Nevin Manimala

Med Intensiva (Engl Ed). 2021 Dec;45(9):516-531. doi: 10.1016/j.medine.2021.03.001.

ABSTRACT

OBJECTIVE: The “Open Lung Approach” (OLA), that includes high levels of positive end-expiratory pressure coupled with limited tidal volumes, is considered optimal for adult patients with ARDS. However, many previous meta-analyses have shown only marginal benefits of OLA on mortality but with statistical heterogeneity. It is crucial to identify the most likely moderators of this effect. To determine the effect of OLA strategy on mortality of ventilated ARDS patients. We hypothesized that the degree of recruitment achieved in the control group (PaO₂/FiO₂ ratio on day 3 of ventilation), and the difference in Mechanical Power (MP) or Driving Pressure (DP) between experimental and control groups will be the most likely sources of heterogeneity.

DESIGN: A Systematic Review and Meta-analysis was performed according to PRISMA statement and registered in PROSPERO database. We searched only for randomized controlled trials (RCTs). GRADE guidelines were used for rating the quality of evidence. Publication bias was assessed. For the Meta-analysis, we used a Random Effects Model. Sources of heterogeneity were explored with Meta-Regression, using a priori proposed set of possible moderators. For model comparison, Akaike’s Information Criterion with the finite sample correction (AICc) was used.

SETTING: Not applicable.

PATIENTS: Fourteen RCTs were included in the study.

INTERVENTIONS: Not applicable.

MAIN VARIABLES OF INTEREST: Not applicable.

RESULTS: Evidence of publication bias was detected, and quality of evidence was downgraded. Pooled analysis did not show a significant difference in the 28-day mortality between OLA strategy and control groups. Overall risk of bias was low. The analysis detected statistical heterogeneity. The two “best” explicative meta-regression models were those that used control PaO₂/FiO₂ on day 3 and difference in MP between experimental and control groups. The DP and MP models were highly correlated.

CONCLUSIONS: There is no clear benefit of OLA strategy on mortality of ARDS patients, with significant heterogeneity among RCTs. Mortality effect of OLA is mediated by lung recruitment and mechanical power.

PMID:34839883 | DOI:10.1016/j.medine.2021.03.001

Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2021 Sep;33(9):1110-1115. doi: 10.3760/cma.j.cn121430-20210402-00497.

ABSTRACT

OBJECTIVE: To investigate the possible mechanism of ultrasound therapy in the rat model of sepsis.

METHODS: Seventy-eight male Sprague-Dawley (SD) rats were randomly divided into Sham group (n = 12), septic model group (n = 22), ultrasound treatment group (n = 22), methyllycaconitine citrate (MLA) combined with ultrasound treatment group (n = 22). In the Sham group, only the abdomen was opened, the cecum was found to be free, without cecal ligation and puncture (CLP). In the septic model group, CLP was used to replicate the septic rat model. After operation, each group of rats were subcutaneously injected with preheated 37 centigrade normal saline. The rats in the ultrasound treatment group were treated with ultrasound [Philips IU22 L9-3 ultrasound instrument and 9 MHz probe were used to break the sequence in the spleen area once every 6 seconds, with 1 second for each time, the mechanical index (MI) was 0.72, and the treatment time was 10 minutes]. In the MLA combined with ultrasound treatment group, α7 nicotinic acetylcholine receptor (α7nAChR) specific blocker MLA 4 mg/kg was injected intraperitoneally 30 minutes before operation, and ultrasound treatment was performed 2 hours after operation. The levels of tumor necrosis factor-α (TNF-α) and interleukin (IL-1β, IL-6) in serum of each group were measured by enzyme-linked immunosorbent assay (ELISA) at 24 hours after operation. The 10-day survival rate of each group was recorded, and the symptoms of each group were evaluated by clinical disease score (CDS). The histopathological changes of lung and colon were observed under light microscope.

RESULTS: Compared with the Sham group, the 10-day survival rate of rats in the septic model group was decreased significantly [40% (4/10) vs. 100% (6/6)], the CDS was (10.73±2.19 vs. 6.17±0.58) and the levels of TNF-α, IL-6, and IL-1β were increased significantly at 24 hours after operation [TNF-α (ng/L): 42.00±8.92 vs. 13.16±3.19, IL-6 (ng/L): 129.37±25.04 vs. 63.99±12.92, IL-1β (ng/L): 254.98±67.27 vs. 76.83±25.39, all P < 0.01]. Compared with the septic model group, the survival rate in the ultrasound treatment group was improved [70% (7/10) vs. 40% (4/10)], but there was no significant difference (P > 0.05). The CDS (7.64±2.68 vs. 10.73±2.19) and the expressions of TNF-α, IL-6, and IL-1β were significantly reduced at 24 hours after operation [TNF-α (ng/L): 16.93±6.02 vs. 42.00±8.92, IL-6 (ng/L): 73.65±24.38 vs. 129.37±25.04, IL-1β (ng/L): 111.86±14.08 vs. 254.98±67.27, all P < 0.01]. Compared with the ultrasound treatment group, the survival rate in the MLA combined with ultrasound treatment group was reduced [60% (6/10) vs. 70% (7/10)], but the difference was not statistically significant (P > 0.05). CDS was significantly increased (9.55±2.72 vs. 7.64±2.68), and the levels of TNF-α, IL-6 and IL-1β were significantly increased at 24 hours after operation [TNF-α (ng/L): 34.61±7.89 vs. 16.93±6.02, IL-6 (ng/L): 112.92±10.42 vs. 73.65±24.38, IL-1β (ng/L): 212.57±32.16 vs. 111.86±14.08, all P < 0.01]. Microscopically, in the septic model group, the alveolar septum was thickened, a large number of inflammatory cells infiltrated, normal pulmonary reticular structure disappeared, and pulmonary interstitium showed obvious hemorrhage and edema, meanwhile, the structure of colonic villi was obviously abnormal, with cells were edema and inflammatory cell infiltration, and the arrangement was disordered, so that the subepithelial space and the top of it fell off. After ultrasound treatment, the thickness of the alveolar interval in rats was similar to that in Sham group, without obvious inflammatory cell infiltration, and the pulmonary reticular structure was relatively intact. At the same time, the morphology of colonic villi was basically normal and orderly, the edema of cell was not obvious, and subcutaneous space and tip fall off were not obvious. After being antagonized by MLA, the rat lung tissue showed thickened alveolar septum, inflammatory cell infiltration, incomplete pulmonary network structure, hemorrhage and edema in the interstitium. The villi structure of the colon was faintly visible, with obvious cell edema and inflammatory cell infiltration, and the arrangement was abnormal.

