1.Multivariate quantitative combined with chemometrics for evaluating the quality of Sophora flavescens from different producing areas
Jiahui CHEN ; Qiong LUO ; Junli ZHAO ; Yan HAI ; Chengdong LIU ; Tuya BAI ; Jun LI ; Yuewu WANG
China Pharmacy 2025;36(19):2404-2408
OBJECTIVE To establish a content determination method for multiple components in Sophora flavescens from different origins and to evaluate its quality by combining with chemometrics. METHODS Thirteen batches (No. K1-K13) of S. flavescens from different origins were selected as test samples. A high-performance liquid chromatography-tandem triple quadrupole mass spectrometry (HPLC-MS/MS) method was established to determine the contents of 12 components, including matrine, oxymatrine, betaine, cytisine, N-methylcytisine, sophoridine, genistein, sophoricoside, sophorone, formononetin, sophorolone Ⅰ and norkurarinone in S. flavescens. Chromatographic separation was performed on a Shim-pack GIST-HP C18 column with a mobile phase consisting of methanol (A) and water containing 0.1% formic acid (B), using gradient elution at a flow rate of 0.25 mL/min, column temperature of 35 ℃, and an injection volume of 3 μL. Mass spectrometry was conducted using an electrospray ionization source with positive and negative ion scanning. Data were collected in segments using the multiple reaction monitoring mode. Technique for order preference by similarity to ideal solution (TOPSIS) and grey relational analysis (GRA)methods were employed to compare and comprehensively evaluate the 13 batches of S. flavescens from different origins. RESULTS The methodological validation for the content determination met the relevant regulatory requirements. The contents of the 12 components were 490.66-1 231.00, 11 088.10- 18 021.50, 7.91-25.38, 903.97-1 713.64, 336.08-1 485.54,1 065.33-2 075.50, 27.52-71.80, 109.36-517.83, 6 034.55-10 632.73, 21.26-145.35, 814.84-1 911.32, 1 040.87-3 446.37 μg/g), respectively. TOPSIS results showed that the top 7 samples in Euclidean distance ranking were K6, K12, K11, K3, K5, K10, K13. The GRA results showed that the top 7 samples in the relative correlation ranking were K12, K11, K10, K6, K13, K5, K3. CONCLUSIONS The established HPLC-MS/MS method is rapid, accurate, highly sensitive, stable and reliable. Combined with chemometrics methods, it can be used for the quality control and evaluation of S. flavescens. The comprehensive quality of samples K3, K5, K6( from Hebei), K10( from Sichuan), K11-K13( from Shanxi), etc. is relatively superior.
2.Mechanism of tannins from Galla chinensis cream in promoting skin wound healing in rats based on FAK/PI3K/Akt/mTOR signaling pathway.
Wen YI ; Zi-Yi YAN ; Meng-Qiong SHI ; Ying ZHANG ; Jie LIU ; Qian YI ; Hai-Ming TANG ; Yi-Wen LIU
China Journal of Chinese Materia Medica 2025;50(2):480-497
This study investigated the effects and action mechanism of tannins from Galla chinensis cream(TGCC) on the skin wound of rat tail. Male Sprague Dawley(SD) rats were randomly divided into a control group, model group, model+low-dose TGCC(50 mg per rat) group, model+high-dose TGCC group(100 mg per rat), and model+TGC+FAK inhibitor(Y15) cream(100 mg+10 mg per rat) group, with 10 rats in each group. After the rat tail skin injury model was successfully constructed, in the treatment group, corresponding drugs were applied to the wound surface, while in the control and model groups, the same amount of cream base as the TGCC group was applied by the same method. Then, sterile gauze was wrapped around the wound edge, and these operations were performed three times a day for 28 consecutive days. The wound healing status at the third, seventh, eleventh, fourteenth, twenty-first, and twenty-eighth days was recorded, and the wound healing rate and healing time were calculated. On the day after the last dose of medication, rat serum and tail skin wound tissue were collected for analyzing