Effects and mechanism of negative pressure microenvironment on the neogenesis of human umbilical vein endothelial cells / 中华烧伤杂志

Objective: To investigate the effects and mechanism of negative pressure microenvironment on the neogenesis of human umbilical vein endothelial cells (HUVECs). Methods: The experimental research methods were adopted. The third to the fifth passage of HUVECs in the logarithmic growth stage were used for the subsequent experiments. Three batches of cells were taken, with each batch of cells being divided into normal control group and negative pressure treatment alone group (both routinely cultured for 24 h), and 17-allylamino-17-demethoxy-geldanamycin (17-AAG) alone group and 17-AAG+negative pressure treatment group (both cultured with 17-AAG for 24 h). In addition, the intermittent negative pressure suction, with the negative pressure value of -5.33 kPa (suction for 30 s, pause for 10 s) was continuously applied for 8 h on cells in the two negative pressure treatment groups using an automatic three-dimensional cell gradient negative pressure loading device designed and developed by ourselves. After the treatment of the first batch of cells, the cell proliferation level was detected by cell counting kit 8 method at 0 (immediately), 24, 48, and 72 h of culture, with the number of samples being 6. After the treatment of the second batch of cells, the scratch experiment was performed. At 12 h after scratching, the cell migration was observed under an inverted phase contrast microscope and the cell migration rate was calculated, with the number of samples being 3. After the treatment of the third batch of cells, the tubule formation experiment was conducted. After 6 h of culture, the tubulogenesis was observed under an inverted phase contrast microscope and the total tubule length and the number of branch nodes of cells were calculated, with the number of samples being 3. The cells were taken and divided into normal control group, negative pressure treatment alone group, and 17-AAG+negative pressure treatment group. The cells were treated the same as in the previous corresponding group. After the treatment, Western blotting was used to detect the protein expressions of heat shock protein 90 (HSP90), caveolin 1, endothelial nitric oxide synthase (eNOS), and eNOS phosphorylation site 1177 in the cells, and the eNOS phosphorylation site 1177/eNOS ratio was calculated, with the number of samples being 3; co-immunoprecipitation (co-precipitating HSP90 and caveolin 1, caveolin 1 and eNOS) and Western blotting were used to detect the protein expressions of caveolin 1 and eNOS in the cells, with the number of samples being 3; the protein co-localization of HSP90 and caveolin 1 and that of caveolin 1 and eNOS in the cells was assessed by immunofluorescence double staining. The molecular docking prediction of caveolin 1 and eNOS was processed by HADDOCK 2.4 protein-protein docking program. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, and least significant difference method. Results: Compared with that in normal control group, the cell proliferation level in 17-AAG alone group was significantly decreased at culture hour of 24, 48, and 72 after the treatment (P<0.01), while the cell proliferation level in negative pressure treatment alone group was significantly increased at culture hour of 24, 48, and 72 after the treatment (P<0.01). Compared with that in 17-AAG alone group, the cell proliferation level in 17-AAG+negative pressure treatment group was significantly increased at culture hour of 48 and 72 after the treatment (P<0.05 or P<0.01). Compared with that in negative pressure treatment alone group, the cell proliferation level in 17-AAG+negative pressure treatment group was significantly decreased at culture hour of 24, 48, and 72 after the treatment (P<0.01). At 12 h after