CONCLUSIONS: Ultrasound treatment improves the prognosis of septic rats, MLA can reverse the anti-inflammatory effect of ultrasound therapy by antagonizing α7nAChR, suggesting that the protective mechanism of ultrasound in sepsis may be related to activating the cholinergic anti-inflammatory pathway mediated by α7nAChR.

PMID:34839871 | DOI:10.3760/cma.j.cn121430-20210402-00497

Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2021 Sep;33(9):1084-1087. doi: 10.3760/cma.j.cn121430-20210425-00608.

ABSTRACT

OBJECTIVE: To observe the clinical effect of the cannula under laparoscopy, percutaneous puncture cannula, and conventional surgery cannula for peritoneal dialysis.

METHODS: From May 3, 2015 to February 14, 2020, 87 patients with end-stage renal disease needing peritoneal dialysis in Ningbo Zhenhai People’s Hospital were enrolled. These patients were divided into three groups including cannula under laparoscopy (23 cases), percutaneous puncture cannula (29 cases), and conventional surgery cannula (35 cases). The baseline characteristics, perioperative conditions (surgical time, post-surgical hospitalization time), the incidence of recent complications (abdominal hemorrhage, direct abdominal hemorrhage, incision pain, leakage, catheter shift, peritonitis), and long-term complications (catheter shift, peritonitis, hernia, thoracic and abdominal fistula, abdominal tube obstruction) among the three groups were compared.

RESULTS: Compared with the group of conventional surgery cannula, the operation time in the group of cannula under laparoscopy and the group of percutaneous puncture cannula were significantly shorter (minutes: 32.5±12.3, 28.9±11.8 vs. 61.3±15.4, both P < 0.05), the in-hospital stay in the group of cannula under laparoscopy and the group of percutaneous puncture cannula were reduced (days: 9.8±3.4, 9.2±2.6 vs. 10.7±3.2), but there was no statistical significance among the three groups (P > 0.05). The incidence of abdominal bleeding, rectus abdominis bleeding, and incision pain in the group of cannula under laparoscopy and the group of percutaneous puncture cannula were significantly lower than those in the group of conventional surgery cannula [incidence of abdominal bleeding: 4.3% (1/23), 3.4% (1/29) vs. 22.9% (8/35), incidence of rectus abdominis bleeding: 4.3% (1/23), 3.4% (1/29) vs. 22.9% (8/35), incidence of incision pain: 8.7% (2/23), 10.3% (3/29) vs. 42.9% (15/35), all P < 0.01]. The difference between the group of cannula under laparoscopy and the group of percutaneous puncture cannula had no statistical significance. Compared with the group of conventional surgery cannula and the group of percutaneous puncture cannula, the incidence of catheter displacement in the group of cannula under laparoscopy was significantly reduced [4.3% (1/23) vs. 27.6% (8/29), 31.4% (11/35), both P < 0.05]. Compared with the group of conventional surgery cannula and the group of percutaneous puncture cannula, the incidence of catheter displacement in long-term complications in the group of cannula under laparoscopy was significantly reduced [4.3% (1/23) vs. 24.1% (7/29), 31.4% (11/35), both P < 0.05], however, the difference of that between the group of conventional surgery cannula and the group of percutaneous puncture cannula was not statistically significant. The incidence of hernia in the group of cannula under laparoscopy was significantly higher than that in the group of percutaneous puncture cannula or in the group of conventional surgery cannula [21.7% (5/23) vs. 3.4% (1/29), 2.8% (1/35), both P < 0.05], and all of that were umbilical hernia, however, the difference of that between the group of percutaneous puncture cannula and the group of conventional surgery cannula was not statistically significant.

CONCLUSIONS: Compared with the traditional conventional surgical cannula placement methods, percutaneous puncture has the advantages of simple operation, short operation time, small trauma, but still cannot reduce the incidence of drift tube; laparoscopic peritoneal dialysis tube has the advantages of short operation time, small trauma and low catheter displacement rate, but increases the risk of umbilical hernia.

PMID:34839866 | DOI:10.3760/cma.j.cn121430-20210425-00608

Zhonghua Wai Ke Za Zhi. 2021 Dec 1;59(12):982-986. doi: 10.3760/cma.j.cn112139-20210902-00415. Online ahead of print.