the activities of serum alanine aminotransferase(ALT), aspartate aminotransferase(AST), creatinine(CREA), urea, reactive oxygen species(ROS), interferon gamma(IFN-γ), interleukin(IL)-1β, IL-6, IL-4, IL-10, tumor necrosis factor(TNF)-α, as well as catalase(CAT), glutathione(GSH), lactate dehydrogenase(LDH), malondialdehyde(MDA), myeloperoxidase(MPO), superoxide dismutase(SOD), total antioxidant capacity(T-AOC), platelet endothelial cell adhesion molecule-1(CD31), and leukocyte differentiation antigen 34(CD34) in the wound tissue of rat tail skin. Hematoxylin-eosin, Masson, and sirius red staining were used to observe the morphological changes in the wound tissue of rat tail skin. The thickness of the epidermis, the number of fibroblasts and blood vessels, and the contents of collagen fibers, typeⅠ collagen(COLⅠ), and COLⅢ were calculated. The mRNA expressions of keratin 10(KRT10), KRT14, vascular endothelial growth factor(VEGF), fibroblast growth factor(FGF), epidermal growth factor(EGF), CD31, CD34, matrix metallopeptidase-2(MMP-2), MMP-9, COLⅠ, COLⅢ, desmin, fibroblast specific protein 1(FSP1), IFN-γ, IL-1β, TNF-α, IL-4, IL-6, and IL-10 in skin wound tissue were determined by quantitative real-time polymerase chain reaction(PCR). Western blot was utilized to detect the protein expressions of KRT10, KRT14, VEGF, FGF, EGF, MMP-2, MMP-9, COLⅠ, COLⅢ, desmin, FSP1, focal adhesion kinase(FAK), phosphorylated focal adhesion kinase(p-FAK), phosphatidylin-ositol-3-kinase(PI3K), phosphorylated phosphatidylin-ositol-3-kinase(p-PI3K), protein kinase B(Akt), phosphorylated protein kinase B(p-Akt), mammalian target of rapamycin(mTOR), and phosphorylated mammalian target of rapamycin(p-mTOR). The results manifest that TGCC can dramatically elevate the healing rate of rat tail wounds and shorten wound healing time. Besides, it can reduce serum ROS levels, the contents of MDA, MPO, and LDH in the rat skin wound tissue, as well as the serum IFN-γ, IL-1β, IL-6, and TNF-α levels and the mRNA expression levels of IFN-γ, IL-1β, IL-6, and TNF-α in the skin wound tissue. It can elevate the activities of CAT, GSH, SOD, and T-AOC in wound tissue, the IL-4 and IL-10 contents in serum, and the mRNA expressions of IL-4 and IL-10 in the wound tissue. In addition, TGGC can inhibit inflammatory cell infiltration and increase the epidermal thickness, counts of fibroblasts and blood vessels, and contents of collagen fibers, COLⅠ, and COLⅢ. Besides, TGCC can elevate the mRNA and protein expressions of epidermal differentiation markers(KRT10 and KRT14), endothelial cell markers(CD31 and CD34), angiogenesis and fibroblast proliferation, differentiation markers(VEGF, FGF, EGF, COLⅠ, COLⅢ, desmin, and FSP1), reduce the mRNA and protein expressions of gelatinases(MMP-2 and MMP-9), and increase protein expressions of p-FAK, p-PI3K, p-Akt, p-mTOR, as well as ratios of p-FAK/FAK, p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR. These results suggest that TGCC can significantly facilitate skin wound healing, and its mechanism may be related to the activation of the FAK/PI3K/Akt/mTOR signaling pathway, inhibition of inflammatory cell infiltration in skin wound tissue, elevation of epidermal thickness, counts of fibroblasts and vessels, and contents of collagen fiber, COLⅠ, and COLⅢ, and reduction of MMP-2 and MMP-9 expressions, thus accelerating wound healing.
Animals
;
Male
;
Wound Healing/drug effects*
;
Rats
;
Rats, Sprague-Dawley
;
Signal Transduction/drug effects*
;
TOR Serine-Threonine Kinases/genetics*
;
Phosphatidylinositol 3-Kinases/genetics*
;
Skin/metabolism*
;
Proto-Oncogene Proteins c-akt/genetics*
;
Tannins/pharmacology*
;
Humans
;
Drugs, Chinese Herbal/administration & dosage*
;
Focal Adhesion Kinase 1/genetics*
3.Clinical study on the treatment of traumatic osteomyelitis of the upper tibia by membrane-induced technique combined with gastrocnemius muscle flap transposition.
