Insulin protects against damage to pulmonary endothelial tight junctions after thermal injury: relationship with zonula occludens-1, F-actin, and AKT activity.

Intensive insulin therapy during critical illness protects the endothelium and thereby prevents organ failure. This study tested the hypothesis that insulin directly affects the attenuation of burn injury-induced damage to pulmonary endothelial tight junction and investigated the underlying mechanisms. Sprague Dawley rats with severe burn injury were randomized to treatment with insulin dissolved in normal saline (maintenance of blood glucose at a level between 5.0 and 7.0 mmol/L) or normal saline alone (in vivo treatment). Pulmonary damage was evaluated. Rat pulmonary microvascular endothelial cells were treated with 20% burn serum or 20% burn serum + insulin (in vitro treatment). Selected cultures were pretreated with phosphatidylinositol 3-kinase/protein kinase B (AKT) inhibitor (LY294002). Permeability was assessed by migration of bovine serum albumin across cell monolayers. Cells were stained with rhodamine phalloidin and were examined. Cell extracts were obtained to assess zonula occludens-1, occludin, and phosphorylated AKT levels by immunoblotting. Treatment with insulin attenuated the pulmonary edema, hemorrhage, and inflammatory cell infiltration of rats with severe burn injury. Burn serum significantly enhanced monolayer permeability to albumin, whereas treatment with insulin (10(-7 ) mol/L) limited this effect. Meanwhile, insulin (10(-7 ) mol/L) reduced burn serum-induced F-actin stress fiber formation and decreased zonula occludens-1 expression. LY294002 decreased cytoplasmic AKT phosphorylation and inhibited the protection effects of insulin. Through the phosphatidylinositol 3-kinase/AKT pathway, insulin independent of glucose toxicity can attenuate increased pulmonary endothelial permeability induced by burn injury. The effect is attributed to the attenuation of the architectural disruption of protein components of the endothelial tight junction. This result is useful in inhibiting multiple organ failure after burn injury.

Screening and identification of hub-gene associated with brain metastasis in breast cancer.

BACKGROUND: The presence of breast cancer in the brain, also known as brain metastasis (BMS), is the primary reason for a bad prognosis in cases of breast cancer. Breast cancer is the most prevalent malignant tumor seen in women in developing nations. At present, there is no effective method to inhibit brain metastasis of breast cancer. Therefore, it is necessary to conduct a systematic study on BMS of breast cancer, which will not provide ideas and sites for follow-up studies on the treatment and inhibition of BMS. METHODS: In this study, data set GSE43837 was screened from gene expression omnibus database, and then R language tool was used for differential analysis of its expression spectrum, The gene ontology functional enrichment and Kyoto encyclopedia of genes and genomes signal pathway enrichment analyses, as well as the interactive gene retrieval tool for hub-gene analysis, were performed. RESULTS: According to the findings, the primary genes linked to breast cancer brain metastases are those that involve interactions between cytokines and their respective receptors and between neuroactive ligands and their respective receptors. The majority of the gene ontology enrichment took place in the extracellular structural tissues, the extracellular matrix tissues, and the second message-mediated signaling. We were able to identify 8 genes that are linked to breast cancer spreading to the brain. The gene score for matrix metallopeptidase1 (MMP-1) was the highest among them, and the genes MMP10, tumor necrosis factor alpha-inducible protein 8, collagen type I alpha 2 chain, vascular cell adhesion molecule 1, and TNF superfamily member 11 were all connected to 1 another in an interaction way. CONCLUSIONS: There is a possibility that the 8 key genes that were identified in this research are connected to the progression of BMS in breast cancer. Among them, MMP1 is 1 that has the potential to have a role in the diagnosis and treatment of BMS in breast cancer.

[Effect of intensive insulin treatment on cardiac myocytes of severely scalded rats].