ABSTRACT

Objective: To examine the therapeutic effects of drug-coated balloon (DCB) and bare metal stent (BMS) on primary femoropopliteal disease (FPAD) in the real world. Methods: This was a retrospective analysis of single-center follow-up results at 12,24,and 36 months of patients with FPAD lesions that were treated with DCB and BMS at Department of Aortic and Vascular Surgery, National Center for Cardiovascular Diseases, Fu Wai Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College.One-to-one propensity score matching(PSM) was performed to balance the covariance between DCB and BMS groups.There were both 71 patients in each group,aged (68.0±9.6) years(range: 46 to 90 years) and (68.8±7.3) years(range: 48 to 87 years),lesion lengths were (119.6±14.2)mm(range:40-380 mm) and (110.8±13.1)mm(range:40-400 mm).The covariates were balanced between DCB and BMS groups.Freedom from clinically driven target lesion reintervention (fCD-TLR) was determined by Kaplan-Meier curve.Log-rank test was used to compare the rates of fCD-TLR between DCB and BMS groups at 12,24,36 months post-operation. Results: The median follow-up period were 24.3 months (range:5.8 to 55.1 months).There was no death,amputation or reintervention within the 30 days after operation.The rates of fCD-TLR for DCB group at 12,24 and 36 months were 97.2%,85.9%,69.1%, and 95.8%,83.1%,59.2% for BMS group.There was no statistical difference between the two groups by Log-rank test (P=0.551). Conclusion: DCB and BMS can both maintain favorable clinical effects in FPAD patients at 12,24,36 months post-operation.

PMID:34839611 | DOI:10.3760/cma.j.cn112139-20210902-00415

Zhonghua Shao Shang Za Zhi. 2021 Nov 24;37:1-7. doi: 10.3760/cma.j.cn501120-20210325-00103. Online ahead of print.

ABSTRACT

Objective: To explore the clinical effects of artificial dermis combined with split-thickness skin for repair of wounds with bone and tendon exposure in the hand and foot. Methods: A prospective randomized controlled study was conducted. From October 2018 to February 2020, 82 patients with bone and tendon exposed wounds in the hand and foot admitted to Zhengzhou First People’s Hospital who met the inclusion criteria were selected. All the patients were divided into flap group (41 cases, including 27 males and 14 females) and artificial dermis+split-thickness skin group (41 cases, including 29 males and 12 females) according to the random number table, both aged (37±7) years. After complete debridement of wounds of patients in the two groups, the wound of patients in flap group was transplanted with anterolateral femoral free flap; the wound of patients in artificial dermis+split-thickness skin group was grafted with artificial dermis and given continuous negative pressure suction, and autologous lateral thigh split-thickness skin was grafted until complete vascularization of artificial dermis. One week after autologous skin graft/flap grafting, the survival of wound graft was observed and the graft survival rate was calculated. The complete wound healing time, number of operation, length of hospital stay, hospitalization cost, the occurrence of surgery-related complications during hospitalization after autologous skin graft/flap grafting were recorded, and the incidence of complication was calculated. Six months after autologous skin graft/flap grafting, the scar hyperplasia of grafting area was evaluated by Vancouver Scar Scale (VSS), while the recovery of hand and foot function were evaluated by Total Action Mobility (TAM) System Rating method and American Orthopaedic Foot and Ankle Society Ankle and Hindfoot Function Scale (AOFAS-AHS), respectively. Data were statistically analyzed with chi-square test, Fisher’s exact probability test, and independent sample t test. Results: One week after autologous skin graft/flap grafting, the survival rates of wound grafts were similar in the two groups (P>0.05). The complete wound healing time and length of hospital stay were (29±5) and (35±5) d for patients in artificial dermis+split-thickness skin group, respectively, which were significantly longer than (22±4) and (28±5) d in flap group (t=6.96, 6.22, P<0.01). Compared with those in flap group, the number of operation was fewer (t=7.39, P<0.01), the incidence of surgery-related complications during hospitalization after autologous skin graft/flap grafting was lower (P<0.01), but there was no significant change in hospitalization cost of patients in artificial dermis+split-thickness skin group (P>0.05). Six months after autologous skin graft/flap grafting, the VSS scores of grafting area of patients in the two groups were similar (t=0.32, P>0.05); the TAM score and AOFAS-AHS score of hand and foot function of patients in artificial dermis+split-thickness skin group were 40±6 and 62±12, respectively, which were significantly higher than 34±6 and 53±11 of flap group (t=4.66, 3.41, P<0.01). Conclusions: Repairing wounds with bone and tendon exposure in hand and foot with artificial dermis and split-thickness skin reduces the incidence of surgery-related complications, improves postoperative hand and foot joint function of patients, and results in fewer number of operation compared with flap repair, without significant scar hyperplasia, but may prolong wound healing time and length of hospital stay.

PMID:34839603 | DOI:10.3760/cma.j.cn501120-20210325-00103

Zhonghua Shao Shang Za Zhi. 2021 Nov 25;37:1-10. doi: 10.3760/cma.j.cn501120-20210601-00208. Online ahead of print.