Yi-Yang LIU ; Yi-Hang LU ; Qiong-Lin CHEN ; Bing-Yuan LIN ; Hai-Yong REN ; Kai HUANG ; Yang ZHANG ; Qiao-Feng GUO
China Journal of Orthopaedics and Traumatology 2025;38(9):937-944
OBJECTIVE:
To explore clinical efficacy of membrane-induced technique combined with gastrocnemius muscle flap transposition in treating traumatic osteomyelitis of the upper tibia.
METHODS:
A retrospective analysis was conducted on 7 patients with traumatic osteomyelitis of the upper tibia who were treated with membrane-induced technique combined with gastrocnemius muscle flap transposition from January 2022 to December 2023. Among them, there were 4 males and 3 females; aged from 29 to 57 years old; 4 patients were treated after open fracture, 2 patients were treated after closed fracture, and 1 patient was treated after scalding; the courses of disease ranges from 2 weeks to 8 years; sinus tracts were present in all patients, and the lesion range of the tibia ranged from 5 to 9 cm. The results of deep tissue bacterial culture showed that 2 patients were negative, 3 patients were staphylococcus aureus, 1 patient was methicillin-resistant staphylococcus aureus, and 1 patient was pseudomonas aeruginosa and 1 patient was klebsiella pneumoniae. After debridement, the range of bone defect ranged from 8 to 12 cm, and the cortical defect accounted for approximately 30% of the circumference. The area of soft tissue defect ranged from 8.0 cm×2.0 cm to 10.0 cm×6.0 cm. At the first stage, vancomycin-loaded/meropenem/gentamicin-loaded bone cement was implanted. The gastrocnemius muscle flap was repositioned to cover the wound surface and free skin grafting was performed. After an interval of 7 to 10 weeks, the stageⅡsurgery was performed to remove bone cement. Autologous iliac bone mixed with vancomycin/gentamicin and calcium sulfate artificial bone was transplanted, and the wound was sutured. One patient retained the original internal plants, one patient removed the internal plants and replaced them with steel plate external fixation, one patient replaced the internal plants and added steel plate external fixation, and three patients were simply fixed with steel plate external fixation. One year after operation, the recovery of knee joint and ankle joint functions was evaluated by using Hospital for Special Surgery (HSS) knee joint score and Kofoed ankle joint function score respectively.
RESULTS:
All patients had their wounds closed simultaneously with bone cement implantation and healed well. All patients were followed up for 12 to 17 months after operation, and satisfactory bone healing was achieved at 6 months after stageⅡsurgery. Twelve months after operation, all patients had good bone healing without obvious limping was observed when walking. At 12 months after operation HSS knee joint score ranged from 93 to 100 points, and Kofoed ankle function score ranged from 96 to 100 points.
CONCLUSION
For traumatic osteomyelitis of the upper tibia, a staged treatment plan combining membrane-induced technique and gastrocnemius flap transposition on the basis of thorough debridement could safely cover the wound surface, effectively control bone infection and achieve satisfactory bone healing, without adverse effects on limb function.
Humans
;
Male
;
Female
;
Middle Aged
;
Osteomyelitis/surgery*
;
Adult
;
Surgical Flaps
;
Retrospective Studies
;
Tibia/injuries*
;
Muscle, Skeletal/surgery*
4.Antithrombotic effect in zebrafish of a fibrinolytic protein EPF3 from Dilong (Pheretima vulgaris Chen) and its transport mechanism in Caco-2 monolayer through cell bypass pathway.
Wan-Ling ZHONG ; Jian-Qiong YANG ; Hai LIU ; Ya-Li WU ; Hui-Juan SHEN ; Peng-Yue LI ; Shou-Ying DU
Journal of Integrative Medicine 2025;23(4):415-428
OBJECTIVE:
EPF3 is a fibrinolysin monomer isolated and purified from Pheretima vulgaris Chen, an earthworm used in traditional Chinese medicine as Dilong for treating blood stasis syndrome. Its composition, anticoagulant and fibrinolytic activities, and relevant mechanisms have been confirmed through in vitro experiments. However, whether it has antithrombotic effects in vivo and can be absorbed by the gastrointestinal tract is unknown. This study evaluates the antithrombotic effect in zebrafish and investigates the gastrointestinal stability and intestinal absorption mechanism of this protein in vitro.
