1.Multicenter machine learning-based construction of a model for predicting potential organ donors and validation with decision curve analysis
Xu WANG ; Wenxiu LI ; Fenghua WANG ; Shuli WU ; Dong JIA ; Xin GE ; Zhihua SHAN ; Tongzuo LI
Organ Transplantation 2026;17(1):106-115
Objective To evaluate the predictive value of different machine learning models constructed in a multicenter environment for potential organ donors and verify their clinical application feasibility. Methods The study included 2 000 inpatients admitted to five domestic tertiary hospitals from January 2020 to December 2023, who met the criteria for potential organ donation assessment. They were randomly divided into a training set and an internal validation set (7∶3). Another 300 similar patients admitted to the First Affiliated Hospital of Harbin Medical University from January 2024 to April 2025 were included as an external validation set. The area under the curve (AUC), sensitivity, specificity, accuracy and F1-score of three models were compared, and the consistency of the potential organ donor determination process was tested. Multivariate logistic regression analysis was used to identify predictive factors of potential organ donors. Decision curve analysis (DCA) was employed to verify the resource efficiency of each model, and the threshold interval and intervention balance point were assessed. Results Apart from age, there were no significant differences in other basic characteristics among the centers (all P>0.05). The consistency of the potential organ donor determination process among researchers in each center was good [all 95% confidence interval (CI) lower limits >0]. In the internal validation set, the XGBoost model had the best predictive performance (AUC=0.92, 95% CI 0.89-0.94) and the best calibration (P=0.441, Brier score 0.099). In the external validation set, the XGBoost model also had the best predictive performance (AUC=0.91, 95% CI 0.88-0.94), outperforming logistic regression and random forest models. Multivariate logistic regression showed that mechanical ventilation had the greatest impact (odds ratio=2.06, 95% CI 1.54-2.76, P<0.001). DCA indicated that the XGBoost model had the highest net benefit in the threshold interval of 0.2-0.6. The “treat all” strategy only had a slight advantage at extremely low thresholds. The recommended threshold interval, which balances intervention costs and clinical benefits, considers ≥50% positive predictive value (PPV) and ≤50 referrals per 100 high-risk patients. Conclusions The XGBoost model established in a multicenter environment is accurate and well-calibrated in predicting potential organ donors. Combined with DCA, it may effectively guide the timing of clinical interventions and resource allocation, providing new ideas for the assessment and management of organ donation after brain death.
2.Predictive model for severe adverse reaction associated with bevacizumab based on the global trigger tool and machine learning
Yongfei FU ; Xin LONG ; Hongzhen XU ; Jian TANG ; Xiangqing LI ; Yucheng LONG ; Dong QIN
China Pharmacy 2026;37(4):497-503
OBJECTIVE To confirm trigger items for adverse drug reaction (ADR) induced by bevacizumab, to identify and analyze the occurrence of related ADR, and to establish a predictive model for severe adverse reaction (SAR) caused by this drug. METHODS Based on the global trigger tool (GTT) theory, and referencing the GTT White Paper, drug package inserts and relevant literature, trigger items for bevacizumab-related ADR were confirmed using a single-round Delphi method. Utilizing these established items, electronic medical records of relevant patients at Guilin People’s Hospital from January 2020 to September 2024 were actively screened via the China Hospital Pharmacovigilance System. Pharmacists then identified and tallied the occurrence of bevacizumab-induced ADR. Data from patients with any positive trigger item served as the study subjects (divided into training and test sets at a ratio of 7∶3), candidate feature variables were selected from 39 related variables using the Boruta algorithm, and the multivariable Logistic regression analysis was performed with the occurrence of SAR as the dependent variable. Based on these candidate features, Logistic Regression, Extreme Gradient Boosting, Light Gradient Boosting Machine, Random