ABSTRACT

Objective: To investigate the effects and cell signaling mechanism of glutamine on rat cardiomyocytes intervened with serum from burned rat (hereinafter referred to as burn serum). Methods: The experimental research method was applied. Ten gender equally distributed Wistar rats aged 7-8 months were taken to prepare normal rat serum (hereinafter referred to as normal serum), another twenty gender equally distributed Wistar rats aged 7-8 months were taken to prepare burn serum after full- thickness burn injury of 30% total body surface area, and cardiomyocytes were isolated from 180 Wistar rats aged 1-3 days by either gender and used in the following experiments. The cells were divided into normal serum group and burn serum group according to the random number table (the same grouping method below), and cultured with the corresponding serum. At post culture hour (PCH) 1, 3, 6, 9, and 12, trypanosoma blue exclusion test was used to detect the cell survival rate. The cells were divided into burn serum alone group, burn serum+4 mmol/L glutamine group, burn serum+8 mmol/L glutamine group, burn serum+12 mmol/L glutamine group, burn serum+16 mmol/L glutamine group, and burn serum+20 mmol/L glutamine group, and were treated with burn serum alone or added with the corresponding final molarity of glutamine and cultured with the time point screened in the experiment before, then the cell survival rate was dected as before. then The cells were divided into normal serum alone group, burn serum alone group, burn serum+12 mmol/L glutamine group, burn serum+16 mmol/L glutamine group, and burn serum+20 mmol/L glutamine group and treated the same as before. After 30 min of culture, phosphorylation level of mammalian target of rapamycin complex 1 (mTORC1), p70 ribosomal protein S6 kinase (P70 S6k), and eIF4E-binding protein 1 (4E-BP1) were detected by Western blotting. Cells were divided into normal serum group, burn serum alone group, burn serum+12 mmol/L glutamine group, burn serum+12 mmol/L glutamine+25 ng/mL rapamycin group, and treated correspondingly. At PCH 1, 3, and 6, the expressions of heat shock protein 70 (HSP70) and metallothionein (MT), and the morphology of microtubule were observed with immunofluorescence method. The number samples in each index at each time point in each group were all 10. Data were statistically analyzed with analysis of variance of factorial design, one-way analysis of variance, least significant difference t test, least significant difference test, and Bonferroni correction. Results: At PCH 1, 3, 6, 9, and 12, the cell survival rates in burn serum group were significantly lower than those in normal serum group (t=4.950, 16.752, 35.484, 34.428, 27.781, P<0.01). Compared within group at PCH 1, the cell survival rate was significantly decreased in burn serum group at PCH 3, 6, 9, and 12 (P<0.05). Compared within group at PCH 3, the cell survival rate was significantly decreased in burn serum group at PCH 6, 9, and 12 (P<0.05). Compared within group at PCH 6 and 9, the cell survival rate was significantly decreased in burn serum group at PCH 12 (P<0.05). There were no statistically significant differences in the cell survival rate among burn serum group at PCH 6 and 9 (P>0.05). Thus PCH 6 was selected as the subsequent intervention time of burn serum. At PCH 6, compared with burn serum alone group, the cell survival rates in burn serum+4 mmol/L glutamine group, burn serum+8 mmol/L glutamine group, burn serum+12 mmol/L glutamine group, burn serum+16 mmol/L glutamine group, and burn serum+20 mmol/L glutamine group were significantly increased (P<0.01). There were no statistically significantl differences in cell survival rate in burn serum+12 mmol/L glutamine group and burn serum+16 mmol/L glutamine group (P>0.05). There were no statistically significantl differences in cell survival rate in burn serum+16 mmol/L glutamine group and burn serum+20 mmol/L glutamine group (P>0.05). Thus 12, 16, and 20 mmol/L were selected as the subsequent intervention concentrations of glutamine. After 30 min of culture, the phosphorylation levels of mTORC1, P70 S6k, and 4E-BP1 of cells were respectively 1.001±0.042, 0.510±0.024, 0.876±0.022, 0.836±0.074, 0.856±0.041, 1.00±0.11, 0.38±0.09, 0.95±0.13, 0.96±0.13, 0.89±0.24, 1.00±0.07, 0.29±0.08, 0.87±0.27, 0.68±0.08, 0.60±0.21 in normal serum group, burn serum alone group, burn serum+12 mmol/L glutamine group, burn serum+16 mmol/L glutamine group, and burn serum+20 mmol/L glutamine group. Compared with normal serum group, the phosphorylation levels of mTORC1, P70 S6k, and 4E-BP1 of cells were decreased in the other 4 groups (P<0.01). Compared with burn serum alone group, the phosphorylation levels of mTORC1, P70 S6k, and 4E-BP1 of cells were increased in the other 3 groups (P<0.01). The phosphorylation levels of mTORC1, P70 S6k, and 4E-BP1 of cells were similar in burn serum+12 mmol/L glutamine group, burn serum+16 mmol/L glutamine group, and burn serum+20 mmol/L glutamine group (P>0.05). The phosphorylation level of 4E-BP1 of cells in burn serum+12 mmol/L glutamine group was significantly higher than the levels in burn serum+16 mmol/L glutamine group and burn serum+20 mmol/L glutamine group (P<0.05). At PCH 1, 3, and 6, the expressions of HSP70 and MT of cells in burn serum alone group were significantly higher than those in normal serum group (P<0.01); the protein expressions of HSP70 and MT of cells in burn serum+12 mmol/L glutamine group were significantly higher than those in burn serum alone group (P<0.05); the protein expressions of HSP70 and MT of cells in burn serum+12 mmol/L glutamine+25 ng/mL rapamycin group were significantly lower than those in burn serum+12 mmol/L glutamine group (P<0.05). The microtubular structure was integral, network alinement, stain uniformity in normal serum group at PCH 1, 3, and 6. In burn serum alone group, some microtubules were broken and the grid arrangement was disordered at PCH 1; the microtubule structure near the nucleus was clear, while the microtubule at the distal end of the nucleus was blurred at PCH 3; the microtubule structure was blurred at PCH 6. The microtubular damage of cells was alleviated in burn serum+12 mmol/L glutamine group compared with burn serum alone group. The morphology of microtubule of cells in burn serum+12 mmol/L glutamine+25 ng/mL rapamycin group at each time point was similar to that of burn serum alone group. Conclusions: The burn serum can lead to cardiomyocyte damage and cell survival rate decrease in mice. Glutamine can exert cell protective function through regulating the mTOR-P70 S6k-4E-BP1 signaling pathway, thus promoting the expressions of HSP70 and MT and stabilize the microtubule structure.

