Acta Universitatis Medicinalis Anhui

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Anhui Provincial Education Department

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Anhui Medical University

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Weng Jianping

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Wall-broken ganoderma lucidum spore powder protects against DEN/CCl4-induced hepatocellular carcinoma in mice by modulating the Nrf2 pathway

Jin Hai1, Wan Shuo1, Liu Qian1, Zhao Shiyu1, Li Jing1, Zhu Jiaxing1, An Jiaxing1, Jin Feng2

DOI: 专辑：医药卫生科技

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Abstract:
Objectivec To investigate the therapeutic effect of wall-broken Ganoderma lucidum spore powder (GLS) on a mouse model of hepatocellular carcinoma and to explore its underlying mechanism. Methods Fifty 14-day-old C57BL/6J mice were divided into a control group, a model group and low, medium and high-dose GLS groups(0.25, 0.5, 1.0 g/kg ), with 10 mice in each group. A DEN/CCL4 mouse hepatocellular carcinoma model was established, and GLS was administered by gavage starting from the 8th week, once a day for 14 weeks. At the end of the final administration, the number of liver nodules was observed , and the serum alpha-fetoprotein (AFP) was detected using enzyme-linked immunosorbent assay. The content of catalase (CAT) in serum and liver tissue was determined by ammonium molybdate method. The content of reduced glutathione (GSH) in serum and liver tissue was measured by microplate assay. The content of glutathione peroxidase (GSH-PX) in serum and liver tissue was detected by colorimetric method. Immunohistochemistry (IHC-P) was employed to detect the protein expression of proliferating cell nuclear antigen (PCNA). R eal-time fluorescence quantitative polymerase chain reaction was utilized to detect the relative expression of nuclear factor E2-related factor 2 (Nrf2), Kelch-like epichlorohydrin-associated protein 1 (Keap1),NAD(P)H quinone oxidoreductase 1 (Nqo1), heme oxygenase-1 ( Ho-1 ) and glutamate cysteine ligase catalytic subunit (Gclc) mRNA. Results Compared with the control group, the content of serum AFP were significantly higher in the model group (P<0.01), while the contents of CAT, GSH and GSH-PX in liver and serum were significantly lower in the model group (P<0.01). The relative expression of Keap1 and Nqo1 mRNA was significantly higher (P<0.01), the relative expression of Nrf2, Ho-1, Gclc mRNA relative expression was significantly lower (P<0.01), and the positive expression rate of PCNA was significantly higher (P<0.01). Compared with the model group, the total number of liver nodules and content of serum AFP in the administered group were significantly lower (P<0.01), and the contents of CAT, GSH, and GSH-PX in the liver and serum of mice significantly increased (P<0.05 or P<0.01). The expression of Keap1 and Nqo1 mRNA significantly decreased (P<0.05). The expression of Nrf2, Ho-1 and Gclc mRNA significantly increased (P<0.05), and positive expression rate of PCNA significantly decreased (P<0.01). Conclusion GLS has a significant protective effect on DEN/CCL4 -induced mouse hepatocellular carcinoma model, and the mechanism of action is related to the regulation of oxidative stress in mouse liver.

4-Phenylbutyrate ameliorates lipopolysaccharide-induced bile acid metabolism disorder in maternal mice

Liang Xinru 1,2*,Sun Ying1,2*, Bao Shuangrui 1,2, Zhan Tong 1,2 ，Wu Hongyan1,2 ，Tao Wenkang3,4， Zhang Lun4 ，Wang Jianqing3,4,5 ，Zhang Cheng1,2

DOI: 专辑：医药卫生科技

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Humanization modification of IL9-2G1 rabbit monoclonal antibody

