1.Early clinical efficacy of Yiqi Tongluo Decoction on ischemic cerebrovascular disease after interventional therapy
Qinghua WANG ; Gesheng WANG ; Ruiping LI ; Can ZHENG ; Wenjing LI ; Jiaqi CHEN ; Kaihang GUO ; Xiaobo DONG ; Wenxin WANG ; Rongjuan GUO ; Le WANG
Journal of Beijing University of Traditional Chinese Medicine 2025;48(1):108-121
		                        		
		                        			Objective:
		                        			To observe the clinical efficacy and safety of Yiqi Tongluo Decoction in the intervention of early traditional Chinese medicine (TCM) syndromes after ischemic cerebrovascular disease (ICVD) intervention. 
		                        		
		                        			Methods:
		                        			From October 2020 to July 2023, a randomized, double-blind, placebo-controlled study was conducted to include 60 patients with qi deficiency, blood stasis, and phlegm obstruction syndrome after ICVD interventional therapy. They were assigned to the Yiqi Tongluo Decoction treatment group (30 cases) and the TCM placebo routine treatment control group (30 cases) according to the randomized block design. Both groups received routine standardized treatment of Western medicine, including dual antiplatelet, lipid regulation, and control of risk factors for cerebrovascular disease. The treatment group was treated with Yiqi Tongluo Decoction based on the control group. The course of treatment was 60 days and follow-up was carried out 2 and 6 months after the operation. The improvement of qi deficiency syndrome, blood stasis syndrome, phlegm syndrome score and TCM syndrome score, modified Rankin score (mRS), Barthel index (BI) score, Fatty acid-binding protein 4 (FABP4) level, incidence of transient ischemic attack (TIA) and ischemic stroke (IS) and incidence of adverse reactions, Head and neck CT angiography (CTA) or digital subtraction angiography (DSA) examination were collected. The clinical efficacy of the patients 2 months after the operation was taken as the main outcome index to preliminarily evaluate the early and long-term efficacy of Yiqi Tongluo Decoction after the ICVD intervention. The early and long-term clinical efficacy and safety of Western medicine standardized treatment combined with TCM Yiqi Tongluo Decoction on patients with qi deficiency, blood stasis and phlegm obstruction syndrome after ICVD intervention were evaluated. The safety of Yiqi Tongluo Decoction in the treatment of patients after ICVD intervention with white blood cell (WBC), C-reactive protein (CRP), fibrinogen (FIB), plasminogen time (PT), recurrence of cerebral ischaemia and restenosis in patients at 2 and 6 months after treatment were evaluated.
		                        		
		                        			Results:
		                        			Compared to the control group, the TCM syndrome scores for qi deficiency, blood stasis and phlegm syndrome in the treatment group reduced significantly, the clinical efficacy improved significantly, the mRS score and FABP4 were reduced, and the BI score was increased. Adverse events such as cerebral ischaemia were fewer in the treatment group than in the control group, but the difference was not statistically significant; levels of CRP, WBC and PT were reduced, and levels of FIB were reduced at 6 months post-treatment, all P<0.01, and images were intuitively compared. The treatment group was superior to the control group.
		                        		
		                        			Conclusion
		                        			Yiqi Tongluo Decoction combined with Western medicine standard treatment can improve the early clinical efficacy of ICVD patients with qi deficiency, blood stasis and phlegm obstruction syndrome after interventional surgery, improve neurological impairment and daily living ability, reduce the state of qi deficiency syndrome, blood stasis syndrome and phlegm syndrome after interventional surgery, and improve the clinical efficacy of TCM. At the same time, it can reduce the level of FABP4, the target of atherosclerosis and restenosis after interventional surgery, reduce the level of inflammation after interventional surgery in patients with ICVD, regulate coagulation function, and reduce the incidence of long-term recurrence of cerebral ischemia after interventional surgery, with good safety. 
		                        		