PMID:34839600 | DOI:10.3760/cma.j.cn501120-20210601-00208

Zhonghua Shao Shang Za Zhi. 2021 Nov 25;37:1-10. doi: 10.3760/cma.j.cn501120-20210424-00154. Online ahead of print.

ABSTRACT

Objective: To investigate the roles and mechanism of exosomes derived from human amniotic epithelial cells (hAEC-Exos) on the proliferation and migration of HaCaT in high glucose environment. Methods: The experimental research method was adopted. The amniotic membrane tissue was collected from 10 healthy full-term pregnant women in the Department of Obstetrics and Gynecology of Fujian Medical University Union Hospital from January to June 2019. The primary human amniotic epithelial cells (hAECs) were isolated, and the growth status and morphological change of the primary hAECs on the 2nd, 4th, and 7th day of culture were observed. The 2nd to 4th passages of hAECs were used in the following experiments after being identified by flow cytometry. The hAEC-Exos were separated by ultracentrifugation method. The HaCaT and hAEC-Exos were co-cultured for 3 h, and the uptaken of hAEC-Exos by HaCaT was observed by inverted fluorescence microscope. The HaCaT were divided into phosphate buffer solution (PBS) group and hAEC-Exos group or dimethyl sulfoxide (DMSO)+PBS group, DMSO+hAEC-Exos group and LY294002+hAEC-Exos group, which were dealt correspondingly, with 3 wells in each group. Cell counting kit 8 (CCK-8) was used to detect cell proliferation activity at 0 (immediately), 12, 24, 36, 48, and 60 h of culture. The scratch test was conducted to detect the scratch healing at 0, 24, 48, and 72 h after the scratch, and the scratch healing rate was calculated, respectively. The Transwell experiment was conducted to detect the number of transmembrane cells after 48 h of culture. The Western blotting was used to detect the protein expressions of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), kinase-protein kinase B (Akt), and phosphorylated Akt (p-Akt) related to phosphatidylinositol 3-kinase-Akt-mTOR (PI3K-Akt-mTOR) pathway after 24 h of culture. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and independent sample t test. Results: Most of the primary hAECs were oval and uniform in size on the 2nd day of culture. The hAECs were arranged in a typical cobblestone-like monolayer on the 4th and 7th day of culture. The primary hAECs were highly expressed CD73, CD90, and CD29 of mesenchymal stem cell related surface markers, and were with no or low expressions of CD34 and human leukocyte antigen DR of hematopoietic stem cell related surface markers. After 3 h of culture, hAEC-Exos were successfully endocytosed by HaCaT into the cytoplasm and gathered around the nucleus. After 12, 24, 36, 48, and 60 h of culture, the proliferation activity of HaCaT in hAEC-Exos group was significantly higher than that in PBS group (t=3.691, 10.861, 12.121, 10.531, 14.931, P<0.01). At 24, 48, and 72 h after scratch, the scratch healing rates of HaCaT in hAEC-Exos group were significantly higher than those in PBS group (t=3.342, 6.427, 5.485, P<0.05 or P<0.01). After 48 h of culture, the number of transmembrane HaCaT in hAEC-Exos group was significantly higher than that in PBS group (t=5.385, P<0.01). After 24 h of culture, the protein expressions of p-mTOR and p-Akt in HaCaT of hAEC-Exos group were significantly higher than those in PBS group (t=4.240, 5.586, P<0.01), while the protein expressions of mTOR and Akt in HaCaT of the two groups were similar (P>0.05). After 24 h of culture, the protein expressions of p-mTOR and p-Akt in HaCaT of DMSO+hAEC-Exos group were significantly higher than those in DMSO+PBS group (t=6.155, 8.338, P<0.01) and LY294002+hAEC-Exos group (t=5.030, 3.960, P<0.01), while the protein expressions of mTOR and Akt in the three groups were similar (P>0.05). The proliferation activity of HaCaT in DMSO+hAEC-Exos group at 12, 24, 36, 48, and 60 h of culture was 0.78±0.05, 1.23±0.07, 1.60±0.09, 1.86±0.09, and 2.03±0.08, which was significantly higher than 0.46±0.04, 0.69±0.07, 0.98±0.08, 1.16±0.08, and 1.26±0.11 in DMSO+PBS group (t=4.376, 7.398, 8.488, 9.766, 10.730, P<0.01). The proliferation activity of HaCaT in DMSO+hAEC-Exos group at 24, 36, 48, and 60 h was significantly higher than 0.96±0.09, 1.20±0.08, 1.39±0.08, and 1.55±0.10 in LY294002+hAEC-Exos group (t=3.639, 5.447, 6.605, 6.693, P<0.05 or P<0.01). The scratch healing rates of HaCaT in DMSO+hAEC-Exos group at 24, 48, and 72 h after scratch were significantly higher than those in DMSO+PBS group (t=4.003, 6.349, 7.714, P<0.01) and LY294002+hAEC-Exos group (t=3.805, 4.676, 4.067, P<0.05 or P<0.01). After 48 h of culture, the number of transmembrane HaCaT in DMSO+hAEC-Exos group was significantly higher than that in DMSO+PBS group and LY294002+hAEC-Exos group, respectively (t=7.464, 1.232, P<0.01). Conclusions: PI3K-Akt-mTOR pathway can promote the proliferation and migration of HaCaT in high glucose environment by mediating hAEC-Exos.