OBJECTIVE: To study the protective effect of intensive insulin treatment on cardiac myocytes of severely scalded rats. METHODS: Eighteen model Sprague-Dawley (SD) rats were subjected to 30% total body surface area (TBSA) full thickness injury, and they were divided into three groups with 6 rats in each group. The right jugular vein was cannulated for fluid resuscitation and administration of drugs. The rats in burn group were injected with normal saline, the intensive insulin group with injection of insulin to maintain plasma glucose content in normal range, and the sham burn group received physiologic dose of saline without burn injury. Plasma glucose was monitored after burn injury. Rats were sacrificed at 6 hours postburn to examine plasma myocardial enzymes spectrum as well as histological and ultrastructure changes in cardiac tissue. The expression of p-Akt was detected by western blotting. RESULTS: Plasma glucose level was significantly elevated in burn group within postburn 6 hours as compared with the sham burn group, and lowered in intensive insulin group (4.5 approximately 5.2 mmol/L vs. 7.6 approximately 8.4 mmol/L, P<0.05 or P<0.01). And the intensive insulin therapy could effectively inhibit the release of cardiac enzymes [lactate dehydrogenase (LDH): (2 369.3+/- 178.9) U/L vs. (2 684.1+/-335.0) U/L, P<0.05; alpha-hydroxybutyrate dehydrogenase (alpha-HBD): (576.7+/-219.2) U/L vs. (1 002.0+/-347.1) U/L, P<0.01; creatine kinase (CK): (1 041.9+/-623.2) U/L vs. (2 447.1+/-1 183.7) U/L, P<0.01]. The expression of p-Akt was significantly strengthened in the intensive insulin group (1.18+/-0.43 vs. 0.24+/-0.11, P<0.01). Light microscopic and electron microscopic examinations showed that intensive insulin therapy could alleviate the injury to myocardial cells and structural changes. CONCLUSION: Intensive insulin treatment possesses protective effect on cardiomyocytes after a severe burn, and it is related to its up-regulation of phosphorylation level of Akt in cardiomyocyte, thus inhibiting the damage to myocytes.

[Analysis of lipid compounds of high-yielded rhizoma pinelliae growing in the west of Hubei province by gas chromatography-mass spectrometry].

OBJECTIVE: To investigate lipid components of high-yielded Pinellia ternata rhizomes growing in the west of Hubei province. METHODS: To determine the lipid chemical components in Pinellia ternata rhizomes with GC-MS method and NIST atlas. RESULTS: Ten components have been found: palmitic acid (I), 9,12-octadecadienoic acid (II), pyrrolidine,1-(1-oxo-7,10-hexadecadienyl) (III), alpha-monpalmitin (IV), 1,3,12-nonadecatriene (V), campesterol (VI), stigmasta-5,22-dien-3-ol (VII), beta-sitosterol (VIII), stigmasta-5,24-dien-3-ol (IX), cycloartenol (X). CONCLUSION: The relative contents of five kinds of phytosterol: campesterol 28.96%, stigmasta-5,22-dien-3-ol 9.24%, beta-sitosterol 50.77%, stigmasta-5,24-dien-3-ol 4.74%, cycloartenol 6.25%. Component II, III, V, VI, IX are the first time reported in Pinellia ternata.

Iodine-125 irradiation inhibits invasion of gastric cancer cells by reactivating microRNA-181c expression.

Iodine-125 (125I) seed implantation has been widely used for the treatment of unresectable advanced tumors. However, the molecular mechanisms underlying the tumor-suppressive effects of 125I irradiation have not been fully elucidated. The present study demonstrated that 125I irradiation suppresses cell viability and inhibits cell invasiveness of gastric cancer KATO-III and MKN45 cells. Further mechanistic analysis suggested the involvement of microRNA (miR)-181c in the inhibitory effects induced by 125I irradiation. Methylated DNA immunoprecipitation coupled with quantitative-polymerase chain reaction demonstrated that treatment with 125I irradiation, at the dose of 4 Gy, induced promoter demethylation of the miR-181c gene in KATO-III and MKN45 cells. Following irradiation, the expression of miR-181c was significantly increased, which may be attributed to the demethylation caused by 125I irradiation. In addition, upregulation of miR-181c by administration of miR-181c mimics decreased cell invasion, suggesting the role of miR-181c as a tumor suppressor. More importantly, the tumor-suppressive effects of 125I irradiation were significantly compromised by the introduction of miR-181c inhibitors. Overall, these results reveal that 125I irradiation inhibits invasiveness of gastric cancer cells by reactivating miR-181c at the epigenetic level, thereby providing important molecular evidence for the anticancer effects of 125I irradiation.

Effect of brachytherapy on NF-κB and VEGF in gastric carcinoma xenografts.