METHODS:
The antithrombotic effect of EPF3 in vivo was verified using the zebrafish thrombus model induced by arachidonic acid and FeCl3. Then, the protein bands of EPF3 incubated with simulated gastric fluid (SGF), simulated intestinal fluid (SIF), and homogenate of Caco-2 cells (HC2C) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to evaluate its gastrointestinal stability. Finally, the transport behavior and absorption mechanism of EPF3 were studied using Caco-2 cell monolayer.
RESULTS:
EPF3 could significantly enhance the returned blood volume and blood flow velocity in zebrafish with platelet aggregation thrombus induced by arachidonic acid. It could also prolong the formation time of tail artery thrombus and increase the blood flow velocity in zebrafish with vessel injury thrombus induced by FeCl3. EPF3 was stable in SIF and HC2C and unstable in SGF. The permeability of EPF3 in Caco-2 monolayer was time-dependent and concentration-dependent. The efflux ratio was less than 1.2 during transport, and the transport behavior was not affected by inhibitors. EPF3 could reversibly reduce the expression of tight junction-related proteins, including zonula occludens-1, occludin, and claudin-1 in Caco-2 cells.
CONCLUSION
EPF3 could play a thrombolytic and antithrombotic role in zebrafish. It could be transported and absorbed into the intestine through cellular bypass pathway by opening the intestinal epithelium tight junction. This study provides a scientific explanation for the antithrombotic effect of earthworm and provides a basis for the feasibility of subsequent development of EPF3 as an antithrombotic enteric-soluble preparation. Please cite this article as: Zhong WL, Yang JQ, Liu H, Wu YL, Shen HJ, Li PY, Du SY. Antithrombotic effect in zebrafish of a fibrinolytic protein EPF3 from Dilong (Pheretima vulgaris Chen) and its transport mechanism in Caco-2 monolayer through cell bypass pathway. J Integr Med. 2025; 23(4): 415-428.
Animals
;
Zebrafish
;
Humans
;
Caco-2 Cells
;
Fibrinolytic Agents/pharmacology*
;
Thrombosis/drug therapy*
;
Intestinal Absorption
5.Clinical characterization and prediction modeling of lung cancer patients with high energy metabolism
Jiang-Shan REN ; Jun-Mei JIA ; Ping SUN ; Mei PING ; Qiong-Qiong ZHANG ; Yan-Yan LIU ; He-Ping ZHAO ; Yan CHEN ; Dong-Wen RONG ; Kang WANG ; Hai-Le QIU ; Chen-An LIU ; Yu-Yu FAN ; De-Gang YU
Medical Journal of Chinese People's Liberation Army 2024;49(9):1004-1010
Objective To analyze the clinical characteristics of high energy metabolism in lung cancer patients and its correlation with body composition,nutritional status,and quality of life,and to develop a corresponding risk prediction model.Methods Retrospectively analyzed 132 primary lung cancer patients admitted to the First Hospital of Shanxi Medical University from January 2022 to May 2023,and categorized into high(n=94)and low energy metabolism group(n=38)based on their metabolic status.Differences in clinical data,body composition,Patient Generated Subjective Global Assessment(PG-SGA)scores,and European Organization for Research and treatment of Cancer(EORTC)Quality of Life Questionnaire-Core 30(QLQ-C30)scores were compared between the two groups.Logistic regression was used to identify the risk factors for high energy metabolism in lung cancer patients,and a risk prediction model was established accordingly;the Hosmer-Lemeshow test was used to assess the model fit,and the ROC curve was used to test the predictive efficacy of the model.Results Of the 132 patients with primary lung cancer,94(71.2%)exhibited high energy metabolism.Compared with low energy metabolism group,patients in high-energy metabolism group had a smoking index of 400 or higher,advanced disease staging of stage Ⅲ or Ⅳ,and higher levels of IL-6 level,low adiposity index,low skeletal muscle index,and malnutrition(P<0.05),and lower levels of total protein,albumin,hemoglobin level,and prognostic nutritional index(PNI)(P<0.05).There was no significant difference in age,gender,height,weight,BMI and disease type between the two groups(P>0.05).Logistic regression analysis showed that smoking index≥400,advanced disease stage,IL-6≥3.775 ng/L,and PNI<46.43 were independent risk factors for high energy metabolism in lung cancer patients.The AUC of the ROC curve for the established prediction model of high energy metabolism in lung cancer patients was 0.834(95%CI 0.763-0.904).Conclusion The high energy metabolic risk prediction model of lung cancer patients established in this study has good fit and prediction efficiency.