Forest, and Categorical Boosting models were constructed. Model performance was evaluated using metrics including the area under the curve (AUC) of receiver operating characteristic curve and recall rate. The Shapley Additive exPlanations (SHAP) method was applied to analyze and interpret the contribution of each variable. A nomogram was constructed based on the optimal model. RESULTS A total of 38 trigger items for active monitoring of bevacizumab-related ADR were determined, comprising 17 laboratory indicators, 13 clinical manifestations, and 8 intervention measures. In total, 483 patients with positive trigger items were included, and 318 patients with bevacizumab-induced ADR were identified, including 83 SARs. The positive predictive values for the trigger items and cases were 43.57% (708/1 625) and 63.84% (318/483), respectively. Bevacizumab-induced ADR involved 7 systems/organs, with the hematological system being the most frequently involved (64.15%). The Boruta algorithm selected 7 vari ables: serum potassium, hematocrit, albumin-to-globulin ratio, prealbumin, hypertension history, age and red blood cell count. Multivariable Logistic regression showed that elevated serum potassium levels were associated with a decreased risk of bevacizumab-induced SAR (OR=0.234, P =0.002), while a history of hypertension (OR=2.642, P =0.006) and increased age (OR=1.040, P =0.025) were associated with an increased risk. The Logistic Regression model demonstrated superior performance with higher AUC, F1 score and recall rate (0.761, 0.447, 0.607), compared to other models. SHAP evaluation results indicated that variables such as serum potassium, hematocrit, and age ranked highest in importance. CONCLUSIONS Totally 38 trigger entries have been successfully identified for active screening of bevacizumab-related ADR. Elevated serum potassium levels are a protective factor against bevacizumab-induced SAR, whereas the hypertension history and increased age are risk factors. The Logistic Regression model is the optimal predictive model.
3.Effects of Huanglian Jiedutang on Neutrophil Infiltration in Brain of MCAO Mice via Regulation of Chemokine Expression in Exosomes
Haojia ZHANG ; Kai WANG ; Zijin SUN ; Chunyu WANG ; Wei SHAO ; Kunjing LIU ; Liyang DONG ; Dan CHEN ; Wenxiu XU ; Chuanzun WANG ; Wen WANG ; Changxiang LI ; Xueqian WANG ; Fafeng CHENG ; Qingguo WANG
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(8):42-53
ObjectiveTo investigate whether Huanglian Jiedutang can inhibit neutrophil infiltration in the brains of middle cerebral artery occlusion (MCAO) mice by regulating the expression of neutrophil-related chemokines in exosomes, thereby achieving therapeutic effects. MethodsA total of 130 male specific pathogen-free (SPF) C57BL/6J mice were randomly divided into four groups: Sham-operated group, MCAO model group, Huanglian Jiedutang group (6 g·kg-1), and Ginaton group (21.6 mg·kg-1), with 10 mice in the Ginaton group and 40 mice in each of the remaining three groups. Mice in the Huanglian Jiedutang group and the Ginaton group were administered the corresponding drugs by oral gavage once daily at a volume of 0.15 mL·(10 g)-1 for 7 consecutive days, while the sham-operated and model groups received an equal volume of saline via the same route. After 7 days, MCAO surgery was performed. The distal and proximal ends of the right common carotid artery (CCA) were ligated, a small incision was made between the two ligatures, and a silicone rubber-coated monofilament with a rounded tip was inserted into the lumen to occlude the CCA. The filament was left in place for 1 h to establish a focal cerebral ischemia model. At 24 h after modeling, mice were evaluated. Neurological function was assessed using the Longa score. Cerebral infarct volume was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Cerebral blood flow was observed by laser speckle imaging. Hematoxylin and eosin (HE) staining and Nissl staining were used to observe pathological changes in brain tissues. Exosomes were isolated from mouse plasma and brain tissues by ultracentrifugation and molecular size exclusion and identified by electron microscopy, particle size analysis, and protein blotting. Long-chain RNA libraries of exosomes were constructed and sequenced. Real-time quantitative reverse transcription polymerase chain reaction (Real-time