1.The cytochrome P4501A1 (CYP1A1) inhibitor bergamottin enhances host tolerance to multidrug-resistant Vibrio vulnificus infection
Ruo-Bai QIAO ; Wei-Hong DAI ; Wei LI ; Xue YANG ; Dong-Mei HE ; Rui GAO ; Yin-Qin CUI ; Ri-Xing WANG ; Xiao-Yuan MA ; Fang-Jie WANG ; Hua-Ping LIANG
Chinese Journal of Traumatology 2024;27(5):295-304
		                        		
		                        			
		                        			Purpose::Vibrio vulnificus ( V. Vulnificus) infection is characterized by rapid onset, aggressive progression, and challenging treatment. Bacterial resistance poses a significant challenge for clinical anti-infection treatment and is thus the subject of research. Enhancing host infection tolerance represents a novel infection prevention strategy to improve patient survival. Our team initially identified cytochrome P4501A1 (CYP1A1) as an important target owing to its negative modulation of the body's infection tolerance. This study explored the superior effects of the CYP1A1 inhibitor bergamottin compared to antibiotic combination therapy on the survival of mice infected with multidrug-resistant V. Vulnificus and the protection of their vital organs. Methods::An increasing concentration gradient method was used to induce multidrug-resistant V. Vulnificus development. We established a lethal infection model in C57BL/6J male mice and evaluated the effect of bergamottin on mouse survival. A mild infection model was established in C57BL/6J male mice, and the serum levels of creatinine, urea nitrogen, aspartate aminotransferase, and alanine aminotransferase were determined using enzyme-linked immunosorbent assay to evaluate the effect of bergamottin on liver and kidney function. The morphological changes induced in the presence of bergamottin in mouse organs were evaluated by hematoxylin and eosin staining of liver and kidney tissues. The bacterial growth curve and organ load determination were used to evaluate whether bergamottin has a direct antibacterial effect on multidrug-resistant V. Vulnificus. Quantification of inflammatory factors in serum by enzyme-linked immunosorbent assay and the expression levels of inflammatory factors in liver and kidney tissues by real-time quantitative polymerase chain reaction were performed to evaluate the effect of bergamottin on inflammatory factor levels. Western blot analysis of IκBα, phosphorylated IκBα, p65, and phosphorylated p65 protein expression in liver and kidney tissues and in human hepatocellular carcinomas-2 and human kidney-2 cell lines was used to evaluate the effect of bergamottin on the nuclear factor kappa-B signaling pathway. One-way ANOVA and Kaplan-Meier analysis were used for statistical analysis. Results::In mice infected with multidrug-resistant V. Vulnificus, bergamottin prolonged survival ( p = 0.014), reduced the serum creatinine ( p = 0.002), urea nitrogen ( p = 0.030), aspartate aminotransferase ( p = 0.029), and alanine aminotransferase ( p = 0.003) levels, and protected the cellular morphology of liver and kidney tissues. Bergamottin inhibited interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α expression in serum (IL-1β: p = 0.010, IL-6: p = 0.029, TNF-α: p = 0.025) and inhibited the protein expression of the inflammatory factors IL-1β, IL-6, TNF-α in liver (IL-1β: p = 0.010, IL-6: p = 0.011, TNF-α: p = 0.037) and kidney (IL-1β: p = 0.016, IL-6: p = 0.011, TNF-α: p = 0.008) tissues. Bergamottin did not affect the proliferation of multidrug-resistant V. Vulnificus or the bacterial load in the mouse peritoneal lavage fluid ( p = 0.225), liver ( p = 0.186), or kidney ( p = 0.637). Conclusion::Bergamottin enhances the tolerance of mice to multidrug-resistant V. Vulnificus infection. This study can serve as a reference and guide the development of novel clinical treatment strategies for V. Vulnificus.
		                        		