		                        		
		                        		
		                        	
2.Advances in the application of physiologically-based pharmacokinetic model in EGFR-TKI precision therapy
Yingying YANG ; Jiaqi SHAO ; Qiulin XIANG ; Guoxing LI ; Xian YU
China Pharmacy 2025;36(8):1013-1018
		                        		
		                        			
		                        			Epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) represent a class of small-molecule targeted therapeutics for oncology treatment, and serve as first-line therapy for advanced non-small cell lung cancer (NSCLC) with EGFR- sensitive mutations, with representative agents including gefitinib, dacomitinib, and osimertinib. In clinical practice, dose adjustment of EGFR-TKI may be required for cancer patients under special circumstances such as drug combinations or hepatic/ renal impairment. Physiologically-based pharmacokinetic (PBPK) model, capable of predicting pharmacokinetic (PK) processes in humans, has emerged as a vital tool for clinical dose optimization. This article sorts the modeling methodologies, workflows, and commonly used software tools for PBPK model, and summarizes the current applications of PBPK model in EGFR-TKI precision therapy as of June 30, 2024. Findings demonstrate that PBPK modeling methods commonly employ the “bottom-up” approach and the middle-out approach. The process typically involves four steps: parameter collection, compartment selection, model validation, and model application. Commonly used software for modeling includes Simcyp, GastroPlus, and open-source software such as PK- Sim. PBPK model can be utilized for predicting drug-drug interactions of EGFR-TKI co-administered with metabolic enzyme inducers or inhibitors, acid-suppressive drugs, or traditional Chinese and Western medicines. It can also adjust dosages in conjunction with genomics, predict PK processes in special populations (such as patients with liver or kidney dysfunction, pediatric patients), evaluate the efficacy and safety of drugs, and extrapolate PK predictions from animal models to humans.
		                        		
		                        		
		                        		
		                        	
3.Construction of Knowledge Graph Based on Literature Data by Taking Treatment of Diabetic Peripheral Neuropathy with Traditional Chinese Medicine as An Example
Jiaqi CHAI ; Yumeng TAN ; Xinghua XIANG ; Miaomiao LI ; Tiancai WEN ; Hui ZHAO
Chinese Journal of Experimental Traditional Medical Formulae 2024;30(6):144-150
		                        		
		                        			
		                        			ObjectiveTo systematically sort out the knowledge framework and conceptual logic relationship of "disease-syndrome-treatment-prescription-medicine" in the existing literature on traditional Chinese medicine(TCM) treatment of diabetic peripheral neuropathy(DPN), to construct of the knowledge map of TCM treatment of DPN, and to promote the explicitation of the implicit knowledge in the literature on the treatment of DPN with TCM. MethodTaking the literature of China National Knowledge Infrastructure about TCM treatment of DPN as the main data source, TCM-related concepts and entities were constructed by manual citation, and the corresponding relationships between the entities were established. Structured data were formed by processing with Python 3.7, and the knowledge graph was constructed based on Neo4j 3.5.34 graph database. ResultThe resulting knowledge graph with TCM diagnosis and treatment logic, defined 12 node labels such as prescriptions, Chinese medicines and syndrome types at the schema layer, as well as 4 types of relationships, such as inclusion, correspondence, selection and composition. It could support the query and discovery of nodes(syndrome elements, syndrome types and treatment methods), as well as the relationship between each node. ConclusionBased on the literature data, this study constructed a knowledge map for TCM treatment of DPN, which brought together various methods of TCM treatment of DPN, including internal and external treatment. The whole chain knowledge structure of syndrome differentiation and classification for DPN treatment is formed from syndrome element analysis, syndrome type composition to treatment method selection, which can provide new ideas and methods for literature data to serve clinical and scientific research work, as well as reference for visualization of TCM literature knowledge, intellectualization of TCM knowledge services and the standardization of TCM diagnosis and treatment. 
		                        		
