1.Myocardial Metabolomics Reveals Mechanism of Shenfu Injection in Ameliorating Energy Metabolism Remodeling in Rat Model of Chronic Heart Failure
Xinyue NING ; Zhenyu ZHAO ; Mengna ZHANG ; Yang GUO ; Zhijia XIANG ; Kun LIAN ; Zhixi HU ; Lin LI
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(5):178-186
ObjectiveTo examine the influences of Shenfu injection on the endogenous metabolic byproducts in the myocardium of the rat model exhibiting chronic heart failure, thus deciphering the therapeutic mechanism of the Qi-reinforcing and Yang-warming method. MethodsSD rats were randomly allocated into a control group and a modeling group. Chronic heart failure with heart-Yang deficiency syndrome in rats was modeled by multi-point subcutaneous injection of isoproterenol, and the rats were fed for 14 days after modeling. The successfully modeled rats were randomized into model, Shenfu injection (6.0 mL·kg-1), and trimetazidine (10 mg·kg-1) groups and treated with corresponding agents for 15 days. The control group and the model group were injected with equal doses of normal saline, and the samples were collected after the intervention was completed. Cardiac color ultrasound was performed. Hematoxylin-eosin (HE) staining was used to observe histopathological morphology, and the serum level of N-terminal pro-brain natriuretic peptide (NT-proBNP) was assessed by enzyme-linked immunosorbent assay (ELISA). The mitochondrial morphological and structural changes of cardiomyocytes were observed by transmission electron microscopy, and the metabolic profiling was carried out by ultra high performance liquid chromatography-quantitative exactive-mass spectrometry (UHPLC-QE-MS). Differential metabolites were screened and identified by orthogonal partial least squares-discriminant analysis (OPLS-DA) and other methods, and then the MetaboAnalyst database was used for further screening. The relevant biological pathways were obtained through pathway enrichment analysis. The receiver operating characteristic (ROC) curve was established to evaluate the diagnostic value of each potential biomarker for myocardial injury and the evaluation value for drug efficacy. ResultsThe results of color ultrasound showed that Shenfu Injection improved the cardiac function indexes of model rats (P<0.05). The results of HE staining showed that Shenfu injection effectively alleviated the pathological phenomena such as myocardial tissue structure disorder and inflammatory cell infiltration in model rats. The results of ELISA showed that Shenfu injection effectively regulated the serum NT-proBNP level in the model rats. Transmission electron microscopy (TEM) showed that Shenfu injection effectively restored the mitochondrial morphological structure. The results of metabolomics showed that the metabolic phenotypes of myocardial samples presented markedly differences between groups. Nine differential metabolites could be significantly reversed in the Shenfu injection group, involving three metabolic pathways: pyruvate metabolism, histidine metabolism, and citric acid cycle (TCA cycle). The results of ROC analysis showed that the area under the curve (AUC) values of all metabolites were between 0.75 and 1.0, indicating that the differential metabolites had high diagnostic accuracy for myocardial injury, and the changes in their expression levels could be used as potential markers for efficacy evaluation. ConclusionShenfu injection significantly alleviated the damage of cardiac function, myocardium, and mitochondrial structure in the rat model of chronic heart failure with heart-Yang deficiency syndrome by ameliorating energy metabolism remodeling. Reinforcing Qi and warming Yang is a key method for treating chronic heart failure with heart-Yang deficiency syndrome.