Zhou Changhong1,2, Xue Zimeng2, Tu Jiajie2, Wang Xinming1,2

DOI: 专辑：医药卫生科技

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Abstract:
Objective To humanize the variable region of the IL9-2G1 rabbit monoclonal antibody (IL9 2G1 mAb) and to evaluate its biological activity. Methods The humanized sequences of IL9-2G1 mAb were designed. The heavy chain variable region (VH) and light chain variable region (VL) of the antibody were humanized and cloned into vector backbones containing the constant region frameworks of human IgG1 and Ig Kappa subtypes. Protein purification was carried out using the Chinese hamster ovary cell (CHO) expression system. Enzyme-linked immunosorbent assay (ELISA) was used to qualitatively analyze the antigen-binding activity ofthe antibodies. After screening the optimal humanized antibody candidate, surface plasmon resonance (SPR) was performed to detect molecular affinity. Western blot was used to examine the effects of the rIL-9 group and the rIL-9 + hIL-9 mAb group on the phosphorylation of Janus kinase 1/signal transducer and activator of transcription 3 (JAK1/STAT3), while ELISA was employed to assess the impact of the Th9 group and the Th9 + hIL-9 mAb group on interleukin-9 (IL-9) secretion. Results Purification and ELISA results showed that the heavy chain OH and light chain FL exhibited good compatibility and expression performance. SPR results indicated that the equilibrium dissociation constant (KD) of M501042-OHFL against the analyte M30903 was 1.33×10_7 M, which was superior to the parental 2G1 antibody. The Western blot results showed that the levels of phosphorylated JAK1 (p-JAK1) and phosphorylated STAT3 (p-STAT3) were significantly decreased in the rIL-9 + hIL-9 mAb group compared with the rIL-9 group (P<0.01). The ELISA results indicated that the secretion of IL-9 was significantly reduced in the Th9 + hIL-9 mAb group compared with the Th9 group (P<0.01). Conclusion Humanization modification is performed on the variable region of IL9-2G1 rabbit monoclonal antibody, and the humanized antibody hIL-9 mAb OHFL with high affinity and strong binding stability is successfully constructed, which provides an experimental basis for subsequent research.

Construction of a COPD risk prediction model based on machine learning and the COPD-SQ questionnaire

Chen Lin1, Zhao Luna1, Zhou Yue2, Wang Panpan3, Li Jingkun2,Zhang Wenwenl,Zhang Xinxin l,Wu Chaol, Liu Dong1

DOI: 专辑：医药卫生科技

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Abstract:
Objective This study aims to construct and evaluate various machine learning models for predicting the risk of chronic obstructive pulmonary disease (COPD) in individuals, thereby providing data support for early screening and intervention.Methods A total of 823 subjects were selected for this study, comprising l42 individuals in the high-risk group for COPD and 68l individuals in the low-risk group. Data collected included demographic characteristics, smoking history, symptoms (such as cough and shortness of breath), and scores from the Chronic obstructive pulmonary disease screening questionnaire. Four machine learning algorithms—Logistic Regression, Random Forest, Support Vector Machine, and XGBoost—were utilized to construct risk prediction models. The performance of these models was assessed using 5-fold cross-validation, with evaluation metrics including accuracy, precision, recall, Fl-score, area under the receiver operating characteristic curve (AUC), and average precision (AP). Furthermore, a feature importance analysis was performed.Results The Logistic Regression model exhibited superior performance, achieving an AUC of 0.982 and an AP of 0.939. This was closely followed by the Random Forest model, which recorded an AUC of 0.975 and an AP of 0.890. Feature importance analysis revealed that smoking history, symptoms of shortness of breath, and body weight were significant predictors. All models demonstrated robust performance in identifying low-risk populations, with precision values exceeding 0.93; however, variations were observed in their efficacy in identifying high-risk populations.Conclusion Machine learning models have proven effective in identifying individuals at high risk for COPD. Among these, the logistic regression model exhibits the best overall performance, efficiently identifying high-risk populations and serving as a valuable clinical auxiliary screening tool. Various models, each with distinct performance characteristics, are suited to different clinical screening scenarios, thereby offering targeted decision-making support for the establishment of a hierarchical and intelligent COPD screening pathway.