		                        		
		                        		
		                        	
4.Prognostic value of serum adenosylhomocysteinase in patients with hepatitis E related acute liver failure
Hong YAN ; Ze XIANG ; Jiaqi CHEN ; Chun JIANG ; Shuxiang LI ; Guanghua ZHAI ; Jian WU
Chinese Journal of Laboratory Medicine 2024;47(8):888-894
		                        		
		                        			
		                        			Objective:To investigate the prognostic value of serum adenosylhomocysteinase (AHCY) in patients with hepatitis E virus acute liver failure (HEV-ALF).Methods:From 1 January 2017 to 31 December 2022, 100 patients each with HEV-ALF and acute hepatitis E (AHE) from the First Affiliated Hospital of Medical School of Zhejiang University and Affiliated Suzhou Hospital of Nanjing Medical University were included in this case-control study. The HEV-ALF group was 58.56±11.16 years old, including 71 men. The AHE group was 56.04±14.30 years old, including 61 men. All serum samples were obtained before the patient had an acute onset and were obtained without treatment. Firstly, the serum AHCY levels in patients with HEV-ALF and AHE were analyzed by ELISA. Secondly, the serum AHCY levels in HEV-ALF patients with different organ failure and disease condition were compared. According to the number of organ failure, 100 HEV-ALF patients were divided into organ failure number=2 group ( n=58), number=3 group ( n=24) and number>3 groups ( n=18). According to the disease condition, 100 patients were divided into improvement group ( n=49), disease fluctuation group ( n=37), and deterioration group ( n=14). Thirdly, the survival times between the high serum AHCY level group ( n=50) and the low serum AHCY level group ( n=50) were compared. Finally, the independent risk factors to predict mortality using the multivariate Logistic regression analysis, and evaluated the predictive and decision-making abilities of serum AHCY levels were explored using the receiver operating characteristic (ROC) curve and decision curve analysis (DCA). Results:Serum AHCY levels in HEV-ALF patients were significantly higher than those in AHE patients [326.92 (295.37-385.84) pg/ml vs. 222.88 (188.04-246.78) pg/ml, Z=-12.217, P<0.001]. Serum AHCY levels in group 2 were significantly lower than those in group 3 [303.44 (284.40-330.15) pg/ml vs. 335.36 (306.30-385.84) pg/ml, Z=-3.353, P=0.001]. Serum AHCY level in group 3 were significantly lowerthan those in group>3 [335.36 (306.30-385.84) pg/ml vs. 549.89 (423.35-660.22) pg/ml, P<0.001]. The serum AHCY levels in the fluctuation group were lower than those in the deterioration group [322.17 (283.92-423.74) pg/ml vs. 458.26 (374.66, 593.89) pg/ml, Z=-4.016, P=0.009]. The survival time of high serum AHCY level group was significantly lower than that of low serum AHCY level group [23.11 (20.25-25.96) days vs. 29.49 (28.79-30.20) days, Z=-2.596, P<0.001]. The multivariate Logistic regression analysis showed that the levels of serum AHCY and total bilirubin were independent risk factors to predict mortality in HEV-ALF patients [AHCY, OR (95% CI): 1.008 (1.002-1.015), P=0.008; total bilirubin, OR (95% CI): 1.011 (1.005-1.018), P=0.001]. Serum AHCY level predicting the area under the curve (AUC) of 30-day mortality in HEV-ALF patients was 0.912, with a sensitivity of 90.00% and a specificity of 93.75%. DCA results demonstrated that serum AHCY level had good decision-making power for predicting 30-day mortality in HEV-ALF patients. Conclusion:Serum AHCY has an important prognostic value for HEV-ALF patients. Higher serum AHCY levels indicate the worse prognosis of HEV-ALF patients.
		                        		