Iodine-125 (125I) seed irradiation can be used as an important supplementary treatment for unresectable advanced gastric cancer. However, the radiobiological mechanism underlying brachytherapy remains unclear. Therefore, we investigated the influence of continuous and low-energy 125I irradiation on the cell cycle distribution, apoptosis, expression of NF-κB and VEGF and tumor growth in a human gastric cancer xenograft model. To create an animal model of gastric cancer, SGC-7901 cells were surgically implanted into mice. The 60 mice bearing SGC-7901 gastric cancer xenografts were randomly separated into 2 groups. Sham seeds (0 mCi) were implanted into the control group (n=30); 125I seeds (0.6 mCi) were implanted into the treatment group (n=30). At 28 days after irradiation, apoptosis was detected by flow cytometry. fluorescence micrograph detected intense VEGF and NF-κB immunofluorescence in the tumor samples, and changes in NF-κB and VEGF mRNA and protein expression were assessed by real-time PCR and western blot analysis, respectively. The tumor volume and weight were measured 0-28 days after 125I seed implantation. 125I seed irradiation induced significant apoptosis and G2/M phase arrest. Reduction in the intensities of VEGF and NF-κB immunofluorescence in tumor vessels was observed after treatment. NF-κB and VEGF mRNA and protein expression levels were substantially lower in the implantation treatment group than in the control group. Consequently, 125I seed implantation inhibited cancer growth and reduced cancer volume. The present study revealed that 125I seed irradiation significantly induced apoptosis and cell cycle arrest in the human gastric cancer xenografts. 125I-induced changes in NF-κB and VEGF expression are suggested as potential mechanisms underlying effective brachytherapy.

[Free superficial iliac circumflex artery skin flap: the clinical application and management of donor site defects].

OBJECTIVE: To investigate the clinical application of free superficial iliac circumflex artery skin flaps, as well as the management of donor site defects. METHODS: 17 free superficial iliac circumflex artery skin flaps were applied for the traumatic defects or deformities on face, neck, foot, hand, ankle and lower leg, respectively. The donor site defects were closed directly or covered by paraumbilical island flaps. RESULTS: The 17 flap size ranged from 5 cm x 3 cm to 19 cm x 14 cm. 16 flaps survived completely except 1 flap with partial necrosis, which was closed by free skin graft. The donor site defects were closed directly in 10 cases, and covered by paraumbilical island flaps in 7 flaps without no flap necrosis. The abdomen had a good appearance. CONCLUSIONS: Good appearance can be achieved with free superficial iliac circumflex artery skin flaps for the defects on face, neck, foot, hand, ankle and lower leg. Paraumbilical island flap can be used for the donor site defects.

A potential skin substitute constructed with hEGF gene modified HaCaT cells for treatment of burn wounds in a rat model.

This study aimed to investigate the feasibility of using an immortal keratinocyte cell line, HaCaT cells, to effectively deliver epidermal growth factor (EGF) in a skin substitute to treat burn wounds. The skin equivalent was constructed with human EGF (hEGF) gene modified HaCaT cells obtained through stable gene transfection; these were applied to full thickness burn wounds in a rat model. The results showed that the hEGF gene modified HaCaT cells produced more than 390ng/l of bioactive hEGF in the culture supernatant. K19 and integrin-ß1 as keratinocyte differentiation markers were elevated in the hEGF gene modified HaCaT cells which were shown to be non-tumorigenic. The skin equivalent constructed with hEGF gene modified HaCaT cells demonstrated improved epidermal morphogenesis with a thick and compact epidermis. Wound healing was accelerated noticeably when applied with this skin substitute seeded with hEGF gene modified HaCaT cells in vivo. The results suggest that HaCaT cells modified with hEGF gene might be promising seed cells for construction of genetically modified skin substitute which can effectively secrete hEGF to accelerate wound repair and regeneration.

Insulin-mediated inhibition of p38 mitogen-activated protein kinase protects cardiomyocytes in severe burns.

Thermal injury inhibits Akt activation and upregulates p38 mitogen-activated protein kinase, which in turn induces inflammation and increases apoptosis. This study aimed to elucidate the mechanism underlying the cytoprotective role of insulin in severe burns by examining the effects of insulin on inflammation and apoptosis mediated by p38 mitogen-activated protein kinase in burn serum-challenged cardiomyocytes. Neonatal rat cardiomyocytes were exposed to burn serum for 6 hours in the presence or absence of insulin and pretreated with inhibitors to p38 mitogen-activated protein kinase (SB203580) and Akt (LY294002). The authors examined expression of myocardial tumor necrosis factor-alpha, cardiac myofilament proteins caspase-3 and Bcl2, and apoptosis. Burn serum-induced upregulation of tumor necrosis factor was inhibited by both SB203580 and insulin. LY294002 reversed insulin-mediated downregulation of tumor necrosis factor. Both SB203580 and insulin inhibited apoptosis, resulting in fewer pyknotic nuclei and inhibition of caspase-3 activation and Bcl2 downregulation. LY294002 reversed insulin-mediated inhibition of apoptosis. Insulin decreases inflammatory cytokine expression and apoptosis via PI3K/Akt-mediated inhibition of p38 mitogen-activated protein kinase. The cytoprotective role of insulin suggests that it may have a potential role in strategies for treating thermal injuries.