PCR) was used to detect the mRNA expression of inflammatory factors and neutrophil-related chemokines in exosomes from plasma and brain tissues of each group. Enzyme-linked immunosorbent assay (ELISA) was used to detect the protein expression of inflammatory factors and neutrophil-related chemokines in exosomes from brain tissues of each group. Immunohistochemistry was used to detect the expression of the neutrophil-specific protein myeloperoxidase (MPO) in the brains of mice in each group. ResultsCompared with the sham-operated group, the model group showed decreased neurological function scores (P<0.01), obvious cerebral infarction (P<0.01), reduced cerebral blood flow (P<0.01), neuronal necrosis in the brain, and decreased numbers of Nissl bodies (P<0.01). The mRNA expression levels of IL-1β, MPO, CXCL1, CXCL2, CXCL3, CXCL10, CCL2, and CCL3 in exosomes from plasma and brain tissues were significantly increased (P<0.05, P<0.01). The protein expression levels of IL-1β, MPO, CXCL2, and CXCL10 in exosomes from brain tissues were increased (P<0.05, P<0.01), and MPO-positive rates and mean optical density values in brain tissues were elevated (P<0.01). Compared with the model group, the Huanglian Jiedutang group and the Ginaton group showed increased neurological function scores (P<0.05), reduced cerebral infarct volume (P<0.01), restored cerebral blood flow (P<0.01), reduced necrotic cells in the brain, and increased numbers of Nissl bodies (P<0.01). In the Huanglian Jiedutang group, the mRNA expression levels of IL-1β, MPO, CXCL1, CXCL2, CXCL3, CXCL10, CCL2, and CCL3 in exosomes from plasma and brain tissues were decreased (P<0.05, P<0.01). The protein expression levels of IL-1β, MPO, CXCL2, and CXCL10 in exosomes from brain tissues were reduced (P<0.05, P<0.01), and MPO-positive rates and mean optical density values in brain tissues were decreased (P<0.01). ConclusionHuanglian Jiedutang can effectively regulate the expression of neutrophil-related chemokines in exosomes from plasma and brain tissues of MCAO mice, thereby reducing neutrophil infiltration in the brain and achieving therapeutic effects.
4.Effects of Huanglian Jiedutang on Neutrophil Infiltration in Brain of MCAO Mice via Regulation of Chemokine Expression in Exosomes
Haojia ZHANG ; Kai WANG ; Zijin SUN ; Chunyu WANG ; Wei SHAO ; Kunjing LIU ; Liyang DONG ; Dan CHEN ; Wenxiu XU ; Chuanzun WANG ; Wen WANG ; Changxiang LI ; Xueqian WANG ; Fafeng CHENG ; Qingguo WANG
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(8):42-53
ObjectiveTo investigate whether Huanglian Jiedutang can inhibit neutrophil infiltration in the brains of middle cerebral artery occlusion (MCAO) mice by regulating the expression of neutrophil-related chemokines in exosomes, thereby achieving therapeutic effects. MethodsA total of 130 male specific pathogen-free (SPF) C57BL/6J mice were randomly divided into four groups: Sham-operated group, MCAO model group, Huanglian Jiedutang group (6 g·kg-1), and Ginaton group (21.6 mg·kg-1), with 10 mice in the Ginaton group and 40 mice in each of the remaining three groups. Mice in the Huanglian Jiedutang group and the Ginaton group were administered the corresponding drugs by oral gavage once daily at a volume of 0.15 mL·(10 g)-1 for 7 consecutive days, while the sham-operated and model groups received an equal volume of saline via the same route. After 7 days, MCAO surgery was performed. The distal and proximal ends of the right common carotid artery (CCA) were ligated, a small incision was made between the two ligatures, and a silicone rubber-coated monofilament with a rounded tip was inserted into the lumen to occlude the CCA. The filament was left in place for 1 h to establish a focal cerebral ischemia model. At 24 h after modeling, mice were evaluated. Neurological function was assessed using the Longa score. Cerebral infarct volume was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Cerebral blood flow was observed by laser speckle imaging. Hematoxylin and eosin (HE) staining and Nissl staining were used to observe pathological changes in brain tissues. Exosomes were isolated from mouse plasma and brain tissues by ultracentrifugation and molecular size exclusion and identified by electron microscopy, particle size analysis, and protein blotting. Long-chain RNA libraries of exosomes were constructed and