		                        		
		                        		
		                        	
2.Effects of Tao Hong Si Wu decoction on circular RNA expression profiles in rats with middle cerebral artery occlusion
Chang-Yi FEI ; Li-Juan ZHANG ; Ni WANG ; Fu-Rui CHU ; Chao YU ; Su-Jun XUE ; Ling-Yu PAN ; Dai-Yin PENG ; Xian-Chun DUAN
Chinese Pharmacological Bulletin 2024;40(5):954-963
		                        		
		                        			
		                        			Aim To screen and study the effects of Tao Hong Si Wu decoction(THSWD)-mediated treat-ment on circular RNA(circRNA)expression profiles in rats with middle cerebral artery occlusion(MCAO),and investigate the possible roles and molecular mecha-nisms of THSWD.Methods Next-generation RNA sequencing was conducted to identify circRNA expres-sion profiles in MCAO rats after treatment with THSWD and compared with the MCAO model group and control group.Bioinformatics analysis was performed to predict the potential target microRNAs and mRNAs.Gene On-tology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses for the potential target mRNAs were applied to explore the potential roles of differentially expressed circRNAs.RT-qPCR was performed to verify circRNAs with significant differences in expression.Results We identified 87 significantly differentially expressed circRNAs between the MCAO group versus the control group,and 86 sig-nificantly differentially expressed circRNAs between the MCAO group versus the THSWD group.respective-ly.Among them,17 circRNAs induced by the MCAO model were reversed via treatment with THSWD.To demonstrate the roles of mRNAs targeted by DECs,the GO and KEGG databases were used.Further analysis revealed that five circRNAs may play important roles in the development of MCAO.Conclusions The com-prehensive expression profile of circRNAs in rats with middle cerebral artery occlusion after THSWD treat-ment is determined for the first time,suggesting that the therapeutic effect of THSWD on MCAO may be a-chieved by regulating the expression of circRNAs.
		                        		
		                        		
		                        		
		                        	
3.UPLC-Q-Orbitrap-MS based metabolomics and analysis of the effect of Suanzaoren Decoction on serum of chronic unpredictable mild stress depression rats
Jiang WU ; Rui HAN ; Ni ZHAO ; Ying-xin CHU ; Yin-jie ZHANG ; Xue-mei QIN ; Chen-hui DU ; Yan YAN
Acta Pharmaceutica Sinica 2023;58(7):1952-1962
		                        		
		                        			
		                        			 A UPLC-Q-Orbitrap-MS based metabolomic approach combined with biochemical assay and histopathological inspection were employed to study the intervention effects of Suanzaoren Decoction (SZRD) on chronic unpredictable mild stress (CUMS) depression rats, and to clarify the metabolic regulation pathway of SZRD. The rats were randomly divided into normal control group, CUMS model group, positive drug venlafaxine group, SZRD high (24 g·kg-1) and low (12 g·kg-1) dose groups, respectively. The CUMS model was replicated by subjecting to a variety of stimulus, such as thermal stimulation, ice water swimming, ultrasonic stimulation, tail clamping, day and night reversal, plantar electric shock and so on for rats. After oral administration of drugs for 28 days, the behavioral indexes of rats in each group were observed and the hippocampus and serum samples of rats were collected for biochemical assay and histopathological inspection. Compared with the CUMS model group, low dose and high dose SZRD groups can significantly reduce the immobility time of forced swimming (
		                        		
		                        	
4.Obesity is positively Associated with Depression in Older Adults: Role of Systemic Inflammation.
Ye Xin GUO ; An Qi WANG ; Xin GAO ; Jun NA ; Wei ZHE ; Yi ZENG ; Jing Rui ZHANG ; Yuan Jing JIANG ; Fei YAN ; Mukaram YUNUS ; Hui WANG ; Zhao Xue YIN
Biomedical and Environmental Sciences 2023;36(6):481-489
		                        		
		                        			OBJECTIVE:
		                        			We aimed to explore the association between obesity and depression and the role of systemic inflammation in older adults.
		                        		