		                        		
		                        		
		                        	
5.Establishment and validation of a multigene model to predict the risk of relapse in hormone receptor-positive early-stage Chinese breast cancer patients.
Jiaxiang LIU ; Shuangtao ZHAO ; Chenxuan YANG ; Li MA ; Qixi WU ; Xiangzhi MENG ; Bo ZHENG ; Changyuan GUO ; Kexin FENG ; Qingyao SHANG ; Jiaqi LIU ; Jie WANG ; Jingbo ZHANG ; Guangyu SHAN ; Bing XU ; Yueping LIU ; Jianming YING ; Xin WANG ; Xiang WANG
Chinese Medical Journal 2023;136(2):184-193
		                        		
		                        			BACKGROUND:
		                        			Breast cancer patients who are positive for hormone receptor typically exhibit a favorable prognosis. It is controversial whether chemotherapy is necessary for them after surgery. Our study aimed to establish a multigene model to predict the relapse of hormone receptor-positive early-stage Chinese breast cancer after surgery and direct individualized application of chemotherapy in breast cancer patients after surgery.
		                        		
		                        			METHODS:
		                        			In this study, differentially expressed genes (DEGs) were identified between relapse and nonrelapse breast cancer groups based on RNA sequencing. Gene set enrichment analysis (GSEA) was performed to identify potential relapse-relevant pathways. CIBERSORT and Microenvironment Cell Populations-counter algorithms were used to analyze immune infiltration. The least absolute shrinkage and selection operator (LASSO) regression, log-rank tests, and multiple Cox regression were performed to identify prognostic signatures. A predictive model was developed and validated based on Kaplan-Meier analysis, receiver operating characteristic curve (ROC).
		                        		
		                        			RESULTS:
		                        			A total of 234 out of 487 patients were enrolled in this study, and 1588 DEGs were identified between the relapse and nonrelapse groups. GSEA results showed that immune-related pathways were enriched in the nonrelapse group, whereas cell cycle- and metabolism-relevant pathways were enriched in the relapse group. A predictive model was developed using three genes ( CKMT1B , SMR3B , and OR11M1P ) generated from the LASSO regression. The model stratified breast cancer patients into high- and low-risk subgroups with significantly different prognostic statuses, and our model was independent of other clinical factors. Time-dependent ROC showed high predictive performance of the model.
		                        		
		                        			CONCLUSIONS
		                        			A multigene model was established from RNA-sequencing data to direct risk classification and predict relapse of hormone receptor-positive breast cancer in Chinese patients. Utilization of the model could provide individualized evaluation of chemotherapy after surgery for breast cancer patients.
		                        		
		                        		
		                        		
		                        			Humans
		                        			;
		                        		
		                        			Female
		                        			;
		                        		
		                        			Breast Neoplasms/genetics*
		                        			;
		                        		
		                        			East Asian People
		                        			;
		                        		
		                        			Neoplasm Recurrence, Local/genetics*
		                        			;
		                        		
		                        			Breast
		                        			;
		                        		
		                        			Algorithms
		                        			;
		                        		
		                        			Chronic Disease
		                        			;
		                        		
		                        			Prognosis
		                        			;
		                        		
		                        			Tumor Microenvironment
		                        			
		                        		
		                        	
6.Clinical, endoscopic and histological characteristics of heterotopic gastric mucosa in upper esophagus
Jiaqi WANG ; Lizhi XIANG ; Xiaoli PAN ; Changsha ZHANG ; Xiaoyun YU ; Xiaohua HOU
Chinese Journal of Digestive Endoscopy 2022;39(9):695-700
		                        		