Experimental study of EGCG-loaded hydrogel scaffold for the treatment of periodontitis

Liu Longge, Mao Jing, Zheng Huimin, Li Wei

DOI: 专辑：医药卫生科技

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Preparation of rapamycin photosensitive nanodiscs and evaluation of their in vitro antitumor activity

Zhang Kuizhi1,2,3, Lu Guangzhao4, Lu Ying4, Yu Shidan5, Zhang Li1,2,3

DOI: 专辑：工程科技Ⅰ辑;医药卫生科技

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Abstract:
Objective To prepare photosensitive nanodiscs loaded with rapamycin and evaluate their in vitro antitumor activity against ovarian cancer. Methods Nanodiscs were prepared by the thin-film hydration method， and their particle size， potential and morphology were characterized by dynamic light scattering and transmission electron microscopy. High performance liquid chromatography was established for the determination of rapamycin content， and the specificity， linearity， precision， repeatability， stability， and recovery rate of the method were verified. The singlet oxygen generation and light-controlled drug release properties of nanodiscs under near-infrared light were evaluated respectively by singlet oxygen detection probe and reverse dialysis method. The cytotoxicity of the nanodiscs against human ovarian cancer cells (OVCAR-8) was evaluated by detecting cell metabolic activity. Confocal microscopy and flow cytometry were used to investigate the cellular uptake and endocytosis mechanisms of the nanodiscs. Results The nanodiscs exhibited a hydrodynamic diameter of (63.66±0.27) nm， a zeta potential of (-18.2 ± 2.4) mV， and demonstrated good stability. The results from transmission electron microscopy revealed that the nanodiscs exhibited spherical and short rod-like morphologies. The peak area of rapamycin showed good linearity in the concentration range of 0.2～40 μg/mL (R=0.999 9)， and the relative standard deviations (RSDs) of precision， repeatability and recovery were all less than 5%. Under near-infrared light irradiation， the nanodiscs could effectively produce singlet oxygen. At the same time， the singlet oxygen generated could induce lipid membrane oxidation， which significantly promoted the responsive release of rapamycin (P<0.001). Nanodiscs had no obvious cytotoxicity， but the cytotoxicity to ovarian cancer cells was significantly enhanced after near-infrared irradiation. Cellular uptake experiments confirmed that the nanodiscs mainly entered cells through clathrin-mediated， energy-dependent endocytosis (P<0.001)， with drug release triggered by light exposure. Conclusion Rapamycin photosensitive nanodiscs were successfully prepared， which showed significant light-controlled release properties and enhanced anti-ovarian cancer activity in vitro.

FGF21 overexpression upregulates SIRT1/GPX4 to inhibit ferroptosis and relieve CCl4 mouse acute liver injury

Ling Xutao1,2, Huang Qianqian3,4, Zhang Lun3,4, Xie Fang1,2, Bai Nan1,2, Wang Yingxia1,2, Hua Haoran1,2, Wang Haoze1,2, Dai Wenjing2,5, Li Kexin1,2, Wang Jianqing1,2,5