PMID:34839599 | DOI:10.3760/cma.j.cn501120-20210424-00154

Zhonghua Shao Shang Za Zhi. 2021 Nov 25;37:1-9. doi: 10.3760/cma.j.cn501120-20200927-00424. Online ahead of print.

ABSTRACT

Objective: To investigate the effects of temperature-sensitive hydroxybutyl chitosan hydrogel on wound healing of full-thickness skin defect in rats. Methods: The experimental research method was used. Fifty-one male and female Sprague-Dawley rats aged 7-10 weeks were selected, and two round full-thickness skin defect wounds with a diameter of 2 cm were made on the back of the rats at a distance of about 1.0 cm to the spine. The wounds were divided into temperature-sensitive hydrogel group, gel group, and blank control group, with 34 wounds in each group. Wounds in the first two groups were smeared with 0.3 mL temperature-sensitive hydroxybutyl chitosan hydrogel and carboxymethyl chitosan hydrogel, respectively immediately after injury, and the wounds in the blank control group were dealt with no treatment. The wounds in the three groups were all covered with vaseline gauze. The state of temperature-sensitive hydroxybutyl chitosan hydrogel and carboxymethyl chitosan hydrogel was observed every day when the dressing was changed, and the difficulty of removing vaseline oil gauze was recorded. On the 3rd, 7th, 10th, 14th, and 21st day after injury, the wound healing of the three groups was observed and the wound healing rate was calculated. On the 3rd, 7th, 10th, 14th, and 21st day after injury, 4 wounds of 2 rats in each group were collected for the following observation and detection. The inflammatory cell infiltration, angiogenesis, and re-epithelialization were observed by hematoxylin eosin staining. The regeneration and remodeling of collagen were observed by Masson staining, and the the collagen volume fraction was calculated.The expressions of interleukin-6 (IL-6), transforming growth factor β₁ (TGF-β₁), and matrix metalloproteinase-1 (MMP-1) were detected by enzyme-linked immunosorbent assay method. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, and Bonferroni test. Results: Chitosan gel was liquid gel and could flow with the body position, while the temperature-sensitive hydroxybutyl chitosan hydrogel was solid gel and could not flow with the body position, and the distribution of the latter was more uniform. The vaseline gauze was easy to remove in wounds of temperature-sensitive hydrogel group, which was not easy to remove in the other two groups. On the 3rd, 7th, 10th, 14th, and 21st day after injury, the wound granulation tissue grew well in temperature-sensitive hydrogel group and gel group, with no obvious infection, and two rats in blank control group died of wound infection on the 3rd and 5th day after injury. On the 7th, 10th, 14th, and 21st day after injury, the wound healing rates in temperature-sensitive hydrogel group and gel group were significantly higher than that in blank control group (P<0.01). On the 10th day after injury, the wound healing rate in temperature-sensitive hydrogel group was significantly higher than that in gel group (P<0.05). A large number of neutrophils and lymphocytes infiltrated into the wounds in the three groups on the 3rd day after injury. The inflammatory cell infiltration was gradually reduced and the wound healed gradually in temperature-sensitive hydrogel group and gel group from the 7th to 21st day after injury, and the epidermis and dermis could be seen, without hair follicles and other skin appendages. The wounds in blank control group did not heal completely on 21st day after injury. From the 3rd to 10th day after injury, the number of newly formed collagen fibers increased gradually in the wounds of the three groups. On the 14th and 21st day after injury, the collagen fibers in the wounds of temperature-sensitive hydrogel group and gel group were denser and more orderly than those in blank control group. On the 10th, 14th, and 21st day after injury, the collagen volume fraction of wounds in temperature-sensitive hydrogel group and gel group was significantly higher than that in blank control group (P<0.01). On the 14th day after injury, the collagen volume fraction of wounds in temperature-sensitive hydrogel group was significantly higher than that in gel group (P<0.01). On the 3rd, 7th, and 10th day after injury, the expressions of IL-6 of wound in temperature-sensitive hydrogel group were significantly higher than those in gel group and blank control group (P<0.01), and the expressions of IL-6 of wound in gel group were significantly lower than those in blank control group (P<0.01). On the 3rd, 7th, and 10th day after injury, the expressions of TGF-β₁ of wound in temperature-sensitive hydrogel group were significantly higher than those in gel group and blank control group (P<0.01). The expressions of TGF-β₁ of wound in gel group were significantly lower than those in blank control group on the 3rd, 7th day after injury (P<0.01), and the expression of TGF-β₁ of wound in gel group was significantly higher than that in blank control group on the 10th day after injury (P<0.01). On the 14th day after injury, the expression of TGF-β₁ of wound in gel group was significantly higher than that in temperature-sensitive hydrogel group and control group (P<0.01). On the 21st day after injury, the expression of TGF-β₁ of wound in temperature-sensitive hydrogel group was significantly lower than that in gel group and blank control group (P<0.01), and the expression of TGF-β₁ of wound in gel group was significantly lower than that in blank control group (P<0.01). On the 7th day after injury, the expression of MMP-1 of wound in gel group was significantly higher than that in temperature-sensitive hydrogel group and blank control group (P<0.01). On the 10th, 14th, and 21st day after injury, the expressions of MMP-1 of wound in temperature-sensitive hydrogel group were significantly higher than those in hydrogel group and blank control group (P<0.01). On the 10th day after injury, the expression of MMP-1 of wound in hydrogel group was significantly lower than that in blank control group (P<0.01). On the 14th and 21st day after injury, the expressions of MMP-1 of wound in hydrogel group were significantly higher than those in blank control group (P<0.01). Conclusions: Temperature-sensitive hydroxybutyl chitosan hydrogel can promote the healing of full-thickness skin defect wounds in rats by increasing the expression of IL-6, TGF-β₁, and MMP-1, regulating the wound healing environment, inhibiting inflammatory reaction, improving the strength of tissue repair, and promoting collagen synthesis and decomposition.

