± 1.2)%] was significantly higher than that of 50%ADSC-CM group [(6.0 ± 0.8)%], group RC [(4.6 ± 0.8)%], or 100%ADSC-CM group [(3.1 ± 0.4)%], with t values respectively 3.02, 4.96, 6.60, P values below 0.01. There was no statistically significant difference in apoptotic rate between group RC and 100% ADSC-CM group (t = 1.50, P > 0.05), while statistically significant difference was found between 100% ADSC-CM group and 50%ADSC-CM group (t = 10.21, P < 0.01). (4) The mRNA level of Bcl-2 of serum-free group (0.34 ± 0.08) was significantly lower than that of group RC, 50%ADSC-CM group, and 100%ADSC-CM group (0.98 ± 0.04, 0.77 ± 0.05, 1.06 ± 0.04, with t values respectively 12.87, 8.07, 14.11, P values below 0.01). Compared with that of 100%ADSC-CM group, the mRNA level of Bcl-2 of group RC was slightly decreased (t = 0.08, P > 0.05) and that of 50%ADSC-CM group was significantly decreased (t = 8.08, P < 0.01). (5) The mRNA level of caspase-3 of serum-free group (1.15 ± 0.05) was obviously higher than that of 50%ADSC-CM group (0.72 ± 0.11), group RC (0.41 ± 0.03), or 100%ADSC-CM group (0.38 ± 0.11), with t values respectively 6.93, 13.97, 22.79, P values below 0.01. Compared with 100%ADSC-CM group, the mRNA level of caspase-3 was slightly increased in group RC (t = 0.05, P > 0.05) and significantly increased in 50%ADSC-CM group (t = 4.77, P < 0.01). (6) The protein level of Bcl-2 was significantly lower in serum-free group (0.93 ± 0.04) than in group RC, 50%ADSC-CM group, and 100%ADSC-CM group (1.74 ± 0.06, 1.32 ± 0.05, 1.90 ± 0.04, with t values respectively 20.45, 11.15, 31.38, P values below 0.01). Compared with that of 100%ADSC-CM group, the protein level of Bcl-2 of group RC was slightly decreased (t = 1.33, P > 0.05), but that of 50%ADSC-CM group was obviously decreased (t = 17.30, P < 0.01). (7) The protein level of caspase-3 was obviously higher in serum-free group (0.63 ± 0.08) than in 50%ADSC-CM group, group RC, and 100%ADSC-CM group (0.46 ± 0.03, 0.29 ± 0.08, 0.21 ± 0.03, with t values respectively 3.28, 5.05, 8.46, P values below 0.01). Compared with that of 100%ADSC-CM group, the protein level of caspase-3 of group RC was slightly increased (t = 0.08, P > 0.05), but that of 50%ADSC-CM group was significantly increased (t = 3.52, P < 0.05). (8) Compared with that of serum-free group, the percentage of cells in G2/M phase of each of the other 3 groups was significantly decreased (with t values respectively 6.88, 4.08, 7.28, P < 0.05 or P < 0.01). Compared with that in serum-free group, the percentage of cells in S phase was significantly increased in group RC and 100% ADSC-CM group (with t values respectively 2.67 and 2.40, P values below 0.05). There was no statistically significant difference in the percentage of cells in G0/G1 phase among all groups (F = 0.70, P > 0.05).</p><p><b>CONCLUSIONS</b>100% xenogeneic ADSC-CM can suppress apoptosis of HaCaT cells induced by thermal injury through regulating the expression of Bcl-2 and caspase-3, and accelerate cell cycle progression by ameliorating the retardation of cell growth in G2/M phase, and all these effects may give rise to some potential in the treatment of burn wounds at early stage.</p>