Intensive insulin treatment attenuates burn-initiated acute lung injury in rats: role of the protective endothelium.

Nonmetabolic effects of intensive insulin therapy in critically ill patients have been reported, but the underlying mechanisms are unclear. This study was designed to test the hypothesis that intensive insulin treatment would attenuate burn-induced acute lung injury by protecting the pulmonary microvascular endothelium. The rat model of burn injury was achieved by exposure to 92°C water for 18 seconds. The rats were randomly allocated into the sham, burn/normal saline (NS), and burn/intensive insulin treatment groups. Blood glucose level was maintained between 5 and 7 mmol/L in rats in the burn/intensive insulin treatment group. Pulmonary injury was assessed by hematoxylin and eosin staining, scanning electron microscopy, bronchoalveolar lavage fluid protein concentrations, the lung wet:dry weight ratio, and lung myeloperoxidase activity. Pulmonary microvascular endothelial cells were examined by transmission electron microscopy. Western blotting was used to determine the protein expression of caspase-3. Intensive insulin treatment markedly attenuated the acute lung injury, revealed by improvements in histological features and significant decreases in bronchoalveolar lavage fluid protein concentrations, pulmonary wet:dry weight ratio, and myeloperoxidase activity at 12 hours after injury (P < .05 or P < .01 vs burn/NS). Moreover, the injured pulmonary microvascular endothelial cells showed significant improvements, whereas caspase-3 was markedly downregulated in the burn/intensive insulin treatment group when compared with the burn/NS group. Overall, intensive insulin treatment efficiently attenuated pulmonary microvascular endothelial cell dysfunction, decreased cell apoptosis, and inhibited acute lung injury after a burn. These findings may be useful in preventing organ failure after burn injury.

[Biological effects of paracrine from insulin stimulated adipose-derived stem cells (ADSC) on human vascular endothelial cells].

OBJECTIVE: To study the biological effects of the paracrine from ADSC after being stimulated by insulin on vascular endothelial cells. METHODS: (1) ADSC was isolated from human adipose tissue and cultured in vitro. The third generation cells were collected and divided into insulin group (I, cultured with serum-free DMEM containing 1 x 10(-7) mol/L insulin) and control group (C, cultured with serum-free DMEM) according to the random number table, with 6 slots in each group. Three days later, ADSC culture medium (ADSC-CM) was collected for determination of levels of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) by ELISA. (2) Human umbilical vein endothelial cells (HUVEC) were cultured to the third generation, and they were cultured with special nutrient solution and divided into ADSC-CM with insulin stimulation group (AI), ADSC-CM without insulin stimulation group (AC), insulin group (I, with same concentration as above), blank control group (BC) according to the random number table. Three days later, proliferation of HUVEC was determined with MTT method (with expression of absorbance value). Another two samples of HUVEC were respectively divided into 4 groups as above for determination of apoptosis rate with Annexin V/FITC double-staining 12 hours after culture, and HUVEC migration with scratch adhesion test at post scratch hour (PSH) 12, 24, 36, 48. Data were processed with t test. RESULTS: (1) Compared with those in C group [(287 +/- 47), (577 +/- 84) pg/mL, respectively], the secretion levels of VEGF and HGF in I group [(643 +/- 64), (930 +/- 68) pg/mL, respectively] were significantly increased (with t value respectively 18.869, 18.475, P values all below 0.05). (2) The absorbance value of HUVEC in AI and AC groups was 0.847 +/- 0.042, 0.798 +/- 0.022, respectively, which were higher than that in I and BC groups [0.665 +/- 0.028 (with t value respectively 4.579, 3.732), 0.674 +/- 0.031 (with t value respectively 3.761, 4.073), P values all below 0.01], and that in AI group was higher than that in AC group (t = 2.576, P < 0.05). The apoptosis rates of HUVEC in AI and AC groups [(5.8 +/- 1.9)%, (9.0 +/- 2.0)%, respectively] were obviously lower as compared with that in I and BC groups [(30.4 +/- 6.0)% (with t value respectively 12.891, 10.417), (31.4 +/- 7.4)% (with t value respectively 11.474, 9.783), P values all below 0.05 ], and that in AC group was higher than that in AI group (t = 8.548, P < 0.05). The distance of migration of HUVEC in AI and AC groups were greater than that in I and BC groups at PSH 36, 48, and that in AI group was greater as compared with that in AC group (with t value respectively 4.076, 4.573, P values all below 0.05). CONCLUSIONS: Paracrine from ADSC after being stimulated by insulin can promote proliferation and migration of HUVEC, and suppress its apoptosis, and it is beneficial for tissue vascularization.