2.Exploration of a new model for the construction of medical institution formulation platforms from the perspective of industry-university-research collaborative innovation theory
Kana LIN ; Anle SHEN ; Yejian WANG ; Yanqiong WANG ; Hao LI ; Yanfang GUO ; Youjun WANG ; Xinyan SUN
China Pharmacy 2026;37(2):137-141
OBJECTIVE To explore a model for constructing a platform for medical institution formulation and provide insights for promoting their development. METHODS By systematically reviewing the development status and challenges of medical institution preparations in China, and based on the theory of industry-university-research collaborative innovation, the organizational structure, collaborative processes, and safeguard mechanisms of the platform were designed. RESULTS & CONCLUSIONS Medical institution formulations in China mainly faced challenges such as weak research and development (R&D) capacity, uneven quality standards, and blocked transformation pathways. This study established a full-chain, whole- industry collaborative innovation network covering the government, medical institutions, universities/research institutes, pharmaceutical enterprises, and the market, forming a new “government-industry-university-research-application” five-in-one platform model for medical institution formulations. By establishing mechanisms such as multi-entity collaborative cooperation, full- chain intellectual property management, contribution-based benefit distribution, staged risk-sharing, and third-party evaluation, the model clarified the responsibilities and collaborative pathways of all parties. The new model highlights the whole-process transformation of clinical experience-based prescriptions, enabling precise alignment between clinical needs and technological R&D, as well as between preparation achievements and industrial transformation. While breaking down the barriers of traditional platform construction, it effectively achieves optimal resource allocation and complementary advantages, addresses problems emerging in the development of medical institution preparations, and provides reference value for the formulation of relevant systems.
3.Expert Consensus on Neurocritical Care Monitoring and Management in Beijing and Tibet(2025)
Drolma PHURBU ; Wenjin CHEN ; Heng ZHANG ; Jian ZHANG ; Xiaomeng WANG ; Guoying LIN ; Wenjun PAN ; Xiying GUI ; Xin CAI ; Chodron TENZIN ; Jianlei FU ; Qianwei LI ; TSEYANG ; Yijun LIU ; Bo LIU ; Tsering DROLMA ; Yudron SONAM ; KYILV ; Samdrup TSERING ; Wa DA ; Juan GUO ; Cheng QIU ; Huan CHEN ; Xiaoting WANG ; Yangong CHAO ; Dawei LIU ; Wenzhao CHAI ; Chenggong HU ; Wanhong YIN ; Shihong ZHU
Medical Journal of Peking Union Medical College Hospital 2026;17(1):59-72
Neurocritical care involves complex pathophysiological mechanisms, and its incidence is higher, injuries are more severe, and treatment is more challenging in high-altitude environments. This consensus, based on the latest domestic and international evidence-based medical data, establishes a standardized, goal-oriented framework for neurocritical care management applicable in high-altitude regions and nationwide. The consensus was developed following international standards for evidence quality assessment and underwent two rounds of Delphi expert consultation, resulting in 32 recommendation statements covering three parts: management systems, monitoring and assessment, and core strategies. Key updates include: advocating for the establishment of independent neurocritical care units and implementing precise tiered diagnosis and treatment based on the "Five Differences in Critical Care" concept; constructing a "trinity" multimodal brain monitoring system centered on cerebral blood flow, cerebral oxygenation, and brain function, emphasizing routine bedside transcranial Doppler ultrasound, cerebral oximetry, and continuous electroencephalography monitoring; shifting management strategies from mild hypothermia therapy to targeted temperature management, and defining the "446" target management pathway for the supercritical stage; emphasizing the assessment of static and dynamic cerebrovascular autoregulation functions through multimodal methods to achieve individualized optimal mean arterial pressure management; elevating cerebrospinal fluid management goals to the level of "glymphatic system" function maintenance; implementing a multidisciplinary collaborative, whole-process management model focusing on patients' long-term neurological functional outcomes; de-escalation criteria include multidimensional indicators such as recovery of brain structure, restoration of cerebrovascular autoregulation, improvement in cerebrospinal fluid dynamics, and reduction in biomarker levels; and integrating cutting-edge technologies like artificial intelligence into post-critical care management and rehabilitation planning. This consensus systematically integrates the entire process of neurocritical care management, reflecting the modern connotation of goal-oriented, dynamic, and multimodal integration in neurocritical care medicine. It aims to adapt to new trends such as deepening understanding of pathophysiological mechanisms, the integration of medicine and engineering, and the empowerment of artificial intelligence, thereby further advancing the discipline of critical care medicine.