6.Clinical Study of Allogeneic Hematopoietic Stem Cell Transplanta-tion Patients with Co-Reactivation of Cytomegalovirus and Epstein-Barr Virus
Zhi-Wei WANG ; Qiong LIU ; Hai-Ying SUN
Journal of Experimental Hematology 2024;32(2):561-567
Objective:To explore the clinical characteristics and risk factors of cytomegalovirus(CMV)and Epstein-Barr virus(EBV)co-reactivation after allogeneic hematopoietic stem cell transplantation(allo-HSCT)and its influence on prognosis.Methods:The clinical data of 222 patients who received allo-HSCT from January 2015 to December 2020 were collected,and the patients were divided into groups according to the occurrence of CMV and EBV infection.Kaplan-Meier method was used for survival analysis,and Cox proportional hazard regression model was used to analyze the risk factors of co-reactivation of CMV and EBV.Results:After allo-HSCT,there were 30 patients with co-reactivation of CMV and EBV(CMV++EBV+group),101 patients with CMV viremia(CMV+group),149 patients with EBV viremia(EBV+group),and 28 patients with CMV and EBV inactivation(CMV-+EBV-group).Compared with the other groups,the incidence of acute graft-versus-host disease(aGVHD)and hemorrhagic cystitis(HC)was higher in CMV++EBV+groups(53.3%vs 42.6%,36.9%,17.9%,P<0.001;36.7%vs 32.7%,22.8%,10.7%,P=0.042).The incidence of post-transplant lymphoproliferative disease(PTLD)in CMV++EBV+group was similar to CMV+group and EBV+group(3.3%vs 3.0%,3.4%,P=0.811).Univariate and multivariate analysis showed that the persistent time of CMV and EBV after transplantation were independent risk factors for co-reactivation of CMV and EBV.Compared with the other groups,the 2-year overall survival(OS)rate and 2-year disease-free survival(DFS)rate of patients inCMV++EBV+group were lower(46.7%vs 74.9%,83.4%,71.4%,P<0.001;46.7%vs 70.9%,79.5%,69.9%,P=0.002),and 2-year non-recurrence mortality(NRM)was higher(48.2%vs 22%,13.6%,18.7%,P<0.001).Conclusion:The persistent time of CMV and EBV after transplantation are independent risk factors for patients with co-reactivation of CMV and EBV.Patients with co-reactivation of CMV and EBV had lower OS and DFS rate and higher NRM,suggesting that the clinical prognosis of the patients are worse.
7.Exploring biological connotation of blood stasis syndrome of rheumatoid arthritis and establishment of improved animal models based on syndrome-symptom mapping
Wen-jia CHEN ; Tao LI ; Ming-zhu XU ; Xun GONG ; Wei-xiang LIU ; Pei-hao LI ; Quan JIANG ; Wei LIU ; Xia MAO ; Xin LI ; Hai-yu XU ; Na LIN ; Yan-qiong ZHANG
Acta Pharmaceutica Sinica 2023;58(8):2434-2441
Blood stasis syndrome is one of the core clinical syndrome of rheumatoid arthritis (RA), but the biological connotation of this syndrome is not clear, and there is a lack of disease improved animal models that match the characteristics of this disease and syndrome. The aim of this study was to screen the candidate biomarker gene set of blood stasis syndrome of RA, reveal the biological connotation of this syndrome, and explore and evaluate the preparation method of the improved animal model based on the characteristics of "disease-syndrome-symptom". The study was approved by the ethics committee of Guang'anmen Hospital, Chinese Academy of Traditional Chinese Medicine (No. 2019-073-KY-01) and the First Affiliated Hospital of Tianjin University of Traditional Chinese Medicine (No. TYLL2021[K]018), and the study subjects gave their informed consent. Animal welfare and experimental procedures followed the regulations of the Experimental Animal Ethics Committee of the Chinese Academy of Traditional Chinese Medicine (No. IBTCMCACMS21-2207-01). The whole blood samples were collected clinically from RA patients with blood stasis syndrome (3 cases) or other syndromes (7 types, 3 cases/type), and healthy volunteers (4 cases), and then transcriptome sequencing, KEGG, gene set enrichment analysis (GSEA) and weighted correlation network analysis (WGCNA) analysis were performed. 126 pivotal genes were screened, and their functional annotation results were significantly enriched in "immune-inflammation" related pathways and lipid metabolism regulation (sphingolipids, ether lipid metabolism and steroid biosynthesis). Syndrome-symptom mapping of hub gene set to the TCM primary and secondary symptoms, Western phenotypic symptoms and pathological links showed that joint tingling, abnormal joint morphology, petechiae and abnormal blood circulation are representative of blood stasis syndrome of RA. The results of the improved animal model showed that the rats in the collagen-induced arthritis + adrenaline hydrochloride (CIA+Adr) 3 model group had increased blood rheology, coagulation, platelet function and endothelial function abnormalities compared with the CIA-alone model group, suggesting that the rats with blood stasis syndrome of RA may be in a state of "blood stasis". The results of the study can help to advance the objective study of the evidence of blood stasis syndrome in RA, and provide new ideas for the establishment of an animal model that reflects the clinical characteristics of the disease and syndrome.