sequenced. Real-time quantitative reverse transcription polymerase chain reaction (Real-time PCR) was used to detect the mRNA expression of inflammatory factors and neutrophil-related chemokines in exosomes from plasma and brain tissues of each group. Enzyme-linked immunosorbent assay (ELISA) was used to detect the protein expression of inflammatory factors and neutrophil-related chemokines in exosomes from brain tissues of each group. Immunohistochemistry was used to detect the expression of the neutrophil-specific protein myeloperoxidase (MPO) in the brains of mice in each group. ResultsCompared with the sham-operated group, the model group showed decreased neurological function scores (P<0.01), obvious cerebral infarction (P<0.01), reduced cerebral blood flow (P<0.01), neuronal necrosis in the brain, and decreased numbers of Nissl bodies (P<0.01). The mRNA expression levels of IL-1β, MPO, CXCL1, CXCL2, CXCL3, CXCL10, CCL2, and CCL3 in exosomes from plasma and brain tissues were significantly increased (P<0.05, P<0.01). The protein expression levels of IL-1β, MPO, CXCL2, and CXCL10 in exosomes from brain tissues were increased (P<0.05, P<0.01), and MPO-positive rates and mean optical density values in brain tissues were elevated (P<0.01). Compared with the model group, the Huanglian Jiedutang group and the Ginaton group showed increased neurological function scores (P<0.05), reduced cerebral infarct volume (P<0.01), restored cerebral blood flow (P<0.01), reduced necrotic cells in the brain, and increased numbers of Nissl bodies (P<0.01). In the Huanglian Jiedutang group, the mRNA expression levels of IL-1β, MPO, CXCL1, CXCL2, CXCL3, CXCL10, CCL2, and CCL3 in exosomes from plasma and brain tissues were decreased (P<0.05, P<0.01). The protein expression levels of IL-1β, MPO, CXCL2, and CXCL10 in exosomes from brain tissues were reduced (P<0.05, P<0.01), and MPO-positive rates and mean optical density values in brain tissues were decreased (P<0.01). ConclusionHuanglian Jiedutang can effectively regulate the expression of neutrophil-related chemokines in exosomes from plasma and brain tissues of MCAO mice, thereby reducing neutrophil infiltration in the brain and achieving therapeutic effects.
5.Anti-frostbite effect of miglitol on cold-exposed mice through UCP1-mediated thermogenic activation
Xiang LI ; Hongyuan LU ; Mingyu ZHANG ; Huan GAO ; Dong YAO ; Zihua XU
Journal of Pharmaceutical Practice and Service 2025;43(1):1-5
Objective To investigate the effect and mechanism of miglitol on regulating the energy metabolism of brown adipocytes by activating UCP1 and preventing cold injury in mice after cold exposure. Methods Primary brown adipocytes were induced into mature adipocytes, the effect of miglitol on the viability of brown adipocytes was investigated by MTT method, the lipid droplet consumption level of cells after drug administration was investigated by Oil Red O staining technology, and the level of UCP1, a key protein of thermogenesis in brown adipocytes, was detected by Western blotting. The activity of anti-frostbite was investigated in cold exposure at 4 ℃ and −20 ℃. KM mice, which were randomly divided into control group, cold exposure group, miglitol group and all-trans retinoic acid group, and after 7 days of repeated administration, the body surface temperature of mice was detected by infrared thermal imaging system, the anal temperature change was detected by anal thermometer, and the expression levels of UCP1 and PGC1-α in adipose tissue were detected by immunoblotting. Results Compared with the control group, the lipid droplet consumption and UCP1 expression levels in brown adipocytes in the miglitol group were significantly increased. The levels of body surface temperature and rectal temperature increased significantly after cold exposure, and the levels of UCP1 and PGC1α in the brown adipose tissue of mice increased significantly, which indicated that the miglitol could activate the critical proteins UCP1 and PGC1α of the thermogenesis pathway, increase the thermogenesis of mice after cold exposure, and thus improve the effect of cold injury for toe swelling. Conclusion Miglitol could play a role in improving cold injury and body temperature in mice by increasing the level of UCP1 and PGC1α, which are key targets of the thermogenesis pathway to promote the thermogenesis of brown fat.