scratching, compared with (39.9±2.7)% in normal control group, the cell migration rate in 17-AAG alone group was significantly decreased ((10.7±2.7)%, P<0.01), while the cell migration rate in negative pressure treatment alone group was significantly increased ((61.9±2.4)%, P<0.01). Compared with those in 17-AAG alone group, the cell migration rate in 17-AAG+negative pressure treatment group was significantly increased ((37.7±3.7)%, P<0.01). Compared with that in negative pressure treatment alone group, the cell migration rate in 17-AAG+negative pressure treatment group was significantly decreased (P<0.01). At culture hour of 6 after the treatment, compared with those in normal control group, the total length of the tube formed by the cells in 17-AAG alone group was significantly shortened (P<0.05) and the number of branch nodes was significantly reduced (P<0.05), while the total length of the tube formed by the cells in negative pressure treatment alone group was significantly prolonged (P<0.01) and the number of branch nodes was dramatically increased (P<0.01). Compared with that in 17-AAG alone group, the number of branch nodes of the tube formed by the cells was significantly increased in 17-AAG+negative pressure treatment group (P<0.05). Compared with those in negative pressure treatment alone group, the total length of the tube formed by the cells in 17-AAG+negative pressure treatment group was significantly shortened (P<0.01) and the number of branch nodes was significantly reduced (P<0.01). Western blotting detection showed that after treatment, the overall comparison of eNOS and caveolin 1 protein expressions among the three groups of cells showed no statistically significant differences (P>0.05). The expression of HSP90 protein and the eNOS phosphorylation site 1177/eNOS ratio in the cells of negative pressure treatment alone group were significantly increased (P<0.01) compared with those in normal control group. Compared with those in negative pressure treatment alone group, the HSP90 protein expression and the eNOS phosphorylation site 1177/eNOS ratio in the cells of 17-AAG+negative pressure treatment group were significantly decreased (P<0.01). Co-immunoprecipitation and Western blotting detection after the treatment showed that compared with those in normal control group, the expression of caveolin 1 protein in the cells of negative pressure treatment alone group was significantly increased (P<0.01), while the protein expression of eNOS was significantly decreased (P<0.05). Compared with those in negative pressure treatment alone group, the expression of caveolin 1 protein in the cells of 17-AAG+negative pressure treatment group was significantly decreased (P<0.01), while the protein expression of eNOS was significantly increased (P<0.01). After the treatment, compared with those in normal control group, the co-localization of HSP90 and caveolin 1 protein in the cells of negative pressure treatment alone group was significantly increased, while the co-localization of caveolin 1 and eNOS protein was significantly decreased. Compared with those in negative pressure treatment alone group, the co-localization of HSP90 and caveolin 1 protein in the cells of 17-AAG+negative pressure treatment group was significantly decreased, while the co-localization of caveolin 1 and eNOS protein was significantly increased. Molecular docking prediction suggested that caveolin 1 interacted strongly with eNOS and inhibited the 1177 site phosphorylation of eNOS. Conclusions: The negative pressure microenvironment may inhibit the binding of caveolin 1 to eNOS by promoting the binding of HSP90 to caveolin 1 in HUVECs, so as to relieve the inhibition of 1177 site phosphorylation of eNOS by caveolin 1, thereby promoting the proliferation, migration, and tubulogenesis of HUVECs, and ultimately promoting the neogenesis of HUVECs.