		                        			METHODS:
		                        			Adults ≥ 65 years old ( n = 1,973) were interviewed at baseline in 2018 and 1,459 were followed up in 2021. General and abdominal obesity were assessed, and serum C-reactive protein (CRP) levels were measured at baseline. Depression status was assessed at baseline and at follow-up. Logistic regression was used to analyze the relationship between obesity and the incidence of depression and worsening of depressive symptoms, as well as the relationship between obesity and CRP levels. The associations of CRP levels with the geriatric depression scale, as well as with its three dimensions, were investigated using multiple linear regressions.
		                        		
		                        			RESULTS:
		                        			General obesity was associated with worsening depression symptoms and incident depression, with an odds ratio ( OR) [95% confidence interval ( CI)] of 1.53 (1.13-2.12) and 1.80 (1.23-2.63), especially among old male subjects, with OR (95% CI) of 2.12 (1.25-3.58) and 2.24 (1.22-4.11), respectively; however, no significant relationship was observed between abdominal obesity and depression. In addition, general obesity was associated with high levels of CRP, with OR (95% CI) of 2.58 (1.75-3.81), especially in subjects free of depression at baseline, with OR (95% CI) of 3.15 (1.97-5.04), and CRP levels were positively correlated with a score of specific dimension (life satisfaction) of depression, P < 0.05.
		                        		
		                        			CONCLUSION
		                        			General obesity, rather than abdominal obesity, was associated with worsening depressive symptoms and incident depression, which can be partly explained by the systemic inflammatory response, and the impact of obesity on depression should be taken more seriously in the older male population.
		                        		
		                        		
		                        		
		                        			Humans
		                        			;
		                        		
		                        			Male
		                        			;
		                        		
		                        			Aged
		                        			;
		                        		
		                        			Depression/etiology*
		                        			;
		                        		
		                        			C-Reactive Protein/metabolism*
		                        			;
		                        		
		                        			Obesity, Abdominal/epidemiology*
		                        			;
		                        		
		                        			Longitudinal Studies
		                        			;
		                        		
		                        			Inflammation/epidemiology*
		                        			;
		                        		
		                        			Obesity/complications*
		                        			
		                        		
		                        	
5.Clinical characteristics of plastic bronchitis and risk factors for recurrence in children.
Xiao-Yin TIAN ; Guang-Li ZHANG ; Chong-Jie WANG ; Rui-Xue GU ; Yuan-Yuan LI ; Qin-Yuan LI ; Jian LUO ; Zheng-Xiu LUO
Chinese Journal of Contemporary Pediatrics 2023;25(6):626-632
		                        		
		                        			OBJECTIVES:
		                        			To study the clinical characteristics of plastic bronchitis (PB) in children and investigate the the risk factors for recurrence of PB.
		                        		
		                        			METHODS:
		                        			This was a retrospective analysis of medical data of children with PB who were hospitalized in Children's Hospital of Chongqing Medical University from January 2012 to July 2022. The children were divided into a single occurrence of PB group and a recurrent PB group and the risk factors for recurrence of PB were analyzed.
		                        		
		                        			RESULTS:
		                        			A total of 107 children with PB were included, including 61 males (57.0%) and 46 females (43.0%), with a median age of 5.0 years, and 78 cases (72.9%) were over 3 years old. All the children had cough, 96 children (89.7%) had fever, with high fever in 90 children. Seventy-three children (68.2%) had shortness of breath, and 64 children (59.8%) had respiratory failure. Sixty-six children (61.7%) had atelectasis and 52 children (48.6%) had pleural effusion. Forty-seven children (43.9%) had Mycoplasma pneumoniae infection, 28 children (26.2%) had adenovirus infection, and 17 children (15.9%) had influenza virus infection. Seventy-one children (66.4%) had a single occurrence of PB, and 36 cases (33.6%) had recurrent occurrence of PB (≥2 times). Multivariate logistic regression analysis showed that involvement of ≥2 lung lobes (OR=3.376) under bronchoscopy, continued need for invasive ventilation after initial removal of plastic casts (OR=3.275), and concomitant multi-organ dysfunction outside the lungs (OR=2.906) were independent risk factors for recurrent occurrence of PB (P<0.05).
		                        		