6.Research and Application of Scalp Surface Laplacian Technique
Rui-Xin LUO ; Si-Ying GUO ; Xin-Yi LI ; Yu-He ZHAO ; Chun-Hou ZHENG ; Min-Peng XU ; Dong MING
Progress in Biochemistry and Biophysics 2025;52(2):425-438
Electroencephalogram (EEG) is a non-invasive, high temporal-resolution technique for monitoring brain activity. However, affected by the volume conduction effect, EEG has a low spatial resolution and is difficult to locate brain neuronal activity precisely. The surface Laplacian (SL) technique obtains the Laplacian EEG (LEEG) by estimating the second-order spatial derivative of the scalp potential. LEEG can reflect the radial current activity under the scalp, with positive values indicating current flow from the brain to the scalp (“source”) and negative values indicating current flow from the scalp to the brain (“sink”). It attenuates signals from volume conduction, effectively improving the spatial resolution of EEG, and is expected to contribute to breakthroughs in neural engineering. This paper provides a systematic overview of the principles and development of SL technology. Currently, there are two implementation paths for SL technology: current source density algorithms (CSD) and concentric ring electrodes (CRE). CSD performs the Laplace transform of the EEG signals acquired by conventional disc electrodes to indirectly estimate the LEEG. It can be mainly classified into local methods, global methods, and realistic Laplacian methods. The global method is the most commonly used approach in CSD, which can achieve more accurate estimation compared with the local method, and it does not require additional imaging equipment compared with the realistic Laplacian method. CRE employs new concentric ring electrodes instead of the traditional disc electrodes, and measures the LEEG directly by differential acquisition of the multi-ring signals. Depending on the structure, it can be divided into bipolar CRE, quasi-bipolar CRE, tripolar CRE, and multi-pole CRE. The tripolar CRE is widely used due to its optimal detection performance. While ensuring the quality of signal acquisition, the complexity of its preamplifier is relatively acceptable. Here, this paper introduces the study of the SL technique in resting rhythms, visual-related potentials, movement-related potentials, and sensorimotor rhythms. These studies demonstrate that SL technology can improve signal quality and enhance signal characteristics, confirming its potential applications in neuroscientific research, disease diagnosis, visual pathway detection, and brain-computer interfaces. CSD is frequently utilized in applications such as neuroscientific research and disease detection, where high-precision estimation of LEEG is required. And CRE tends to be used in brain-computer interfaces, that have stringent requirements for real-time data processing. Finally, this paper summarizes the strengths and weaknesses of SL technology and envisages its future development. SL technology boasts advantages such as reference independence, high spatial resolution, high temporal resolution, enhanced source connectivity analysis, and noise suppression. However, it also has shortcomings that can be further improved. Theoretically, simulation experiments should be conducted to investigate the theoretical characteristics of SL technology. For CSD methods, the algorithm needs to be optimized to improve the precision of LEEG estimation, reduce dependence on the number of channels, and decrease computational complexity and time consumption. For CRE methods, the electrodes need to be designed with appropriate structures and sizes, and the low-noise, high common-mode rejection ratio preamplifier should be developed. We hope that this paper can promote the in-depth research and wide application of SL technology.