[Inhibitory effect of insulin on nuclear factor-kappa B nuclear translocation of vascular endothelial cells induced by burn serum].

OBJECTIVE: To study the inhibitory effects of insulin on nuclear factor-kappa B (NF-kappaB) nuclear translocation of vascular endothelial cells induced by burn serum and its correlative mechanism. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and divided into 5 groups: blank control group (BC, ordinary culture without any stimulation), normal serum control group (NS, cultured with nutrient solution containing 20% healthy human serum), burn serum stimulation group (BS, cultured with nutrient solution containing 20% burn human serum), burn serum+insulin treatment group (BI, cultured with nutrient solution containing 20% burn human serum and 1x10(-7) mol/L insulin), inhibitor pretreatment group [IP, pretreated with 50 micromol/L protein kinase B (Akt) specific inhibitor LY-294002, then cultured with the same medium as used in BI group 30 minutes later] according to the random number table. Six hours later, the injury and apoptosis of HUVECs was respectively observed by the scanning electron microscope and determined by the flow cytometry. Meanwhile, the phosphorylation of inhibitor kappa B-alpha (p-IkappaB-alpha) and Akt (p-Akt) in cytoplasm, and the content of NF-kappaB-p65 in nucleus were determined with Western blot. RESULTS: (1) Compared with those in BC group, HUVECs in BS group shrank obviously with irregular nuclear structure, and intercellular links jagged or vanished. Slight change was observed in HUVECs structure in NS and BI groups, with the cell ductility and nuclear structure much better than those in BS group. (2) The apoptosis rates of HUVECs in BS group [(28.5+/-2.3)%], BI group [(22.3+/-1.8)%], and IP group [(29.7+/-2.4)%] were all obviously higher than that in BC group [(15.7+/-2.2)%, F=14.288, P<0.05 or P<0.01]. There was no significant statistical difference between NS group [(17.0+/-2.5)%] and BC group in apoptosis rate (F=14.288, P>0.05). The apoptosis rate of HUVECs in BI group was obviously lower than that in BS group (F=14.288, P<0.05). (3) Compared with those in BC group, the protein expressions of p-IkappaB-alpha in cytoplasm and NF-kappaB-p65 in nucleus were up-regulated, and the protein expression of p-Akt in cytoplasm was down-regulated in BS and IP groups. The expression levels of the three proteins in NS and BI groups were close to those in BC group. CONCLUSIONS: Insulin could inhibit the IkappaB phosphorylation, and then restrict NF-kappaB nuclear translocation and improve the vascular endothelial cells function accordingly through regulating phosphatidylinositol 3 kinase/Akt pathway.

[Effect of heat injured keratinocytes supernatant on biological behavior of fibroblasts].

OBJECTIVE: To observe the effect of the supernatant of heat injured keratinocytes (KC) on biological behavior of the dermal fibroblasts (Fb). METHODS: Human dermal Fb were isolated and cultured. A model of heat injured KC (HaCaT) was reproduced in vitro. Supernatant of normal KC and the supernatant of KC culture 12 hours after heat injury were collected and diluted with non-serum DMEM in 1:1 volume ratio to make normal KC conditioned medium (NKCM) and heat injury KC conditioned medium (HKCM) respectively. Fb was respectively treated with non-serum DMEM and 2 kinds of conditioned medium. (1) The proliferation of Fb was detected with MTT method at post culture hour (PCH) 12, 24, 36, 48. (2) The apoptosis of Fb was determined by flow cytometry at PCH 12 (Fb were heat injured in advance; Fb without heat treatment was used as control). (3) At PCH 24, expression of a-SMA in Fb cytoplasm was determined with immunofluorescence method; expression of a-SMA mRNA in Fb was determined with real-time quantified PCR. Data were processed with one-way analysis of variance, and pairwise comparison among groups with LSD-t test. RESULTS: (1) The proliferation of Fb: the absorbance value of Fb cultured with HKCM at PCH 12, 24, 36, 48 was respectively higher than that of Fb cultured with non-serum DMEM (with t value respectively 1.89, 2.35, 2.02, 1.94, and P values all below 0.01). There were significant statistical differences between the absorbance values of Fb cultured with HKCM and those of Fb cultured with NKCM at PCH 12, 24, and 48 (at PCH 12, t = 1.83, P < 0.01; at PCH 24, t = 2.91, P < 0.05; at PCH 48, t = 1.83, P < 0.05). (2) Apoptosis of Fb cultured with HKCM was diminished as compared with that of Fb cultured with NKCM and of Fb without treatment (t = 3.31, P < 0.05; t = 1.47, P < 0.01). (3) The expression of alpha-SMA (red fluorescence) in Fb cultured with non-serum DMEM or NKCM was less as seen under fluorescence scope, and it was obviously increased in Fb cultured with HKCM. (4) The relative expression amount of alpha-SMA mRNA in Fb cultured with HKCM was 1.32 +/- 0.06, which was higher than that both in Fb cultured with NKCM (1.14 +/- 0.07, t = 2.51, P < 0.05) and in Fb cultured with non-serum DMEM (1.00 +/- 0.09, t = 1.77, P < 0.05). CONCLUSIONS: The supernatant of KC 12 hours after heat injury can obviously promote the proliferation of Fb, inhibit its apoptosis and accelerate transdifferentiation of Fb to myofibroblasts.