Redox regulation of c-JNK signaling pathway on myocardial potassium channel reconstruction in diabetic rats / 解放军医学杂志

Objective To investigate the role of c-Jun NH2-terminal kinase (c-JNK) signaling pathway on voltage-gated potassium channel (Kv) remodeling in left ventricular myocytes of diabetic rats,and explore the intrinsic regulatory mechanism.Methods Forty-five SD rats were randomly divided into DM group (n=25,modeling with streptozotocin induction) and control group (n=20,fed with normal diet).Transient outward potassium current (Ito) of rats' ventricular myocytes in DM group and control group was recorded by whole-cell patch-clamp method.The c-Jun activity was detected using a non-radioactive JNK kinase assay kit (Cell Signaling Technology).JNK inhibitor SP600125 was used to incubate the cardiomyocytes of diabetes rats in vitro,and then the changes of I,o in cardiomyocytes were observed.Thioredoxin reductase (TrxR) inhibitor--auranofin (AF) was used to treat the rats' cardiomyocytes incubated with SP600125,and then the changes of Ito in cardiomyocytes were observed.The content of Kv4.2 was tested using anti-Kv4.2 antibody,and the results were analyzed using a UVP bioimaging system.Results The JNK activity in DM group rose more than 1 times compared with control group,while the density of Ito decreased significantly (Control:30.2 ± 3.3pA/pF,n=16;DM:15.3 ± 2.1pA/pF,n=17;P＜0.05).The ventricular myocytes of DM rats were treated with SP600125 (10μmol/Lol/L) for 4 hours,then the Ito density increased to control group level (DM+SP600125:32.3 ± 3.7pA/pF,n=18;Control:30.2 ± 3.3pA/pF,n=16;P＜0.05).There was no significant difference in the maximum Ito density between the treated with SP600125 (Control+SP600125:31.6 ± 3.4pA/pF,n=18) and untreated control groups.The Ito density in DM myocardial cells significantly increased after treatment with the membrane permeable protein inhibitor JNKI-1 (10μmol/L),and no changes were found in control group after the same treatment.The augmentation effect of SP600125 on Ito current in DM myocytes was significantly inhibited by TrxR inhibitor auranofin (lμmol/L) (DM+SP600125+AF:15.7 ± 3.3pA/pF,n=15),while AF did not change the Ito density in control group.The expression of Kv4.2 protein was significantly increased in DM rats after administration of SP600125,which was consistent with the changes of Ito current observed in the myocardium of DM rats,although not fully restored to the level of control group myocardium.JNK inhibitor did not markedly alter the expression of Kv4.2 protein in control group myocardium.Conclusions Kv channel remodeling in DM rat's myocardium is redox-regulated,and the Ito remodeling might be assisted with the persistent activation of c-JNK signaling pathway.It has showed that c-JNK activity is significantly increased in DM rat heart and the current density of Kv channels is reduced.The inhibition of JNK signaling pathway can markedly improve Kv channel reconstruction and the process may be regulated by thioredoxin system.

Observation on the fluid resuscitation and the change in blood sodium of severely burned patients in the early stage / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the necessary amount of fluid consisting of electrolyte and colloid, the ratio of electrolyte and colloid used, and the change of blood sodium during early resuscitation in severely burned patients.</p><p><b>METHODS</b>Sixty-seven patients with total burn surface area (TBSA) equal to or over 70% and full-thickness area equal to or over 50%TBSA, hospitalized from March 2004 to March 2009, were resuscitated with fluid. The infusion amount of electrolyte, colloid, and water, and urinary output of patients at post injury hour (PIH) 24, 48, and 72 were analyzed retrospectively. The variation in blood sodium and fluid infusion at different time points was recorded. Data were processed with SPSS 13.0 software.</p><p><b>RESULTS</b>Among the 67 patients, hyponatremia occurred in 9 cases, hypernatremia occurred in 5 cases, and 53 patients had normal blood sodium level. The urinary output of patients within PIH 72 was above 70 mL/h. K value was calculated through the formula: actual total infusion amount of electrolyte and colloid (mL) = burn area (%TBSA) x body weight (kg) x K. In the first 24 PIH, K value was about 1.7, and the ratio of electrolyte and colloid was 1.4. In the second 24 PIH, K value was about 1.3 with electrolyte and colloid ratio 1.6. K value in the third 24 PIH was about 0.9 with electrolyte and colloid ratio 2.0.</p><p><b>CONCLUSIONS</b>The actual amount of resuscitation fluid is slightly larger than that calculated from traditional formula during the early stage in severely burned patients. The amount of electrolytes and the proportion of electrolyte and colloid will influence blood sodium level of patients.</p>