		                        			CONCLUSIONS
		                        			Children with pneumonia accompanied by persistent high fever, shortness of breath, respiratory failure, atelectasis or pleural effusion should be highly suspected with PB. Involvement of ≥2 lung lobes under bronchoscopy, continued need for invasive ventilation after initial removal of plastic casts, and concomitant multi-organ dysfunction outside the lungs may be risk factors for recurrent occurrence of PB.
		                        		
		                        		
		                        		
		                        			Female
		                        			;
		                        		
		                        			Male
		                        			;
		                        		
		                        			Child
		                        			;
		                        		
		                        			Humans
		                        			;
		                        		
		                        			Child, Preschool
		                        			;
		                        		
		                        			Multiple Organ Failure
		                        			;
		                        		
		                        			Retrospective Studies
		                        			;
		                        		
		                        			Bronchitis/etiology*
		                        			;
		                        		
		                        			Dyspnea
		                        			;
		                        		
		                        			Pleural Effusion
		                        			;
		                        		
		                        			Pulmonary Atelectasis
		                        			;
		                        		
		                        			Plastics
		                        			;
		                        		
		                        			Respiratory Insufficiency
		                        			
		                        		
		                        	
6.Optimization of perioperative treatment strategies for locally advanced esophageal squamous cell carcinoma from the perspective of tumor heterogeneity.
Xiao Zheng KANG ; Rui Xiang ZHANG ; Zhen WANG ; Xian Kai CHEN ; Jian Jun QIN ; Yin LI ; Qi XUE ; Jie HE
Chinese Journal of Gastrointestinal Surgery 2023;26(4):334-338
		                        		
		                        			
		                        			Recent advances in multimodality treatment offer excellent opportunities to rethink the paradigm of perioperative management for locally advanced esophageal squamous cell carcinoma. One treatment clearly doesn't fit all in terms of a broad disease spectrum. Individualized treatment of local control of bulky primary tumor burden (advanced T stage) or systemic control of nodal metastatic tumor burden (advanced N stage) is essential. Given that clinically applicable predictive biomarkers are still awaited, therapy selection guided by diverse phenotypes of tumor burden (T vs. N) is promising. Potential challenges regarding the use of immunotherapy may also boost this novel strategy in the future.
		                        		
		                        		
		                        		
		                        			Humans
		                        			;
		                        		
		                        			Esophageal Squamous Cell Carcinoma/surgery*
		                        			;
		                        		
		                        			Carcinoma, Squamous Cell/pathology*
		                        			;
		                        		
		                        			Esophageal Neoplasms/pathology*
		                        			;
		                        		
		                        			Combined Modality Therapy
		                        			;
		                        		
		                        			Immunotherapy
		                        			
		                        		
		                        	
7.Effect of High-Concentration Uric Acid on Nitric Oxide.
Si-Yu QIN ; Rong-Yu LAN ; Jia ZENG ; Xue BAI ; Jing-Tao WANG ; Xiang-Lin YIN ; Rui-Jie QU ; Ming-Hai QU ; Hao JIANG ; Wen-Long LI ; Si-Ying PEI ; Zhi-Ling HOU ; Bao-Sheng GUAN ; Hong-Bin QIU
Acta Academiae Medicinae Sinicae 2023;45(4):666-671
		                        		
		                        			
		                        			Uric acid (UA) is the final product of purine metabolism in human body,and its metabolic disorder will induce hyperuricemia (HUA).The occurrence and development of HUA are associated with a variety of pathological mechanisms such as oxidative stress injury,activation of inflammatory cytokines,and activation of renin-angiotensin-aldosterone system.These mechanisms directly or indirectly affect the bioavailability of endogenous nitric oxide (NO).The decrease in NO bioavailability is common in the diseases with high concentration of UA as an independent risk factor.In this review,we summarize the mechanisms by which high concentrations of UA affect the endogenous NO bioavailability,with a focus on the mechanisms of high-concentration UA in decreasing the synthesis and/or increasing the consumption of NO.This review aims to provide references for alleviating the multisystem symptoms and improving the prognosis of HUA,and lay a theoretical foundation for in-depth study of the correlations between HUA and other metabolic diseases.
		                        		