[Effects of insulin on the growth factor secreting function of adipose-derived stem cells].

OBJECTIVE: To study the effect of insulin in different concentrations on secretion function of growth factors of adipose-derived stem cells (ADSCs). METHODS: ADSCs were isolated from human abdominal adipose tissue and cultured. The immunophenotype and adipose induced-differentiation were identified, and the third generation cells were collected. The collected cells were assigned to 1 x 10(-8), 1 x 10(-7), 1 x 10(-6) mol/L insulin groups according to the concentration of added insulin. When cells grew into 70% confluence in conventional medium, ADSCs were cultured further in serum-free DMEM containing insulin in different concentrations for 3 days. ADSCs cultured in medium without insulin were used as control group. Secretion amount of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) of ADSCs were determined by enzyme-linked immunosorbent assay. The effects of the supernatant fluid of ADSCs' nutrient solution on the proliferation and collagen synthesis of the cultured fibroblast were detected by MTT chromatometry and hydroxyproline chromatometry. RESULTS: The secretion amounts of VEGF and HGF of ADSCs in 1 x 10(-8) and 1 x 10(-7) mol/L insulin groups [(471 +/- 41, 762 +/- 66 ng/L), (643 +/- 64, 930 +/- 67 ng/L), respectively] were significantly higher as compared with those in control group (286 +/- 47, 577 +/- 84 ng/L) (P < 0.05 or P < 0.01). No change occurred in the secretion amount of VEGF and HGF of ADSCs in 1 x l0(-6) mol/L insulin group (P > 0.05). The supernatant fluid of ADSCs' nutrient medium of 1 x 10(-8), 1 x 10(-7) mol/L insulin groups showed obvious stimulative effect on the proliferation and collagen synthesis of fibroblasts, and it was most obvious in the 1 x 10(-7) mol/L group (P < 0.05 or P < 0.01). CONCLUSIONS: Insulin in the concentrations of 1 x 10(-8) and 1 x 10(-7) mol/L can notably promote ADSCs' function of secreting VEGF and HGF.

[The anti-apoptosis effect of intensive insulin treatment on cardiac myocytes in severe scald rats].

OBJECTIVE: To investigate the anti-apoptosis effect of intensive insulin treatment on cardiac myocytes and its underlying mechanism in severe scald rats. METHODS: Twelve SD rats were suffered from 30% TBSA full thickness scald, and they were divided into: IT group [with intravenous injection of isotonic saline including insulin (15 mU x kg(-1) x min(-1)) and 100 g/L glucose], B group [with treatment of isotonic saline (2 mL x kg(-1) x %TBSA(-1) x 8 h(-1)]. Six SD rats received sham burn as controls[sham(S)group, with treatment of fluid at physiologic dose]. + dp/ dtmax (the rate of the rise of left ventricular pressure) and -dp/ dtmax (the rate of the fall of left ventricular pressure)at 6 post burn hour (PBH)were recorded. Apoptosis were determined by TUNEL staining and DNA ladder. The phosphorylation f Akt and protein expression of Bcl-2 in cardiomyocyte were assayed by Western blotting. RESULTS: The + dp/ dtmax in the S group, IT group and B group at6 PBH were respectively (5.5 +/- 0.5) x 10(3) mm Hg/s, (3.4 +/- 0.4) x 10(3 mm Hg/s and (2.5 +/- 0.5) x 10(3) mm Hg/s (1 mm Hg = 0.133 kPa), the - dp/ dtmax were respectively (4.55 +/- 0.34) x 10(3) mmHg/s, (2.94 +/- 0.22) x 10(3) mm Hg/s and (2.05 +/- 0.19) x 10(3) mmHg/s.The +/- dp/dtmax in IT group was significantly higher than those in B group( P < 0.01). The apoptosis index in B group was (13.1 +/- 3.4)%, which was obviously higher than that in IT group (6.7 +/- 1.8)% and S group (0.6 +/- 0.4)% (P < 0.01). DNA ladder showed that no DNA fragmentation in S group, but obvious DNA fragmentation forming ladder pattern in B group, and no obvious ladder pattern in IT group. The phosphorylation of Akt and level of Bcl-2 protein in B group were markedly higher than those in IT group ( P < 0.05 or P < 0.01). CONCLUSION: Intensive insulin treatment can upregulate the activity of Akt and enhance the expression of Bcl-2, and they might constitute the mechanisms for anti-apoptosis in cardiomyocyte and protection of cardiac function.