		                        			
		                        			Objective:To study the clinical, endoscopic and histological characteristics of heterotopic gastric mucosa in upper esophagus (HGMUE).Methods:A Total of 177 patients who underwent gastroscopy and were diagnosed as having HGMUE at the Endoscopy Center of Wuhan Union Hospital from January 2017 to December 2017 were included in the study. According to the gastroesophageal reflux disease symptom questionnaire (GERD-Q) scores, patients were divided into the HGMUE group (GERD-Q<8, n=101) and GERD+HGMUE group (GERD-Q≥8, n=76). The data of clinical, endoscopic and histological characteristics were analyzed. Results:Among the 177 HGMUE cases, there were 111 males (62.71%) and 66 females (37.29%), 76 (42.94%) with GERD, and 101 (57.06%) without GERD. The most common symptom was continuous clearing throat [54.24% (96/177)], followed by foreign body sensations of throat [48.59% (86/177)], and gastroesophageal reflux symptoms such as heartburn, chest pain, indigestion, acid reflux [48.59% (86/177)]. In the HGMUE group, the occurrence rate of clearing throat was the highest [42.57% (43/101)], then foreign body sensations of throat accounted for 33.66% (34/101), and gastroesophageal reflux symptoms was 27.72% (28/101). In the HGUME+GERD group, the most common symptom was gastroesophageal reflux symptoms [76.32% (58/76)], then clearing throat [69.74% (53/76)] and foreign body sensations of throat [68.42% (52/76)]. Under gastroscopy, 177 heterotopic gastric lesions were found under gastroscopy with orange-red round, oval or elongated island like ones, most of which were flat and a few slightly protruded from the peripheral plane. There were 132 (74.58%) single-lesion cases, 38 (21.47%) 2-lesion, and 7 (3.95%) 3- or more-lesion cases; there were 37 (20.90%) small lesions (maximum diameter <0.5 cm), and 74 (41.81%) median-size lesions (maximum diameter of 0.5-1.0 cm), and 66 (37.3%) larger lesions (maximum diameter >1.0 cm). Among the 30 [16.95% (30/177)] samples of mucosal tissue, 15 [50.00% (15/30)] were mainly cardia gland, 8 [26.67% (8/30)] were mainly pyloric gland, 6 [20.00% (6/30)] were mixed type, and 1 [3.33% (1/30)] was squamous epithelium. In the immunohistochemical test, 20 cases [66.67% (20/30)] showed positive of H +/K +-ATPase, and 10 cases [33.33% (10/30)] were negative. Conclusion:HGMUE is more common in male patients, and may be combined with GERD. Among them, patients with combined GERD are more likely to develop laryngopharyngeal reflux. The heterotopic gastric mucosas lesions are orange-red round, oval or elongated island-like under gastroscopy, and most of them are flat, single and median- or large-sized. Histological types are mostly fundic glands, and H +/K +-ATPase positive is more common. It is speculated that acid secretion may be an important factor leading to throat symptoms.
		                        		
		                        		
		                        		
		                        	
7.Progress of mass spectrometry-based proteomics technology andits application of radiation
Jiaqi XIANG ; Qingjie LIU ; Chengfang WANG ; Mei TIAN
Chinese Journal of Radiological Health 2021;30(1):110-116
		                        		
		                        			
		                        			Proteomics was first proposed by Marc Wikins, et al in 1995. It refers to the investigation of all the proteins expressed in a set of genomes. With the development of mass spectrometry technology, it more and more extensively apply to the field of radiation. In particular, due to the widespread application of nuclear and radiation technologies has greatly increased, the probability of nuclear and radiation accidents, and the exposure of large-scale populations are increased. Therefore, simple, rapid, and high-throughput measurement of acute radiation exposure is necessary. The application of proteomics technology to study the molecular biological effects of radiation and the discovery of radiation biomarkers provide the possibility for rapid high-through put dose assessment. We reviewed the recent development of proteomics and its application in the field of radiation.
		                        		
		                        		
		                        		
		                        	
8.Nomogram for predicting axillary lymph node pathological response in node-positive breast cancer patients after neoadjuvant chemotherapy.
Wenyan WANG ; Xin WANG ; Jiaqi LIU ; Qiang ZHU ; Xiang WANG ; Pilin WANG
Chinese Medical Journal 2021;135(3):333-340
		                        		
		                        			BACKGROUND:
		                        			Pathological complete response (pCR) of axillary lymph nodes (ALNs) is frequently achieved in patients with clinically node-positive breast cancer after neoadjuvant chemotherapy (NAC), and ALN status is an important prognostic factor for breast cancer patients. This study aims to develop a new predictive clinical model to assess the ALN pCR rate after NAC.
		                        		