		                        		
		                        		
		                        			Humans
		                        			;
		                        		
		                        			Nitric Oxide
		                        			;
		                        		
		                        			Uric Acid
		                        			;
		                        		
		                        			Hyperuricemia
		                        			;
		                        		
		                        			Biological Availability
		                        			;
		                        		
		                        			Cytokines
		                        			
		                        		
		                        	
8.Prediction and analysis of Q-markers of Elephantopus scaber based on its UPLC fingerprint, content determination of components, and in vitro a nti-tumor activity.
Can-Chao JIA ; Ling-Jie LI ; Zhi-Hao ZENG ; Rui-Yin TANG ; De-Zheng JIA ; Min-Juan YANG ; Jin-Yan QIU ; Dong-Mei LI ; Can-Hui XIE ; Guang-Ying WU ; Yang-Xue LI ; Jie-Yi JIANG ; Hong HUANG ; Guan-Lin XIAO ; Da-Ke CAI ; Xiao-Li BI
China Journal of Chinese Materia Medica 2023;48(16):4421-4428
		                        		
		                        			
		                        			This study aimed to provide scientific evidence for predicting quality markers(Q-markers) of Elephantopus scaber by establishing UPLC fingerprint of E. scaber from different geographical origins and determining the content of 13 major components, as well as conducting in vitro anti-cancer activity investigation of the main components. The chromatographic column used was Waters CORTECS UPLC C_(18)(2.1 mm×150 mm, 1.6 μm), and the mobile phase consisted of acetonitrile and 0.1% formic acid solution(gradient elution). The column temperature was set at 30 ℃, and the flow rate was 0.2 mL·min~(-1). The injection volume was 1 μL, and the detection wavelength was 240 nm. The UPLC fingerprint of E. scaber was fitted using the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine(2012 edition) to determine common peaks, evaluate similarity, identify and determine the content of major components. The CCK-8 assay was used to explore the inhibitory effect of the main components on the proliferation of lung cancer cells. The results showed that in the established UPLC fingerprint of E. scaber, 35 common peaks were identified. Thirteen major components, including neochlorogenic acid(peak 1), chlorogenic acid(peak 2), cryptochlorogenic acid(peak 3), caffeic acid(peak 4), schaftoside(peak 6), galuteolin(peak 9), isochlorogenic acid B(peak 10), isochlorogenic acid A(peak 12), isochlorogenic acid C(peak 18), deoxyelephantopin(peak 28), isodeoxyelephantopin(peak 29), isoscabertopin(peak 31), and scabertopin(peak 32) were identified and quantified, and a quantitative analysis method was established. The results of the in vitro anti-cancer activity study showed that deoxyelephantopin, isodeoxyelephantopin, isoscabertopin, and scabertopin in E. scaber exhibited inhibition rates of lung cancer cell proliferation exceeding 80% at a concentration of 10 μmol·L~(-1), higher than the positive drug paclitaxel. These results indicate that the fingerprint of E. scaber is highly characteristic, and the quantitative analysis method is accurate and stable, providing references for the research on quality standards of E. scaber. Four sesquiterpene lactones in E. scaber show significant anti-cancer activity and can serve as Q-markers for E. scaber.
		                        		