[Study on crosstalk between phosphatidylinositol 3 -kinase/Akt pathway and p38 mitogen-activated protein kinase pathway in cardiomyocyte with challenge of burn serum].

OBJECTIVE: To investigate the possibility of crosstalk between phosphatidylinositol 3-kinase (PI3-K)/Akt pathway and p38 mitogen-activated protein kinase (p38MAPK) pathway in cardiomyocyte with challenge of burn serum, and to explore their influence on cardiomyocyte injury after burn. METHODS: The model of murine cardiomyocyte with stimulation of burn serum was established. (1) The level of Akt and p38 phosphorylation in cardiomyocyte were examined with stimulation of 10% burn serum before stimulation and 1, 3, 6, 12, 24 hour after stimulation. (2) The levels of Akt and p38 phosphorylation in cardiomyocyte were determined with stimulation of burn serum (at concentration of 5%, 10%, 20%) or 10% burn serum plus insulin (at concentration of 1 x 10(-8), 1 x 10(-7), 1 x 10(-6)mol/L). The content of creatine kinase (CK) in supernate was also detected. (3) Addition to the inhibitor of p38 MAPK pathway (SB203580) and PI3K/Akt pathway (LY294002), the level of p38MAPK, PI3K/Akt and the content of CK in supernate were determined. RESULTS: (1) The level of p-p38 in cardiomyocyte was 4.0 +/- 0.8, 3.6 +/- 0.8, 5.1 +/- 1.6, 2.4 +/- 0.5, 3.0 +/- 0.6 at 1, 3, 6, 12, 24 hour (s) after stimulation of burn serum, which was obviously higher than that immediate after stimulation (1.0, P < 0.01). The level of p-Akt was 0.15 +/- 0.07, 0.64 +/- 0.10, 0.26 +/- 0.08, 0.38 +/- 0.11, 0.59 +/- 0.13, which was obviously lower than that before stimulation (1.00, P < 0.01). (2) With stimulation of different concentration of burn serum or burn serum plus insulin, the level of p-Akt and p-p38 changed in the opposite directions comparatively. The content of CK increased along with increase of burn serum concentration, but decreased obviously with treatment of insulin (P < 0.05 or 0.01). (3) Low level of p38 induced by burn serum was increased after treatment of LY294002, which neutralized the protection of insulin (P < 0.01). Low level of p-Akt induced by burn serum increased after treatment of SB203580 (P < 0.01), which inhibited the release of CK induced by burn serum. CONCLUSION: There is being crosstalk between PI3K/Akt pathway and p38 MAPK pathway in cardiomyocytes with challenge of burn serum, which may regulate cardiomyocytes.

[The protective effect of early insulin treatment on vascular endothelial cells in severely scalded rats].

OBJECTIVE: To investigate the protective effect of insulin on vascular endothelial cells of rats at early post-burn stage,and its mechanism. METHODS: Adult male Sprague-Dawley rats were randomly divided into 3 groups: i. e, sham scald group (n = 7), scald group (n = 7) and treatment group (n = 7). The rats in the latter 2 groups were subjected to 30% TBSA full-thickness burns with 94 degrees C water, and the sham scald rats were treated with 37 degrees C water. Intra-peritoneal injection of 40 ml/kg isotonic saline solution and subcutaneous injection of 3 units/kg insulin were given to the rats in treatment group after being subjected to 30% TBSA full-thickness burns. Subcutaneous injection of equal amount of isotonic saline was given to the sham and burn groups. The changes in vascular endothelial cell structure were observed with electron microscopy at 24 post-scald hours(PSH). Meanwhile, the blood glucose contents, the serum levels of nitric oxide (NO) and nitric oxide synthetase (NOS) were determined with oxidase method and colorimetric method, respectively. RESULTS: The injury of arterial endothelial cells in the treatment group was obviously alleviated compared with that in burn group. The blood glucose content in the treatment group (7.1 +/- 0.7 mmol/L) was significantly lower than that in scald group (8.2 +/- 1.0 mmol/L, P < 0.05), though it was much higher in both groups than that in sham scald group (4.9 +/- 0.8 mmol/L, P < 0.01) at 24 PBH. The serum content of NO, total NOS and cNOS in treatment group were obviously higher than those in scald group (P < 0.01), but there was no obvious difference in iNOS content between the two groups(P > 0.05). CONCLUSION: Insulin exhibits protective effect on vascular endothelial cells in severely scalded rats at the early post-burn stage, and it is attributed to its promotion of cNOS level leading to NO production.