		                        			METHODS:
		                        			This was a retrospective series of 467 patients who had biopsy-proven positive ALNs at diagnosis and underwent ALN dissection from 2007 to 2014 at the National Cancer Center/Cancer Hospital of the Chinese Academy of Medical Sciences. We analyzed the clinicopathologic features of the patients and developed a nomogram to predict the probability of ALN pCR. A multivariable logistic regression stepwise model was used to construct a nomogram to predict ALN pCR in node-positive patients. The adjusted area under the receiver operating characteristic curve (AUC) was calculated to quantify the ability to rank patients by risk. Internal validation was performed using the 50/50 hold-out validation method. The nomogram was externally validated with prospective cohorts of 167 patients from 2016 to 2018 at the Cancer Hospital of the Chinese Academy of Medical Sciences and 114 patients from 2018 to 2020 at Beijing Tiantan Hospital.
		                        		
		                        			RESULTS:
		                        			In this retrospective study, 115 (24.6%) patients achieved ALN pCR after NAC. Multivariate analysis showed that clinical tumor stage (Odds ratio [OR]: 0.321, 95% confidence interval [CI]: 0.121-0.856; P = 0.023); primary tumor response (OR: 0.189; 95% CI: 0.123-0.292; P < 0.001), and estrogen receptor status (OR: 0.530, 95% CI: 0.304-0.925; P = 0.025) were independent predictors of ALN pCR. The nomogram was constructed based on the result of multivariate analysis. In the internal validation of performance of nomogram, the AUCs for the training and test sets were 0.719 and 0.753, respectively. The nomogram was validated in external cohorts with AUCs of 0.720, which demonstrated good discriminatory power in these data sets.
		                        		
		                        			CONCLUSION:
		                        			We developed a nomogram to predict the likelihood of axillary pCR in node-positive breast cancer patients after NAC. The predictive model performed well in multicenter prospective external validation. This practical tool could provide information to surgeons regarding whether to perform additional ALN dissection after NAC.
		                        		
		                        			TRIAL REGISTRATION
		                        			ChiCTR.org.cn, ChiCTR1800014968.
		                        		
		                        		
		                        		
		                        			Breast Neoplasms/drug therapy*
		                        			;
		                        		
		                        			Female
		                        			;
		                        		
		                        			Humans
		                        			;
		                        		
		                        			Lymph Nodes
		                        			;
		                        		
		                        			Lymphatic Metastasis
		                        			;
		                        		
		                        			Neoadjuvant Therapy
		                        			;
		                        		
		                        			Nomograms
		                        			;
		                        		
		                        			Prospective Studies
		                        			;
		                        		
		                        			Retrospective Studies
		                        			
		                        		
		                        	
9.Study on the role of FoxO1 in the regulation of osteoblastic metabolism by 1,25(OH) 2 D 3 in a high glucose envi⁃ronment
ZHOU Jiaqi ; SHU Linjing ; XIONG Yi ; ZHANG Yixin ; XIANG Lin
Journal of Prevention and Treatment for Stomatological Diseases 2020;28(1):24-29
		                        		
		                        			Objective:
		                        			Yingying, Email: yywdentist@163.com, Tel: 86⁃28⁃85503579
【Abstract】 Objective To explore the effect of 1,25(OH) 2 D 3 on the regulation of bone metabolism in a high⁃glucose
environment and to provide evidence for the possible regulatory mechanism of 1,25(OH) 2 D 3 on osteoblasts in a high⁃glu⁃
cose environment.
		                        		