8.Bis (2-butoxyethyl) Phthalate Delays Puberty Onset by Increasing Oxidative Stress and Apoptosis in Leydig Cells in Rats.
Miao Qing LIU ; Hai Qiong CHEN ; Hai Peng DAI ; Jing Jing LI ; Fu Hong TIAN ; Yi Yan WANG ; Cong De CHEN ; Xiao Heng LI ; Jun Wei LI ; Zhong Rong LI ; Ren Shan GE
Biomedical and Environmental Sciences 2023;36(1):60-75
OBJECTIVE:
This study investigated the effects of bis (2-butoxyethyl) phthalate (BBOP) on the onset of male puberty by affecting Leydig cell development in rats.
METHODS:
Thirty 35-day-old male Sprague-Dawley rats were randomly allocated to five groups mg/kg bw per day that were gavaged for 21 days with BBOP at 0, 10, 100, 250, or 500 mg/kg bw per day. The hormone profiles; Leydig cell morphological metrics; mRNA and protein levels; oxidative stress; and AKT, mTOR, ERK1/2, and GSK3β pathways were assessed.
RESULTS:
BBOP at 250 and/or 500 mg/kg bw per day decreased serum testosterone, luteinizing hormone, and follicle-stimulating hormone levels mg/kg bw per day (P < 0.05). BBOP at 500 mg/kg bw per day decreased Leydig cell number mg/kg bw per day and downregulated Cyp11a1, Insl3, Hsd11b1, and Dhh in the testes, and Lhb and Fshb mRNAs in the pituitary gland (P < 0.05). The malondialdehyde content in the testis significantly increased, while Sod1 and Sod2 mRNAs were markedly down-regulated, by BBOP treatment at 250-500 mg/kg bw per day (P < 0.05). Furthermore, BBOP at 500 mg/kg bw per day decreased AKT1/AKT2, mTOR, and ERK1/2 phosphorylation, and GSK3β and SIRT1 levels mg/kg bw per day (P < 0.05). Finally, BBOP at 100 or 500 μmol/L induced ROS and apoptosis in Leydig cells after 24 h of treatment in vitro (P < 0.05).
CONCLUSION:
BBOP delays puberty onset by increasing oxidative stress and apoptosis in Leydig cells in rats.
UNLABELLED
The graphical abstract is available on the website www.besjournal.com.