Effects of activating silent information regulator 1 on early myocardial damage in severely burned rats / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the effects of activating silent information regulator 1 (SIRT1) on early myocardial damage in severely burned rats.</p><p><b>METHODS</b>Twenty-four healthy male SD rats were divided into sham injury group (SI), scald group (S), and resveratrol (RSV) treatment group (RT) according to the random number table, with 8 rats in each group. Rats in groups S and RT were inflicted with 30% TBSA full-thickness scald on the back by immersing in 95 °C water for 18 s. Immediately after injury, rats in group S were intraperitoneally injected with 10 mL normal saline (50 mL/kg) and those in group RT with 10 mL normal saline (50 mL/kg)+10 µL RSV in the concentration of 1 g/mL (50 mg/kg). Backs of rats in group SI were immersed in 20 °C room temperature water for 18 s to simulate the scald process. Heart tissues and aorta abdominalis blood samples were collected at post injury hour (PIH) 6. The histomorphology of heart tissues was observed with HE staining. The serum contents of creatine kinase (CK) and lactate dehydrogenase (LDH) were determined with ELISA. The protein expressions of SIRT1 and caspase-3 and mRNA expressions of SIRT1, caspase-3, IL-1β, and TNF-α in heart tissue specimens were determined with Western blotting and real-time fluorescent quantitative RT-PCR (with protein level denoted as gray value). Data were processed with one-way analysis of variance and LSD- t test.</p><p><b>RESULTS</b>(1) In group SI, myocardial fibers were in irregularly cylindrical shape, neatly arranged, and the transverse striation were distinct. In group S, myocardial interstitial edema, disorder of myocardial fiber arrangement, and cytoplasm destruction were observed. In group RT, the degrees of myocardial interstitial edema, disorder of myocardial fiber arrangement, and cytoplasm destruction were alleviated in comparison with those of group S. (2) The serum contents of CK and LDH of rats in group S were respectively (2 385 ± 712) and (2 551 ± 196) U/L, which were significantly higher than those in the group SI [(290 ± 59) and (759 ± 60) U/L, with t values respectively 9.466 and 25.452, P values below 0.01]. The serum contents of CK and LDH of rats in group RT were respectively (1 336 ± 149) and (2 209 ± 133) U/L, which were significantly lower than those of group S (with t values respectively -4.506 and -4.860, P values below 0.01). (3) The protein expressions of SIRT1 and caspase-3 in heart tissue of rats in group S were respectively 0.47 ± 0.11 and 0.48 ± 0.12, which were significantly higher than those in group SI [0.18 ± 0.06 and 0.09 ± 0.05, with t values respectively 4.813 and 9.014, P values below 0.01]. The protein expression of SIRT1 in heart tissue of rats in group RT was 0.74 ± 0.18, which was significantly higher than that of group S (t = 4.561, P < 0.01); the protein expression of caspase-3 in heart tissue of rats in group RT was 0.21 ± 0.08, which was significantly lower than that of group S (t = -6.239, P < 0.01). (4) The mRNA expressions of SIRT1, caspase-3, IL-1β, and TNF-α in heart tissue of rats in group S were respectively 2.33 ± 0.24, 1.96 ± 0.20, 2.46 ± 0.21, 1.89 ± 0.37, which were significantly higher than those in group SI (1.00 ± 0.07, 1.00 ± 0.06, 1.00 ± 0.08, 1.00 ± 0.09, with t values respectively 14.961, 12.823, 18.559, 6.679, P values below 0.01). The mRNA expression of SIRT1 in heart tissue of rats in group RT was 2.89 ± 0.31, which was significantly higher than that of group S (t = 3.997, P < 0.01). The mRNA expressions of caspase-3, IL-1β, and TNF-α in heart tissue of rats in group RT were respectively 1.31 ± 0.08, 1.64 ± 0.09, 1.25 ± 0.08, which were significantly lower than those of group S (with t values respectively -8.264, -10.245, -4.818, P values below 0.01).</p><p><b>CONCLUSIONS</b>The expression of SIRT1 in heart tissue is upregulated in the early stage of severely burned rats. Activation of SIRT1 by RSV can alleviate myocardial tissue injury and reduce apoptosis of cardiac myocytes and secretion of IL-1β and TNF-α.</p>

Isolation, culture and multiple differentiations of rabbit bone marrow-derived mesenchymal stem cells in vitro / 中国组织工程研究

BACKGROUND: Mesenchymal stem cells (MSCs), with low immunogenicity, can regulate cellular immunity and mitigate graft rejection, which has a good prospect in tissue engineering. However, it is rarely present in bone marrow. OBJECTIVE: To explore an isolation and culture method of the rabbit bone marrow-derived MSCs, to observe the biological characteristics and differentiation potential of bone marrow-derived MSCs.METHODS: MSCs were isolated from rabbit tibia bone marrow by combination of gradient centrifugation and different adherent method, then proliferation in vitro. Morphology was examined by phase contrast microscopy, and the growth curve of cultured MSCs was drawn via MTT results. MSCs were treated with osteogenetic inductor (L-DMEM/F12, 10% fetal bovine serum, 0.1 μmol/L dexamethasone, 200 μmol/L vitamin C, 10 mmol/L β-phosphoglycerol), adipose inductor (L-DMEM/F12, 10% FBS, 1 μmol/L dexamethasone, 200 μmol/L antifani, 0.5 mmol/L IBMX, 10 μg/mL insulin), and chondrocytes inductor (L-DMEM/F12, 10% FBS, 10 μg/L TGF-β1, 0.1 μmol/L dexamethasone, 50 μmol/L vitamin C, 6.25 mg/L insulin) to differentiated into osteoblast, dipocytes and chondrocytes. And the differentiated cells were identified by alkaline phosphatase staining, oil red O staining, and toluidine blue staining, respectively.RESULTS AND CONCLUSION: Bone marrow-derived MSCs can be isolated and cultured by the combination of gradient centrifugation and different adherent method in vitro, which have the better potentiality of proliferation and multi-directional differentiation. Mostly of the primary and passaged cells were spindle-shaped. After osteogenetic induction, cells were positive to alkaline phosphatase staining. Oil red O staining showed that red lipid droplet existed in adipose cells, and toluidine blue staining showed that toluidine blue was positive after chondrocytes induction.