7.Role and Mechanism of Cucurbitacin B in Suppressing Proliferation of Breast Cancer 4T1 Cells via Inducing Ferroptosis
Yidan RUAN ; Huizhong ZHANG ; Huating HUANG ; Pingzhi ZHANG ; Aina YAO ; Yongqiang ZHANG ; Xiaohan XU ; Shiman LI ; Jian NI ; Xiaoxu DONG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):91-97
ObjectiveTo explore the role of cucurbitacin B (CuB) in inducing ferroptosis in 4T1 cells and its mechanism. MethodsThe effects of CuB(0.2, 0.4, 0.8 μmol·L-1)on the proliferation ability of 4T1 cells in vitro were detected using the methyl thiazolyl tetrazolium (MTT) assay. The clonogenic ability of 4T1 cells was detected by the plate cloning assay, and the levels of lactate dehydrogenase (LDH) in 4T1 cells were detected by the use of a kit. The mitochondrial membrane potential and reactive oxygen species (ROS) levels in 4T1 cells were detected by flow cytometry, and the mitochondrial ultrastructure of 4T1 cells was observed by transmission electron microscopy. The western blot was used to detect the expression of ferroptosis-related protein p53 in 4T1 cells, solute carrier family 7 member 11 (SCL7A11), glutathione peroxidase 4 (GPX4), long-chain acyl-CoA synthetase 4 (ACSL4), transferrin receptor protein 1 (TFR1), and ferritin heavy chain 1 (FTH1). ResultsCompared with that in the blank group, the survival rate of 4T1 cells in CuB groups was significantly decreased (P<0.05), and the number of cell clones in CuB groups was significantly reduced (P<0.01). In addition, compared with that in the blank group, the leakage of LDH in cells in CuB groups was significantly increased (P<0.01), and the mitochondrial membrane potential of cells in CuB groups decreased significantly (P<0.01). Cellular ROS levels were significantly elevated in CuB groups (P<0.01). The mitochondria of cells in CuB groups were obviously wrinkled, and the mitochondrial cristae were reduced or even disappeared. Compared with that in the blank group, the protein expression of p53, ACSL4, and TFR1 were significantly up-regulated in CuB groups (P<0.05), and that of SLC7A11, GPX4, and FTH1 were significantly down-regulated (P<0.05). ConclusionCuB may inhibit SLC7A11 and GPX4 expression by up-regulating the expression of p53, which in turn regulates the p53/SLC7A11/GPX4 signaling pathway axis and accelerates the generation of lipid peroxidation substrate by up-regulating the expression of ACSL4. It up-regulates TFR1 expression to promote cellular uptake of Fe3+ and down-regulates the expression of FTH1 to reduce the ability of iron storage, resulting in an elevated free Fe2+ level. It catalyzes the Fenton reaction, generates excess ROS, imbalances the antioxidant system and iron metabolism, and then induces ferroptosis in 4T1 cells.
8.Clinical Efficacy of Tonifying Kidney and Replenishing Essence on Asthenospermia Patients with Syndrome of Kidney Essence Deficiency and Effect of This Method on Expression Levels of AMPK/mTORC1 Signaling Pathway-associated Proteins
Yuanjie FU ; Fuhao LI ; Chenghua PENG ; Dong XU ; Guoan YIN ; Xiaopeng HUANG ; Degui CHANG ; Liang DONG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):141-147
ObjectiveTo observe the clinical efficacy of tonifying kidney and replenishing essence on asthenozoospermia patients with the syndrome of kidney essence deficiency and the effects of this method on the adenosine 5′-monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. MethodsSeventy-two eligible asthenozoospermia patients with the syndrome of kidney essence deficiency treated in the Affiliated Hospital of Chengdu University of Traditional Chinese Medicine from February 2023 to January 2024 were selected and randomly assigned into an observation group and a control group, with 36 patients in each group. The observation group received oral administration of Guilu Tianjing capsules, while the control group received oral administration of L-carnitine oral solution. The treatment course lasted for 4 weeks in both groups. The observed indicators included sperm progressive motility rate (PR), total sperm motility (PR+NP), percentage of normal mitochondrial membrane potential (MMP), and traditional Chinese medicine (TCM) symptom scores before and after treatment in both groups. A three-month follow-up was instituted to record the conception status of the patients’ spouses. Additionally, eight patients were randomly selected from the eligible patients in the observation group, and four healthy males with normal semen routine examination results were included as the control group for the determination