Therapeutic effect of vacuum-assisted closure technology on infected explosion wound of pig / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the effect of vacuum-assisted closure (V. A. C.) technology in the treatment of infected wound of skin and soft tissue as a result of explosion injury in pig.</p><p><b>METHODS</b>Sixteen explosion wounds were established by electric detonators on the shoulders and hips on both sides of 4 small white domestic pigs ,and they were divided into A group [(without treatment and infection occurred on 1-2 post burn day (PBD), then treated with vaseline gauze on 3 (PBD)], and B group (with the same treatment as in A group, except for treatment of vacuum assisted closure (V. A. C) with pressure of - 15 kPa after 3 PBD). The data of wound depth, wound area, wound healing time were collected and analyzed at 3 PAD and 1, 3, 6, 9, 14, 19, 24 days after treatment. Specimens from wounds were collected for histopathology observation, including also cell proliferation index, the number of vascular endothelial cells, the activity of myeloperoxidase (MPO) and the number of bacteria.</p><p><b>RESULTS</b>Compared with those in A group on land 3 days after treatment, wound area, wound depth were not enlarged or deepened in B group, while the number of inflammatory cells, vascular endothelial cells, proliferative cells were increased, the activity of MPO was enhanced and the number of bacteria was decreased. There were obvious differences between two groups in following indices: wound area ,wound depth, the number of vascular endothelial cells and bacteria during 1 to 19 days after treatment (P < 0.01)), the number of cell proliferation from 1 - 9 days after treatment (P < 0.01)), and the activity of MPO on 3, 6 days after treatment (P < 0. 01). The wound healing time was (32.8 +/- 1.6) d in A group, which was longer than that in B group (25.8 +/- 1.0 d, P < 0.01).</p><p><b>CONCLUSION</b>Compared with conventional dressing change, V. A. C can decrease bacteria load, lessen secondary necrosis, prompt the inflammatory response, accelerate the formation of granulation tissue, shorten wound healing time in infectious wound of porcine skin and soft tissue resulted from explosion injury.</p>

Effect of strengthening Pi and activating blood circulation therapy on serum levels of adrenocorticotrophic hormone and vascular endothelial growth factor in rheumatoid arthritis patients / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the effect of strengthening Pi and activating blood circulation therapy (SPAB) on serum levels of adrenocorticotrophic hormone (ACTH) and vascular endothelial growth factor (VEGF) in rheumatoid arthritis (RA) patients.</p><p><b>METHODS</b>Seventy RA patients were randomly assigned to 3 groups. The 30 in the Chinese medicine (CM) group were treated with SPAB; the 20 in the Western medicine (WM) group were treated with indomethacin and methopterin; and the 20 in the CM-WM group were treated with the combination of the therapy in the above two groups, and the course of treatment was 3 months for all. Serum levels of ACTH and VEGF were determined before and after treatment.</p><p><b>RESULTS</b>The total effective rate in the CM group was 80.0%, WM group 85.0% and CM-WM group 95.0%, the last one showed the best efficacy (P<0.05). Serum level of ACTH increased and level of VEGF decreased after treatment in all groups (P<0.05 or P<0.01), but the increment/decrement in the CM-WM group was higher than that in the other two groups (P<0.01), while comparison between that in the CW group and WM group showed insignificant difference.</p><p><b>CONCLUSION</b>SPAB therapy has the effect in alleviating the condition of RA, similar to that of Western medicine, it can increase the serum level of ACTH and decrease the serum level of VEGF remarkably in RA patients.</p>

The changes in blood flow in sciatic nerve after electrical injury in rabbit / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study the changes in sciatic nerve blood flow and the expression of collagen type I after electric injury of rabbit nerve with different voltages.</p><p><b>METHODS</b>Thirty-six healty rabbits were randomized into 3 groups before receiving injury with electricity in voltages, i.e. 50 v, 75 v, and 100 v groups. The changes in blood flow of sciatic nerve were observed with Laser Doppler Flowmeter immediately after injury and 1, 4, 8 weeks after injury. The changes in the expression of collagen type I was observed by immunohistochemical method, and the positive expression rate was calculated.</p><p><b>RESULTS</b>The sciatic nerve blood flow increased in all groups immediately after electric injury. In the 75 v and 100v groups, the nerve blood flow [(53 +/- 3 ), (48 +/- 5) PU] was obviously lower than that of normal value [(62 +/- 4) PU, P < 0.05]. There was little collagen type I deposition in 50 v group, while brown collagenous fibers in epineurium and perineurium were observed in 75 v and 100v groups 4 and 8 weeks after injury. The expression of collagen type I in all groups were obviously higher than that of normal value, and that in 75v and 100 v groups were higher than that in 50 v group at bachl time-point (P < 0.01).</p><p><b>CONCLUSION</b>The restoration of sciatic nerve blood flow is postponed following by the injury with increase of the electrical voltage. The collagen deposition after electrical injury may be one of the reasons for nerve blood flow decrease.</p>