DOI: 专辑：医药卫生科技

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Abstract:
Objective To investigate the protective effect of liver-targeted fibroblast growth factor 21 (FGF21) overexpression against CCl₄-induced acute liver injury (ALI) and its association with silent information regulator 1/glutathione peroxidase 4 (SIRT1/GPX4) expression and ferroptosis-related markers. Methods Female ICR mice (8 weeks old) were randomly divided into Control, CCl₄, adeno- associated virus (AAV)-FGF21, and AAV-FGF21 + CCl4 groups. Liver-targeted FGF21 overexpression was achieved via tail vein injection of AAV8-FGF21 virus with green fluorescent protein (GFP) labeling. ALI was induced by intraperitoneal injection of CCl₄ (0.3 mL/kg), with sample collection at 12 hours. Body weight and liver weight were recorded to calculate the liver weight/body weight. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured using a biochemical analyzer. FGF21 expression in liver tissues was detected by immunofluorescence. Hepatic histopathology was evaluated by HE staining. RT-qPCR was performed to assess mRNA expression of ferroptosis and iron metabolism-related genes (ACSL4, PTGS2, HJV, FPN, GPX4), FGF21, and SIRT1. Protein expression ofFGF21, SIRT1, and GPX4 were determined by Western blot. Iron content, malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) activity in liver homogenate were quantified using assay kits. Results Compared with the Control group, the FGF21 mRNA and protein expression elevated in CCl4 group;serum ALT/AST levels and liver weight/body weigh increased. The HE staining results showed that the liver cell membranes were ruptured. The mRNA levels of ACSL4 and PTGS2, the iron content and MDA levels all increased (P<0.01). The mRNA levels of SIRT1, GPX4, HJV, and FPN, the GSH level and SOD activity all decreased (P<0.05), and the protein levels ofSIRT1 and GPX4 both decreased (P<0.05). Compared with the CCl4 group, the serum levels of ALT and AST in the AAV- FGF21 + CCl4 group were decreased (P<0.01), and the liver/body ratio of mice decreased (P<0.01). The results of HE staining showed that the degree of liver injury was alleviated. The mRNA levels of ACSL4 and PTGS2, iron content and MDA level all decreased (P<0.01), while the mRNA levels ofSIRT1, GPX4, HJV, FPN, GSH level and SOD activity all increased (P<0.05). The protein levels of SIRT1 and GPX4 both increased (P<0.05). Conclusion FGF21 overexpression is associated with enhanced SIRT1/GPX4 expression and suppression of ferroptosis features, thereby contributing to the alleviation of CCl4-induced acute liver injury.

Semaphorin 3A promotes pathological cardiac hypertrophy: an experimental study

Wu Lifei1,2, Zhang Jiaojiao1,3, Zhang Xing1,3, Li Donghang1,2, Wang Hongbo1,2, Cao Jimin1,3

DOI: 专辑：医药卫生科技

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Abstract:
Objective To investigate the effect of Semaphorin 3A (Sema3A) on the progression of pathological cardiac hypertrophy. Methods Wild-type C57BL 6J mice, Sema3A global overexpressing (Sema3A/Cre-ERTM) mice, and their littermate controls (Cre-ERTM) were used, with 6 mice per group. A pressure-overload induced cardiac hypertrophy model was established via transverse aortic constriction (TAC), with a sham-operated group (Sham) as the control. Cardiac hypertrophy was assessed by gross morphology, heart weight/body weight ratio (HW/BW), heart weight/tibia length ratio (HW/HL), and heart weight/lung weight ratio (HW/PW). Cardiac function was evaluated by measuring ejection fraction (EF) and fractional shortening (FS) via echocardiography. In heart tissues, the mRNA level of atrial natriuretic peptide (ANP) and Sema3A were detected by RT-qPCR, Sema3A protein level was detected by western blot, morphological structure was detected by hematoxylin and eosin (HE) staining, level of fibrosis was detected by Masson’s trichrome staining, and angiogenesis was detected by immunohistochemical detection of CD31.Forin vitroexperiments, rat H9C2 cardiomyocytes were treated with angiotensin II (Ang II) to induce hypertrophy, with PBS-treated cells as the control. After 48 hours, the mRNA expression of ANP, B-type natriuretic peptide (BNP) and Sema3A were measured by RT-qPCR, and Sema3A protein level was assessed by western blot.Results At the animal level, compared with the sham group, mice subjected to TAC showed significantly increased mRNA levels of ANP in cardiac tissue, as well as markedly elevated mRNA and protein levels of Sema3A (P<0.05). Compared with the Cre-ERTM + TAC group, the Sema3A/Cre-ERTM + TAC group exhibited more pronounced cardiac hypertrophy, greater ventricular wall thickening, higher HW/BW, HW/HL, and HW/PW ratios, more significant reductions in EF and FS, more severe cardiac fibrosis, and a significant decrease in cardiac CD31 expression levels (P<0.05).At the cellular level, compared with the control group, H9C2 cells treated with Ang II displayed significantly increased mRNA levels ofANP and BNP, along with markedly elevated mRNA and protein levels of Sema3A (P<0.05).ConclusionSema3A promotes the progression of pressure overload-induced cardiac hypertrophy by inhibiting angiogenesis.

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