of protein expression. Western blotting was conducted to assess the expression of AMPK, phosphorylated (p)-AMPK, regulatory-associated protein of mTOR (RAPTOR) and p-RAPTOR, and PTEN-induced putative kinase 1 (PINK1) in sperms from the observation group before and after treatment, as well as in the sperms of the control group. ResultsThe pregnancy rate of spouses in the observation group was 9.09% (3/33), which was higher than that (3.33%, 1/30) in the control group. The total response rate was 84.8% (28/33) in the observation group and 66.7% (20/30) in the control group, with no statistically significant difference. After treatment, both groups were improved considering PR, PR+NP, MMP, and TCM symptom scores (P<0.01). Moreover, the observation group exhibited more pronounced decreases in TCM symptom scores than the control group (P<0.05), while the changes in PR, PR+NP, and MMP showed no statistical significance between groups. Compared with the control group, the asthenozoospermia group exhibited upregulations in phosphorylation levels of AMPK and RAPTOR and protein level of PINK (P<0.01). The administration of Guilu Tianjing Capsules led to downregulations in the phosphorylation levels of AMPK and RAPTOR and protein level of PINK1 (P<0.01). However, the protein levels of AMPK and RAPTOR demonstrated no significant difference between before and after treatment. During the study period, neither group of patients exhibited any notable adverse reactions. ConclusionGuilu Tianjing capsules can enhance the sperm motility and percentage of normal mitochondrial membrane potential in asthenozoospermia patients with the syndrome of kidney essence deficiency by downregulating the AMPK/mTORC1 signaling pathway, lowering the protein level of PINK1, and inhibiting excessive activation of mitophagy.
9.Role and Mechanism of Cucurbitacin B in Suppressing Proliferation of Breast Cancer 4T1 Cells via Inducing Ferroptosis
Yidan RUAN ; Huizhong ZHANG ; Huating HUANG ; Pingzhi ZHANG ; Aina YAO ; Yongqiang ZHANG ; Xiaohan XU ; Shiman LI ; Jian NI ; Xiaoxu DONG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):91-97
ObjectiveTo explore the role of cucurbitacin B (CuB) in inducing ferroptosis in 4T1 cells and its mechanism. MethodsThe effects of CuB(0.2, 0.4, 0.8 μmol·L-1)on the proliferation ability of 4T1 cells in vitro were detected using the methyl thiazolyl tetrazolium (MTT) assay. The clonogenic ability of 4T1 cells was detected by the plate cloning assay, and the levels of lactate dehydrogenase (LDH) in 4T1 cells were detected by the use of a kit. The mitochondrial membrane potential and reactive oxygen species (ROS) levels in 4T1 cells were detected by flow cytometry, and the mitochondrial ultrastructure of 4T1 cells was observed by transmission electron microscopy. The western blot was used to detect the expression of ferroptosis-related protein p53 in 4T1 cells, solute carrier family 7 member 11 (SCL7A11), glutathione peroxidase 4 (GPX4), long-chain acyl-CoA synthetase 4 (ACSL4), transferrin receptor protein 1 (TFR1), and ferritin heavy chain 1 (FTH1). ResultsCompared with that in the blank group, the survival rate of 4T1 cells in CuB groups was significantly decreased (P<0.05), and the number of cell clones in CuB groups was significantly reduced (P<0.01). In addition, compared with that in the blank group, the leakage of LDH in cells in CuB groups was significantly increased (P<0.01), and the mitochondrial membrane potential of cells in CuB groups decreased significantly (P<0.01). Cellular ROS levels were significantly elevated in CuB groups (P<0.01). The mitochondria of cells in CuB groups were obviously wrinkled, and the mitochondrial cristae were reduced or even disappeared. Compared with that in the blank group, the protein expression of p53, ACSL4, and TFR1 were significantly up-regulated in CuB groups (P<0.05), and that of SLC7A11, GPX4, and FTH1 were significantly down-regulated (P<0.05). ConclusionCuB may inhibit SLC7A11 and GPX4 expression by up-regulating the expression of p53, which in turn regulates the p53/SLC7A11/GPX4 signaling pathway axis and accelerates the generation of lipid peroxidation substrate by up-regulating the expression of ACSL4. It up-regulates TFR1 expression to promote cellular uptake of Fe3+ and down-regulates the expression of FTH1 to reduce the ability of iron storage, resulting in an elevated free Fe2+ level. It catalyzes the Fenton reaction, generates excess ROS, imbalances the antioxidant system and iron metabolism, and then induces ferroptosis in 4T1 cells.