		                        			 Methods:
		                        			The osteoblast cell line MC3T3⁃E1 was cultured in 3 groups: ① control group, cultured
in low⁃glucose (5.5 mmol/L) DMEM; ② high⁃glucose group: cultured in high⁃glucose (22 mmol/L) DMEM; ③ high⁃glu⁃
cose +1,25(OH) 2 D 3 group: high⁃glucose DMEM + 1,25(OH) 2 D 3 medium culture. The CCK⁃8 method was used to detect cell proliferation in each group; Annexin V and FITC apoptosis kits were used to detect apoptosis; Alizarin red was used
to semiquantitatively analyze cell differentiation; qRT⁃PCR was used to detect forkhead transcription factor⁃1 (forkhead
transcription factor 1, FoxO1) mRNA expression. Immunofluorescence was used to observe the changes in FoxO1 pro⁃
tein expression and its relative position in the nucleus.
		                        		
		                        			 Results:
		                        			ence was used to observe the changes in FoxO1 pro⁃
tein expression and its relative position in the nucleus. Results Our analysis showed that compared with those in the
control group, the osteoblast apoptosis and proliferation in the high⁃glucose group were improved, while differentiation
was inhibited (P < 0.05); at the same time, the mRNA expression of FoxO1(P = 0.006) was reduced. The immunofluores⁃
cence results showed that more FoxO1 was inside the nucleus (P < 0.001). Compared with those in the high⁃glucose
group, excessive proliferation was inhibited, apoptosis was reduced, and osteogenic differentiation was improved in the
high⁃glucose +1,25(OH) 2 D 3 group (P < 0.05); furthermore, FoxO1 mRNA was decreased (P = 0.006), and the transfer of
FoxO1 protein was blocked (P < 0.001).
		                        		
		                        			 Conclusion 
		                        			re, FoxO1 mRNA was decreased (P = 0.006), and the transfer of
FoxO1 protein was blocked (P < 0.001). Conclusion We found that 1,25(OH) 2 D 3 may prevent the transfer of FoxO1 to
the cell nucleus, inhibit the abnormal proliferation and apoptosis of osteoblasts in a high⁃glucose environment, and re⁃
verse the inhibitory effect of high glucose on the differentiation of osteoblasts.
		                        		
		                        		
		                        		
		                        	
10.Expressions and effects of autophagy-related genes in bleomycin-induced skin fibrosis of mice
Xiang HE ; Jie LI ; Jiaqi LIU ; Zhao ZHENG ; Dahai HU
Chinese Journal of Burns 2020;36(5):346-356
		                        		