		                        		
		                        		
		                        			Humans
		                        			;
		                        		
		                        			Chromatography, High Pressure Liquid
		                        			;
		                        		
		                        			Drugs, Chinese Herbal/chemistry*
		                        			;
		                        		
		                        			Asteraceae/chemistry*
		                        			;
		                        		
		                        			Lung Neoplasms/drug therapy*
		                        			
		                        		
		                        	
9.Effects of low-dose photodynamic therapy on the function of human adipose mesenchymal stem cells and its mechanism.
Yang YANG ; Lei LI ; Zeng Jun YANG ; Meng Xue ZHENG ; Wei Feng HE ; Rui YIN
Chinese Journal of Burns 2022;38(9):830-838
		                        		
		                        			
		                        			Objective: To investigate the effects of low-dose photodynamic therapy on the proliferation, regulation, and secretion functions of human adipose mesenchymal stem cells (ADSCs) and the related mechanism, so as to explore a new method for the repair of chronic wounds. Methods: The experimental research methods were adopted. From February to April 2021, 10 patients (5 males and 5 females, aged 23 to 47 years) who underwent cutaneous surgery in the Department of Dermatology of the First Affiliated Hospital of Army Medical University (the Third Military Medical University) donated postoperative waste adipose tissue. The cells were extracted from the adipose tissue and the phenotype was identified. Three batches of ADSCs were taken, with each batch of cells being divided into normal control group with conventional culture only, photosensitizer alone group with conventional culture after being treated with Hemoporfin, irradiation alone group with conventional culture after being treated with red light irradiation, and photosensitizer+irradiation group with conventional culture after being treated with Hemoporfin and red light irradiation, with sample number of 3 in each group. At culture hour of 24 after the treatment of the first and second batches of cells, the ADSC proliferation level was evaluated by 5-ethynyl-2'-deoxyuridine staining method and the migration percentage of HaCaT cells cocultured with ADSCs was detected by Transwell experiment, respectively. On culture day of 7 after the treatment of the third batch of cells, the extracellular matrix protein expression of ADSCs was detected by immunofluorescence method. The ADSCs were divided into 0 min post-photodynamic therapy group, 15 min post-photodynamic therapy group, 30 min post-photodynamic therapy group, and 60 min post-photodynamic therapy group, with 3 wells in each group. Western blotting was used to detect the protein expressions and calculate the phosphorylated mammalian target of rapamycin complex (p-mTOR)/mammalian target of rapamycin (mTOR), phosphorylated p70 ribosomal protein S6 kinase (p-p70 S6K)/p70 ribosomal protein S6 kinase (p70 S6K) ratio at the corresponding time points after photodynamic therapy. Two batches of ADSCs were taken, and each batch was divided into normal control group, photodynamic therapy alone group, and photodynamic therapy+rapamycin group, with 3 wells in each group. At culture minute of 15 after the treatment, p-mTOR/mTOR and p-p70 S6K/p70 S6K ratios of cells from the first batch were calculated and detected as before. On culture day of 7 after the treatment, extracellular matrix protein expression of cells from the second batch was detected as before. Data were statistically analyzed with one-way analysis of variance and least significant difference test. Results: After 12 d of culture, the cells were verified as ADSCs. At culture hour of 24 after the treatment, the ADSC proliferation level ((4.0±1.0)% and (4.1±0.4)%, respectively) and HaCaT cell migration percentages (1.17±0.14 and 1.13±0.12, respectively) in photosensitizer alone group and irradiation alone group were similar to those of normal control group ((3.7±0.6)% and 1.00±0.16, respectively, P>0.05), and were significantly lower than those of photosensitizer+irradiation group ((34.2±7.0)% and 2.55±0.13, respectively, P<0.01). On culture day of 7 after the treatment, compared with those in normal control group, the expression of collagen Ⅲ in ADSCs of photosensitizer alone group was significantly increased (P<0.05), and the expressions of collagen Ⅰ and collagen Ⅲ in ADSCs of irradiation alone group were significantly increased (P<0.01). Compared with those in photosensitizer alone group and irradiation alone group, the expressions of collagen Ⅰ, collagen Ⅲ, and fibronectin of ADSCs in photosensitizer+irradiation group were significantly increased (P<0.01). Compared with those in 0 min post-photodynamic therapy group, the ratios of p-mTOR/mTOR and p-p70 S6K/p70 S6K of ADSCs in 15 min post-photodynamic therapy group were significantly increased (P<0.01), the ratios of p-p70 S6K/p70 S6K of ADSCs in 30 min post-photodynamic therapy group and 60 min post-photodynamic therapy group were both significantly increased (P<0.01). At culture minute of 15 after the treatment, compared with those in normal control group, the ratios of p-mTOR/mTOR and p-p70 S6K/p70 S6K of ADSCs in photodynamic therapy alone group were significantly increased (P<0.05 or P<0.01). Compared with those in photodynamic therapy alone group, the ratios of p-mTOR/mTOR and p-p70 S6K/p70 S6K of ADSCs in photodynamic therapy+rapamycin group were significantly decreased (P<0.05). On culture day of 7 after the treatment, compared with those in normal control group, the expressions of collagen Ⅰ, collagen Ⅲ, and fibronectin of ADSCs in photodynamic therapy alone group were significantly increased (P<0.01). Compared with those in photodynamic therapy alone group, the expressions of collagen Ⅰ, collagen Ⅲ, and fibronectin of ADSCs in photodynamic therapy+rapamycin group were significantly decreased (P<0.01). Conclusions: Low-dose photodynamic therapy can promote the proliferation of ADSCs, improve the ability of ADSCs to regulate the migration of HaCaT cells, and enhance the secretion of extracellular matrix protein by rapidly activating mTOR signaling pathway.
		                        		