10.Clinical Efficacy of Tonifying Kidney and Replenishing Essence on Asthenospermia Patients with Syndrome of Kidney Essence Deficiency and Effect of This Method on Expression Levels of AMPK/mTORC1 Signaling Pathway-associated Proteins
Yuanjie FU ; Fuhao LI ; Chenghua PENG ; Dong XU ; Guoan YIN ; Xiaopeng HUANG ; Degui CHANG ; Liang DONG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):141-147
ObjectiveTo observe the clinical efficacy of tonifying kidney and replenishing essence on asthenozoospermia patients with the syndrome of kidney essence deficiency and the effects of this method on the adenosine 5′-monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. MethodsSeventy-two eligible asthenozoospermia patients with the syndrome of kidney essence deficiency treated in the Affiliated Hospital of Chengdu University of Traditional Chinese Medicine from February 2023 to January 2024 were selected and randomly assigned into an observation group and a control group, with 36 patients in each group. The observation group received oral administration of Guilu Tianjing capsules, while the control group received oral administration of L-carnitine oral solution. The treatment course lasted for 4 weeks in both groups. The observed indicators included sperm progressive motility rate (PR), total sperm motility (PR+NP), percentage of normal mitochondrial membrane potential (MMP), and traditional Chinese medicine (TCM) symptom scores before and after treatment in both groups. A three-month follow-up was instituted to record the conception status of the patients’ spouses. Additionally, eight patients were randomly selected from the eligible patients in the observation group, and four healthy males with normal semen routine examination results were included as the control group for the determination of protein expression. Western blotting was conducted to assess the expression of AMPK, phosphorylated (p)-AMPK, regulatory-associated protein of mTOR (RAPTOR) and p-RAPTOR, and PTEN-induced putative kinase 1 (PINK1) in sperms from the observation group before and after treatment, as well as in the sperms of the control group. ResultsThe pregnancy rate of spouses in the observation group was 9.09% (3/33), which was higher than that (3.33%, 1/30) in the control group. The total response rate was 84.8% (28/33) in the observation group and 66.7% (20/30) in the control group, with no statistically significant difference. After treatment, both groups were improved considering PR, PR+NP, MMP, and TCM symptom scores (P<0.01). Moreover, the observation group exhibited more pronounced decreases in TCM symptom scores than the control group (P<0.05), while the changes in PR, PR+NP, and MMP showed no statistical significance between groups. Compared with the control group, the asthenozoospermia group exhibited upregulations in phosphorylation levels of AMPK and RAPTOR and protein level of PINK (P<0.01). The administration of Guilu Tianjing Capsules led to downregulations in the phosphorylation levels of AMPK and RAPTOR and protein level of PINK1 (P<0.01). However, the protein levels of AMPK and RAPTOR demonstrated no significant difference between before and after treatment. During the study period, neither group of patients exhibited any notable adverse reactions. ConclusionGuilu Tianjing capsules can enhance the sperm motility and percentage of normal mitochondrial membrane potential in asthenozoospermia patients with the syndrome of kidney essence deficiency by downregulating the AMPK/mTORC1 signaling pathway, lowering the protein level of PINK1, and inhibiting excessive activation of mitophagy.

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