		                        			
		                        			Objective:To investigate the expressions and effects of autophagy-related genes in bleomycin-induced skin fibrosis of mice.Methods:(1) Totally 72 male BALB/c mice aged 6 weeks were divided into blank control group, simple phosphate buffer solution (PBS) group, and bleomycin group according to the random number table, with 24 mice in each group. Mice in blank control group received no treatment, and 100 μL of PBS and bleomycin (1 mg/mL) were respectively injected subcutaneously in the back skin of mice in simple PBS and bleomycin group, once a day for 28 days. On injection day (ID) 7, 14, 21, and 28, 6 mice in each group were collected to observe the skin change on the back of mice with naked eyes. After the observation, the mice were sacrificed and skin tissue on the back was taken. Skin tissue of mice on ID 28 was collected to measure the thickness of skin tissue by routine hematoxylin-eosin staining and observe skin tissue morphology by Masson staining. Skin tissue on ID 7, 14, 21, and 28 was taken to detect content of hydroxyproline by enzyme linked immunosorbent assay, and mRNA and protein expressions of p62, microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ) and Beclin-1 were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction and Western blotting, respectively. (2) Skin tissue of mice in blank control group in experiment (1) was taken to culture fibroblasts (Fbs) in 3rd-6th passages. The cells were divided into blank control group, simple PBS group, and bleomycin group according to the random number table, with 6 wells in each group. Cells in blank control group were not stimulated, and cells in simple PBS group and bleomycin group were stimulated with 20 μL of PBS and bleomycin (1 mg/mL) for 72 h, respectively. Cellular immunofluorescence staining was used to observe the expression of LC3 Ⅱ. Data were statistically analyzed with analysis of variance of factorial design, one-way analysis of variance, t test, and Bonferroni correction. Results:(1) Skin on the back of mice in blank control group and simple PBS group was thin and ruddy, and the veins were clear on ID 7, 14, 21, and 28. Several raised ridges were visible on the puncture site of mice in simple PBS group from ID 14. Skin on the back of mice was ruddy, with several raised ridges visible on the puncture site of mice in bleomycin group on ID 7, the skin turned slightly white on ID 14, the skin turned white obviously with unclear surrounding blood vessels on ID 21, and the skin turned white and the surrounding blood vessels could not be recognized on ID 28. (2) On ID 28, the skin thicknesses of mice in blank control group and simple PBS group were similar ( t=0.79, P>0.05). Compared with that in blank control group and simple PBS group, the skin thickness of mice in bleomycin group was significantly increased ( t=0.50, 0.50, P<0.01). (3) On ID 28, the skin tissue structure of mice in blank control group and simple PBS group was similar, with a small amount of orderly arranged collagen and evenly distributed hair follicle; the number of collagen of skin in mice of bleomycin group was increased obviously and arranged disorderly, and the number of hair follicle was decreased significantly. (4) On ID 7, 14, 21, and 28, the content of hydroxyproline in the skin tissue of mice in bleomycin group was significantly higher than that in blank control group and simple PBS group ( t=0.99, 0.98, 0.50, 0.51, 0.50, 0.50, 0.52, 0.51, P<0.05 or P<0.01). (5) On ID 7, p62 mRNA expression in the skin tissue of mice in bleomycin group was significantly lower than that in simple PBS group ( t=0.93, P<0.05). On ID 14 and 21, the mRNA expressions of p62, LC3 Ⅱ, and Beclin-1 in the skin tissue of mice in bleomycin group were significantly higher than those in blank control group ( t=0.74, 0.70, 0.58, 0.49, 0.51, 0.74, P<0.05) and simple PBS group ( t=0.94, 0.65, 0.65, 0.77, 0.49, 0.51, P<0.05). On ID 28, the mRNA expressions of p62 and Beclin-1 in the skin tissue of mice in bleomycin group were significantly lower than those in blank control group ( t=0.50, 0.44, P<0.05) and simple PBS group ( t=0.97, 0.55, P<0.05), and that of LC3 Ⅱ was significantly higher than that in blank control group and simple PBS group, respectively ( t=0.51, 0.98, P <0.01). (6) On ID 7, 14, 21, and 28, the protein expressions of LC3 Ⅱ in blank control group, simple PBS group, and bleomycin group were 0.167±0.042, 0.122±0.016, 0.553±0.078, 0.118±0.035, 0.120±0.023, 0.117±0.061, 0.581±0.039, 0.159±0.065, 0.233±0.027, 0.304±0.031, 1.020±0.010, 0.089±0.045. On ID 14, the protein expressions of p62 and Beclin-1 in the skin tissue of mice in bleomycin group were significantly higher than those in blank control group ( t=0.86, 0.89, P<0.05) and simple PBS group ( t=0.42, 0.89, P<0.05). On ID 21, the protein expressions of p62, LC3 Ⅱ, and Beclin-1 in the skin tissue of mice in bleomycin group were significantly higher than those in blank control group and simple PBS group ( t=0.82, 0.45, 0.50, 0.79, 0.51, 0.50, P<0.01). On ID 28, the protein expressions of p62, LC3 Ⅱ, and Beclin-1 in the skin tissue of mice in bleomycin group were significantly lower than those in blank control group and simple PBS group ( t=0.77, 0.54, 0.52, 0.50, 0.51, 0.50, P<0.05). (7) After culture for 72 h, the expression of LC3 Ⅱ in Fbs of bleomycin group was significantly lower than that of blank control group and simple PBS group, respectively. Conclusions:In the process of bleomycin stimulating skin fibrosis, autophagy-related genes increase firstly and then decrease. When the autophagy process is activated, it is expected to reverse the process of skin fibrosis.
		                        		
		                        		
		                        		
		                        	
            

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