PMID:34839597 | DOI:10.3760/cma.j.cn501120-20200927-00424

Zhonghua Shao Shang Za Zhi. 2021 Nov 25;37:1-12. doi: 10.3760/cma.j.cn501120-20200920-00418. Online ahead of print.

ABSTRACT

Objective: To explore the effects of porcine acellular dermal matrix (ADM) combined with human epidermal stem cells (ESCs) on wound healing of full-thickness skin defect in nude mice. Methods: Experimental research methods were applied. The physical properties of ADM were analyzed by photograph of digital camera, hematoxylin eosin (HE) staining, scanning electron microscope, infrared spectrometer, particle size analyzer and nano-particle size potentiometer. The biological properties of ADM were analyzed by fluorescence inverted microscope, weighing method (ADM was divided into 5 min group, 10 min group, 20 min group, 30 min group, 60 min group, and 120 min group according to the random number table (the same grouping method as below) and in static state at normal temperature for the corresponding time to calculate the water absorption, with sample number of 3), cytotoxicity test and rating (mouse embryonic fibroblasts (Fbs) were divided into blank control group (culture medium only), 50.0 g/L ADM extract group, 37.5 g/L ADM extract group, 25.0 g/L ADM extract group, 12.5 g/L ADM extract group, and 6.5 g/L ADM extract group (the latter 5 groups were added with the corresponding final concentrations of AMD extract respectively). At post culture hour (PCH) 24, 48, and 72, the cell survival rate was detected by cell counting kit 8, with sample number of 5), and blood compatibility test (the erythrocytes of a 6-week-old male Sprague-Dayley rat were divided into normal saline group, ultra-pure water group, 5 mg/mL ADM extract group, 10 mg/mL ADM extract group, and 15 mg/mL ADM extract group and treated with the corresponding reagents of corresponding concentration. After reaction for 3 h, the absorbance value was detected by microplate reader, with sample number of 3). ESCs were isolated, cultured, and identified from the discarded prepuce of a 6-year-old boy who was treated in the Department of Urology of the First Affiliated Hospital of Army Medical University (the Third Military Medical University) in July 2020. ADM particles of composite ESC (hereinafter referred to as ESC/ADM) were constructed by mixed culture. After 3 days of culture, the composite effect of ESC/ADM was observed by HE staining and laser confocal microscope. Thirty-six 7-8-week-old male non-thymic nude mice were divided into phosphate buffer solution (PBS) alone group, ADM alone group, ESC alone group, and ESC/ADM group, with 9 mice in each group, and the wound model of full-thickness skin defect was established. Immediately after injury, the wounds were treated with the corresponding reagents at one time. On post injury day (PID) 7, 11, and 15, the wound healing was observed and the wound healing rate was counted (with sample number of 3). On PID 7, the epithelialization of wounds was observed by HE staining and the epithelialization length was measured (with following sample numbers of 4). On PID 11, the dermis area and collagen deposition of each group were observed by Masson staining and calculated, the number of cells expressing CD49f, a specific marker of ESC, was calculated with immunofluorescence staining, the mRNA expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene in ESC after wound transplantation with detected by real-time fluorescence quantitative PCR. Data were statistically analyzed with independent sample t test, one-way analysis of variance, analysis of variance for repeated measurement, and least signigicant difference t test. Results: ADM was white particles and composed of reticular structure, with disordered structure, rough surface, and no cells inside. The absorption peak of ADM appears at the wave numbers of 1 659, 1 549 and 1 239 cm^-1, respectively. The main particle size distribution of ADM in solution was 500 to 700 nm, with negative charge on the surface. The morphology of ADM in static state at 30 min and on 1 and 5 d were relatively stable. The water absorption of ADM reached its peak in static state at 30 min and remained relatively stable after that. The cytotoxicity of mouse Fbs in 6.5 g/L ADM extract group, 12.5 g/L ADM extract group, and 25.0 g/L ADM extract group was grade 1 at PCH 24, and the cytotoxicity of the other groups was 0 grade at each time point. After reaction for 3 h, the absorbance value of hemoglobin in ultrapure water group was significantly higher than the values in normal saline group and 15 mg/mL ADM extract group (t=8.14, 7.96, P<0.01). The results showed that hemolysis did not occur in 5 mg/mL ADM extract, 10 mg/mL ADM extract, nor 15 mg/mL ADM extract. The cells of the fourth passage showed pebble-like morphology, low expression of CD71 and high expression of CD49f, which were identified as ESCs. There was ESC attachment and growth on ADM particles. On PID 1, the wound sizes of nude mice were almost the same in PBS alone group, ADM alone group, ESC alone group, and ESC/ADM group. On PID 7 and 15, the wound contraction of nude mice in each group was observed, especially in ADM alone group, ESC alone group, and ESC/ADM group. On PID 7, the wound healing rates of nude mice in ESC alone group and ESC/ADM group were significantly higher than the rate in PBS alone group (P<0.05 or P<0.01). On PID 11, the wound healing rate of nude mice in ESC/ADM group was significantly higher than that in PBS alone group (P<0.01). On PID 15, the wound healing rates of nude mice in ADM alone group, ESC alone group, and ESC/ADM group were significantly higher than the rate in PBS alone group (P<0.05). On PID 7, the length of un-epithelialized wound of nude mice in ADM alone group, ESC alone group, and ESC/ADM group was (816±85), (635±66), (163±32) μm, respectively, which were significantly lower than (1 199±43) μm in PBS alone group (t=5.69, 10.19, 27.54, P<0.01). On PID 11, the dermal areas of wounds of nude mice in ADM alone group, ESC alone group, and ESC/ADM group were significantly larger than those in PBS alone group (t=27.14, 5.29, 15.90, P<0.01); the collagen production of nude mice in ADM alone group and ESC/ADM group was more obvious than that in PBS alone group, and that in ESC alone group and PBS alone group was similar. On PID 11, the expression of CD49f and mRNA of GAPDH gene were positive in the wounds of nude mice in ESC alone group and ESC/ADM group. Conclusions: ESC/ADM particles can promote the healing of full-thickness skin defects in nude mice, which may be related to the fact that particles can improve the survial rate of ESC after transplant and ADM can promote rearrangement and vascularization of dermis.