		                        		
		                        		
		                        			Adipose Tissue
		                        			;
		                        		
		                        			Female
		                        			;
		                        		
		                        			Fibronectins
		                        			;
		                        		
		                        			Humans
		                        			;
		                        		
		                        			Male
		                        			;
		                        		
		                        			Mesenchymal Stem Cells
		                        			;
		                        		
		                        			Photochemotherapy
		                        			;
		                        		
		                        			Photosensitizing Agents/pharmacology*
		                        			;
		                        		
		                        			Sirolimus/pharmacology*
		                        			;
		                        		
		                        			TOR Serine-Threonine Kinases
		                        			
		                        		
		                        	
10.Effects of porcine acellular dermal matrix combined with human epidermal stem cells on wound healing of full-thickness skin defect in nude mice.
Xiao Hong ZHAO ; Yu Cheng GUO ; Hong Hao CHEN ; Xue LI ; Ying WANG ; Wen Qiang NI ; Meng Qiu XING ; Rui ZHANG ; Shi Cang YU ; Yin Gen PAN ; Ri Xing ZHAN ; Gao Xing LUO
Chinese Journal of Burns 2022;38(1):45-56
		                        		
		                        			
		                        			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: The morphology of porcine ADM was analyzed by photograph of digital camera, the cell residues in porcine ADM were observed by hematoxylin-eosin (HE) staining, the surface structure of porcine ADM was observed by scanning electron microscope, the secondary structure of porcine ADM was analyzed by infrared spectrometer, the porcine ADM particle size was analyzed by dynamic light scattering particle size analyzer, and the porcine ADM potential was analyzed by nano-particle size potentiometer. The morphology of porcine ADM was observed by inverted fluorescence microscope when it was placed in culture medium for 30 min, 1 d, and 5 d (n=2). The porcine 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 below) in static state at normal temperature for the corresponding time to calculate the water absorption by weighing method (n=3). Swiss white mouse embryonic fibroblasts (Fbs) were divided into blank control group (culture medium only), and 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 which were added with the corresponding final concentrations of ADM extract respectively. At post culture hour (PCH) 24, 48, and 72, the cell survival rate was detected by cell counting kit 8 and the cytotoxicity was graded (n=5). The erythrocytes of a 6-week-old male Sprague-Dawley male rat were divided into normal saline group, ultra-pure water group, and 5 mg/mL ADM extract group, 10 mg/mL ADM extract group, and 15 mg/mL ADM extract group which were treated with the corresponding final concentrations of porcine ADM extract respectively. After reaction for 3 h, the absorbance value of hemoglobin was detected by microplate reader to represent the blood compatibility of porcine ADM (n=3). ESCs were isolated and cultured from the discarded prepuce of a 6-year-old healthy 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, and then identified by flow cytometry. The porcine 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 scanning 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) 1, 7, 11, and 15, the wound healing was observed and the wound healing rate was counted (n=3). On PID 7, the epithelialization of wounds was observed by HE staining and the length of un-epithelialized wound was measured (with this and the following sample numbers of 4). On PID 11, the dermal area and collagen deposition of wounds were observed by Masson staining and the dermal area of wound section was 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) in ESC after wound transplantation was detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction. Data were statistically analyzed with independent sample t test, one-way analysis of variance, analysis of variance for repeated measurement, and least significant difference t test. Results: The porcine ADM was white particles and composed of reticular structure, with no cells inside, disordered structure, and rough surface. The absorption peak of porcine ADM appeared at the wave numbers of 1 659, 1 549, and 1 239 cm-1, respectively. The main particle size distribution of porcine ADM in solution was 500 to 700 nm, with negative charge on the surface. The morphology of porcine ADM in static state at 30 min and on 1 and 5 d was relatively stable. The water absorption of porcine ADM remained relatively high level in static state from 30 min to 120 min. The cytotoxicity of mouse embryonic 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 of erythrocytes in ultra-pure water group was significantly higher than the values in normal saline group and 15 mg/mL ADM extract group (with t values of 8.14 and 7.96, respectively, P<0.01). After 3 days of culture, the cells of the fourth passage showed pebble-like morphology, with low expression of CD71 and high expression of CD49f, which were identified as ESCs. There was ESC attachment and growth on porcine 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, 11, 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 (with t values of 2.83 and 4.72 respectively, 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 (t=4.86, 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 (with t values of 2.71, 2.90, and 3.23 respectively, 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), and (163±32) μm, respectively, which were significantly shorter than (1 199±43) μm in PBS alone group (with t values of 5.69, 10.19, and 27.54 respectively, P<0.01). On PID 11, the dermal areas of wound section of nude mice in ADM alone group, ESC alone group, and ESC/ADM group were significantly larger than the area in PBS alone group (with t values of 27.14, 5.29, and 15.90 respectively, 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 the collagen production of nude mice in ESC alone group and PBS alone group was similar. On PID 11, in the wounds of nude mice in ESC alone group and ESC/ADM group, the cells with positive expression of CD49f were respectively 135±7 and 185±15, and the mRNA expressions of GAPDH were positive; while there were no expressions of CD49f nor mRNA of GAPDH in the wounds of nude mice in PBS alone group and ADM alone group. Conclusions: ESC/ADM particles can promote the wound healing of full-thickness skin defects in nude mice, which may be related to the improved survival rate of ESCs after transplantation and the promotion of dermal structure rearrangement and angiogenesis by ADM.
		                        		
		                        		
		                        		
		                        			Acellular Dermis
		                        			;
		                        		
		                        			Animals
		                        			;
		                        		
		                        			Fibroblasts
		                        			;
		                        		
		                        			Humans
		                        			;
		                        		
		                        			Male
		                        			;
		                        		
		                        			Mice
		                        			;
		                        		
		                        			Mice, Nude
		                        			;
		                        		
		                        			Rats
		                        			;
		                        		
		                        			Rats, Sprague-Dawley
		                        			;
		                        		
		                        			Stem Cells
		                        			;
		                        		
		                        			Swine
		                        			;
		                        		
		                        			Wound Healing
		                        			
		                        		
		                        	
            
Result Analysis
Print
Save
E-mail