The influence of vacuum-assisted drainage on the growth of capillaries in the wound produced by explosion in pig / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the influence of vacuum-assisted closure (VAC) technique on the growth of capillaries in the wound of the pig produced by explosion.</p><p><b>METHODS</b>Four small white pigs were inflicted with 16 explosion wounds [(7.3 +/- 1.0) cm2 in area] on both sides of the buttocks, shoulders and hips by detonation of a specific type of explosive, and the wounds were randomly divided into 2 groups, i. e, control (C, with conventional treatment from 2 post-injury day (PID) on and treatment (T, with VAC treatment after debridement from 2 PID on) groups, with 8 wounds in each group. Wound tissues of 2mm x 2mm x 2mm in size were harvested for pathological examination before treatment and on 1 and 3 post-treatment day (PTU). The differentiation of adventitial cells were examined with light microscope, and the pixel value of desmin positive particles and the luminal area of newly formed capillaries were assessed with Image C software.</p><p><b>RESULTS</b>Most of vessels in the wound of both groups were in elliptic shape when observed in longitudinal section. In C group, few newly formed capillaries vessels with lack of pericytes were observed before treatment and on 1, 3 PTD, then the number began to increase on 6 PTD. In T group, the number of newly formed capillaries with pericytes was increased on 1 PTD, and it continued to increase thereafter. The pixel values of desmin positive particles in C group on 1, 3, and 6 PTD were (91 +/- 54), (199 +/- 85), and (1552 +/- 298), respectively, which were obviously higher than those in T group [(2569 +/- 330), (3984 +/- 377), (9611 +/- 960), P < 0.01]. The area of vessel lumen in C group was (59 +/- 36), (250 +/- 70), and (938 +/- 287) microm2, respectively on 1, 3, and 6 PTD, which was also smaller than those in T group [(818 +/- 234), (4518 +/- 1080), and (9058 +/- 1656) microm2, P < 0.01].</p><p><b>CONCLUSION</b>Compared with conventional therapy, VAC can not only accelerate the formation of new capillaries, but also enhance the differentiation of pericytes and the process of enwrapping them around the vessels, and increase the luminal area of newly formed capillaries.</p>

500 cases of external ultrasound-assisted liposuction / 中华整形外科杂志

<p><b>OBJECTIVE</b>To discuss the therapeutic effects and safety of external ultrasound-assisted liposuction and to find out the rules of circumference variation in different body areas after liposuction.</p><p><b>METHODS</b>External ultrasound liposuction was performed on 500 patients (595 sites). We recorded the volume of anesthetic drugs and the aspirated pure fat as well as the body contour variations of every area at one to three months postoperatively. The decreased dimension at different body sites after liposuction were compared and analyzed.</p><p><b>RESULTS</b>The volumes of the anesthetic liquid and the aspirated pure fat were the most in the waist-abdomen and the thigh. After liposuction, the circumstances in different body areas were all decreased to certain degrees. Along with the severity of obesity, changes in the waist-abdomen became more obvious and the decreases of the umbilical circumference, the minimum and the maximum waist circumferences were prone to be less. But when the, minimum waist circumference was over 111 cm, obvious changes occurred in the umbilical. Meanwhile, the upper-middle part of the thigh and the upper arm showed larger variations than the lower part. The ultrasound-assisted liposuction had less blood loss and pain. It resulted in smooth skin without severe complications.</p><p><b>CONCLUSIONS</b>External ultrasound-assisted liposuction is a safe, effective and easily acceptable operation for body contour remodeling. The body circumference variation may have its intrinsic rule, which, to some extent, is meaningful for conducting clinical inquiry and forecasting the operation results.</p>