PMID:34839596 | DOI:10.3760/cma.j.cn501120-20200920-00418

Zhonghua Shao Shang Za Zhi. 2021 Nov 25;37:1-9. doi: 10.3760/cma.j.cn501120-20200916-00414. Online ahead of print.

ABSTRACT

Objective: To analyze the changes and predict the metabolic function of intestinal microflora in severe burn patients at early stage by 16S ribosomal RNA (rRNA) sequencing. Methods: In the prospective observational study, 48 patients with severe burns who met the inclusion criteria and were admitted to Department of Burns and Plastic Surgery of Affiliated Hospital of Jiangsu University from January 2018 to December 2019 were included the burn group, and 40 healthy volunteers who met the inclusion criteria and underwent health examination at the Physical Examination Center of Affiliated Hospital of Jiangsu University in the same period were included in healthy group. Stool samples were collected from patients in burn group in about 1 week after admission and from healthy volunteers on the day of physical examination. The 16S rRNA V4 gene sequencing was performed in the stool of patients in burn group and volunteers in healthy group to analyze the relative abundance of various bacteria. The operational classification unit (OTU) was divided by Mothur software, and the thermal map of fecal micro flora structure was drawn. The OTU number, Chao1 index, Ace index, and Shannon index of stool microflora were analyzed by QIIME1.9.0 software. The principal component analysis (PCA) for relative abundance of stool microflora was preformed by Canoco Software 50. The metabolic function of stool microflora was predicted by Kyoto Encyclopedia of Genes and Genomes. Data were statistically analyzed with independent sample t test, Mann-Whitney U test, and Bonferroni correction. Results: The relative abundance of Bacteroides, Enterococcus, Acinetobacter, Macrococcus, and Staphylococcus in feces of patients in burn group was significantly higher than that of volunteers in healthy group (Z=-5.20, -2.37, -5.17, -4.41, -6.03, P<0.05 or P<0.01), and the relative abundance of unclassified-Helicobacillae, Prevotella, Cecobacteria, unclassified-Rumencocci, Pseudobutyrivibrio, Brautia, unclassified-Streptococcidae, unclassified-Digiestive and other 13 species of bacteria in the feces of volunteers in healthy group was significantly higher than that of patients in burn group (Z=-8.03, -3.21, -7.63, -5.88, -8.05, -8.05, -6.77, P＜0.01). The diversity of fecal microflora of volunteers in healthy group was better than that of patients in burn group, the dominant microflora of volunteers in healthy group were Bacteroides, unclassified-Helicobacillae, Prevotella, unclassified-Enterobacteriaceae, Brucella, Parabacteroides, Escherichia coli, etc., and the dominant microflora of patients in burn group were Bacteroides, Prevotella, Enterobacteriaceae, and Parabacteroides. The OTU number, Ace index, Chao1 index, and Shannon index of fecal microflora of patients in burn group were 149±47, 199±45, 190±45, 2.0±0.9, which were significantly lower than 266±57, 323±51, 318±51, 3.8±0.5 of volunteers in healthy group (t=10.325, 11.972, 12.224, 11.662, P<0.01). The relative abundance of fecal microflora of patients in burn group and volunteers in healthy group was clearly divided into two groups by principal component 1, and the contribution rate of principal component 1 was 32.50%, P<0.01. The fecal samples of volunteers in healthy group were more concentrated on principal component 2, the fecal samples of patients in burn group were dispersed in principal component 2, and the contribution rate of principal component 2 was 13.44%, P>0.05. The metabolic levels of alanine-aspartate-glutamate, arginine-proline, cysteine-methionine, glycine-serine-threonine, phenylalanine, tryptophan, and tyrosine in amino acid, tricarboxylic acid cycle, glucose and mannose, galactolipin, glycolysis/gluconiogenesis, starch and sucrose in carbohydrate of fecal microflora of patients in burn group were significantly lower than those of volunteers in healthy group (Z=-4.75, -4.54, -4.75, -4.62, -3.71, -3.28, -4.19, -3.82, -4.72, -4.35, -4.75, -4.71, P<0.01). The levels of lipoic acid metabolism and coenzyme Q synthesis of fecal microflora of patients in burn group were significantly higher than those of volunteers in healthy group (Z=-6.07, -4.51, P<0.01). The metabolic level of arachidonic acid of fecal microflora of patients in burn group was similar to that of volunteers in healthy group (P>0.05). Conclusions: There were significant differences in intestinal microflora between severe burn patients at the early stage and healthy people. In burn patients, the species and diversity of microflora were decreased, and the nutrient metabolism level was decreased.

PMID:34839595 | DOI:10.3760/cma.j.cn501120-20200916-00414