Rats
;
Male
;
Animals
;
Leydig Cells/metabolism*
;
Testosterone
;
Glycogen Synthase Kinase 3 beta/pharmacology*
;
Rats, Sprague-Dawley
;
Sexual Maturation
;
Testis
;
Oxidative Stress
;
TOR Serine-Threonine Kinases/metabolism*
;
Apoptosis
9.Effect of MG53 on Cardiac Function Affected by Acute Doxorubicin-Induced Cardiotoxicity in Mice and Its Mechanism
Wei-zhe LU ; Hai-qiong LIU ; Han-yan YANG ; Xu-dong SONG ; Ai-hua CHEN
Journal of Sun Yat-sen University(Medical Sciences) 2023;44(1):34-43
ObjectiveThis study was designed to explore the effect of MG53 on cardiac function affected by acute doxorubicin (DOX)-induced cardiotoxicity (DIC) in mice and its possible mechanism. MethodsIn vivo, C57BL/6 mice were injected intraperitoneally with twenty mg/kg DOX for one week to induce the acute DIC. In vitro, neonatal rat cardiomyocytes (NRCs) were treated with 1 μmol/L DOX to induce DIC. A small animal ultrasound imaging system was used to evaluate cardiac function, and the left ventricular changes in ejection fraction (EF) and fraction shortening (FS) were measured. qPCR technology was used to evaluate cardiac remodeling related factors ANP, BNP and α-MHC, autophagy-related factors Beclin1 and LC3, and apoptosis-related factor CASPASE3. Autophagy-related protein levels of Beclin1, LC3 and apoptosis-related protein levels of caspase3 were assessed by Western Blot. Transmission electron microscopy (TEM) was used to detect autophagosomes in heart tissues. TUNEL assay kit was used to detect apoptosis in neonatal murine cardiomyocytes. ResultsThe small animal ultrasound imaging revealed cardiac function was significantly reduced by doxorubicin in the DOX group and DOX+AAV9-NC group compared with the sham group (EF: Sham: 86.06 ± 2.08 vs. DOX:58.97 ± 1.62, P < 0.000 1; Sham: 86.06 ± 2.08 vs. DOX+AAV9-NC: 59.00 ± 1.86, P < 0.000 1. FS: Sham: 45.47 ± 1.95 vs. DOX:30.68 ± 1.21, P < 0.000 1; Sham: 45.47 ± 1.95 vs. DOX+AAV9-NC: 30.79 ± 1.13, P < 0.000 1). However, the overexpression of MG53 with adeno-associated virus9 (AAV9) ameliorated cardiac dysfunction (EF: DOX+AAV9-MG53: 66.93 ± 1.78 vs. DOX+AAV9-NC: 59.00 ± 1.86, P < 0.000 1. FS: DOX+AAV9-MG53: 36.35 ± 1.33 vs. DOX+AAV9-NC: 30.79 ± 1.13, P < 0.000 1). TEM showed autophagosomes were increased in the DOX+AAV9-MG53 group compared with the DOX group and DOX+AAV9-NC. qPCR results suggested that MG53 down-regulated the mRNA expression of cardiac remodeling related genes. Additionally, Western blot results confirmed that the protein level of caspases3 was decreased and Beclin1 and LC3 expression was increased in the DOX+AAV9-MG53 group compared with those in the DOX group and DOX+AAV9-NC group (caspase: DOX+AAV9-MG53: 1.49 ± 0.13 vs. DOX+AAV9-NC: 2.49 ± 0.46, P = 0.000 2; Beclin-1: DOX+AAV9-MG53:0.82 ± 0.02 vs. DOX+AAV9-NC: 0.62 ± 0.05, P < 0.000 1; LC3: DOX+AAV9-MG53: 0.83 ± 0.04 vs. DOX+AAV9-NC: 0.40 ± 0.05, P < 0.000 1). In contrast, knockdown of MG53 significantly up-regulated the protein level of Caspase3 and significantly down-regulated the protein level of Beclin1 and LC3 (caspase: DOX+si-MG53: 4.52 ± 0.28 vs. DOX+si-NC: 3.37 ± 0.08, P < 0.000 1; Beclin-1: DOX+si-MG53: 0.34 ± 0.06 vs. DOX+si-NC: 0.54 ± 0.07, P = 0.026 2; LC3: DOX+si-MG53: 0.41 ± 0.12 vs. DOX+si-NC: 0.70 ± 0.07, P = 0.001 5). TUNEL analysis showed overexpression of MG53 significantly inhibited the apoptosis of cardiomyocytes (DOX+Ad-MG53: 9.41 ± 0.53 vs. DOX+Ad-NC: 29.34 ± 7.29, P < 0.000 1), and knockdown of MG53 significantly facilitate the apoptosis of cardiomyocytes (DOX+si-MG53: 71.34 ± 5.90 vs. DOX+si-NC: 32.19 ± 9.91, P < 0.000 1). ConclusionMG53 inhibits cardiac apoptosis and enhances autophagy, which delays cardiac remodeling and ameliorates cardiac dysfunction.

Result Analysis
Print
Save
E-mail