[Protective effect of insulin on oxygen-radical induced hepatic injury in severely scalded rats].

OBJECTIVE: To investigate the protective effect of insulin on oxygen-radical induced hepatic injury in severely scalded rats in early stage of severe scald. METHODS: Eighty-four male Sprague-Dawley rats were randomly divided into three groups: i. e, normal group, saline group, and insulin group, with 28 rat in each group. The rats in the latter two groups were subjected to 30% TBSA full-thickness scald on the back, and received intra-peritoneal injection of 40ml/kg isotonic saline, and subcutaneous injection of 3 IU/kg insulin, respectively. The total anti-oxygen capability (T-AOC), the expression of superoxide dismutase (SOD), reactive oxygen species (ROS) and intercellular adhesion molecule (ICAM-1) in hepatic tissue, and serum alanine transaminase (ALT) were determined in each group at 6, 12, 24, 48 post-scald hours (PSH) with corresponding methods. RESULTS: The hepatic T-AOC and SOD content were obviously decreased, while the ROS content were markedly increased at 6 PSH in saline group compared with that in normal group (P < 0.05 or P < 0.01). The expression of ICAM-1 and serum content of ALT were significantly higher than that in normal group at 12 PSH and 48 PSH (P < 0.01). At 24 PSH, the hepatic T-AOC (386 +/- 75) U/g and SOD content (210 +/- 39 ) U/g were obviously higher in insulin group than those in saline group [(124 +/- 18), (111 +/- 9) U/g, respectively, P < 0.01), but the ROS content (154 +/- 29 ) U/g was much lower than that in saline group [(351 +/- 41) U/g, respectively, P < 0.01]. At 48 PSH, the serum content of ALT and hepatic expression of ICAM-1 in insulin group exhibited obvious difference when compared with those in saline group (P < 0.01). Meanwhile, Pathological examination showed that hepatic injury was alleviated by insulin administration after scald. CONCLUSION: Insulin administration early after severe scald exhibits protective effect on liver function by improving anti-oxygen radical ability of rat liver.

[The protective effect of intensive insulin treatment on the myocardium in severely scalded rats].

OBJECTIVE: To study the protective effect of intensive insulin treatment on the myocardium of severely scalded rats, and to primarily explore its mechanism. METHODS: Eighteen SD rats were divided into three groups, with 6 rats in each group. The rats in burn and intensive insulin group were inflicted with 30% TBSA full-thickness injury on the back. Isotonic saline containing 0.12 U/ml insulin solution, and 100 g/L glucose solution were infused into the rats in the intensive insulin group to keep plasma glucose at the level of 4.0 - 6.6 mmol/L (the total fluid amount was 2 ml x kg(-1) x 8h(-1)). In sham burn group,fluid was given according to physiological demand. The same amount of isotonic saline was infused into the rats in burn group. The venous blood was obtained for the detection of plasma glucose contents, and the left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP) were recorded via aortic ventricle cannula before scald and at 1, 2, 3, 4, 5, 6 post-scald hours (PSH). The tissue of the left ventricle was harvested at 6 PSH for the detection of troponin T expression in myocardiocytes. RESULTS: Plasma glucose level was increased to (7.6 +/- 1.7) mmol/L - (8.4 +/- 4.7) mmol/L in burn group during 1-6 PSH, which was significantly higher than that in intensive insulin group (4.5 +/- 0.9) mmol/L - (5.2 +/- 1.3) mmol/L, P < 0.01). Compared with the intensive insulin group, LVSP was markedly decreased in the burn group (60 +/- 11 mm Hg vs 72 +/- 8 mm Hg, P < 0.05) at 1 PSH,whereas LVEDP was increased significantly (21.3 +/- 11.3 mmHg vs 11.7 +/- 5.2 mmHg, P < 0.05). Intensive insulin treatment could significantly inhibit the loss of troponin T protein in myofilaments of myocardium. CONCLUSION: Intensive insulin treatment possesses a protective effect on myocardia function after severe burns, and it may be related to its preventive effect on the loss of contractile protein in cardiocytes.