4.Influencing Factors of Urate Crystal Deposition in Patients with Hyperuricemia and Prediction Model of TCM Syndrome Types-inflammatory Indicators
Jiaqi XU ; Bin AI ; Chao LIN ; Qiaoxuan LIN ; Changning LI ; Jing CAI ; Yan XIAO ; Jiemei GUO ; Youxin SU
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(7):66-73
ObjectiveTo identify potential influencing factors of urate crystal deposition at ankle/foot in patients with hyperuricemia (HUA), and to analyze the predictive value of inflammatory indicators for urate crystal deposition in patients with different traditional Chinese medicine (TCM) syndromes, so as to provide potential reference for clinical risk assessment and individualized TCM intervention. MethodsA retrospective study was carried out with the enrollment of 231 HUA patients from The Third Affiliated People's Hospital of Fujian University of Traditional Chinese Medicine between January 2021 and December 2024. The enrolled patients were further divided into a crystal deposition-positive group (143 cases) and a crystal deposition-negative group (88 cases) according to the results of dual-energy computed tomography (CT). Sociodemographic data, living habits, serum uric acid levels, and inflammatory indicators of the enrolled patients were collcted, and TCM syndrome differentiation was performed. Furthermore, univariate analysis was used to compare inter-group differences in clinical characteristics. MMultivariate Logistic regression was applied to identify the influencing factors of urate crystal deposition. In addition, the receiver operating characteristic (ROC) curves were plotted to evaluate the predictive efficacy of inflammatory indicators for crystal deposition across different TCM syndromes. ResultsThere were statistically significant inter-group differences in the proportion of males, age, body mass index, proportion of mental labor, rate of low water intake, and rate of high-sugar beverage consumption (P<0.05),whereas no significant difference in low exercise intensity was found between the two groups. Furthermore, compared with the negative group, the positive group had higher serum uric acid level, neutrophil-to-lymphocyte ratio (NLR), and platelet-to-lymphocyte ratio (PLR), but lower systemic immune-inflammation index (SIRI) (P<0.05). Regarding the distribution of TCM syndromes, the positive group was dominated by the dampness-heat accumulation syndrome (55/143,38.46%), while the negative group was mainly characterized by the phlegm-turbidity obstruction syndrome (44/88,50.00%). Multivariate Logistic regression analysis revealed that high-sugar beverage consumption, elevated NLR, and elevated PLR were risk factors for urate crystal deposition [odd ratio (OR) = 8.002, 5.377, 1.034, respectively; 95% CI 1.572-40.732, 2.179-13.270, 1.013-1.054,all P<0.05], while SIRI was a protective factor (OR = 0.869, 95% CI 0.778-0.971, P<0.05). In the positive group, patients with the dampness-heat accumulation syndrome exhibited the highest NLR, while the lowest PLR and SIRI, showing statistically significant differences with those of other syndromes (all P<0.05). In addition, ROC curve analysis indicated that for the dampness-heat accumulation syndrome, the combined "NLR + PLR" model had an area under the curve (AUC) of 0.901 (95% CI 0.850-0.951, P<0.01), with a sensitivity of 89.1% and a specificity of 79.5%; for the blood stasis-heat obstruction syndrome, the combined "NLR + PLR" model had an AUC of 0.880 (95% CI 0.825-0.934, P<0.01), with a sensitivity of 100.0% and a specificity of 67.3%; for the liver-kidney Yin-deficiency syndrome, the single PLR model had an AUC of 0.842 (95% CI 0.731-0.952, P<0.01), with a sensitivity of 83.3% and a specificity of 84.0%. ConclusionUrate crystal deposition in HUA patients exhibits intimate associations with high-sugar beverage consumption as well as elevated NLR and PLR levels. Meanwhile, TCM syndrome differentiation has potential correlation with inflammatory characteristics. The inflammatory indicator-based prediction model constructed based on TCM syndromes exhibits good predictive value.
5.Mechanism of Huazhuo Sanjie Chubi Presciption in Regulating Macrophage Polarization and Improving Low-grade Inflammation in Rats with Chronic Gouty Arthritis
Yuwan LI ; Yingjie ZHANG ; Siyuan LIN ; Xiaohua CHEN ; Qianglong CHEN ; Fan YANG ; Jun LIU ; Bingyan CHEN ; Peng CHEN ; Jiemei GUO ; Youxin SU ; Yan XIAO
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(7):93-104
ObjectiveTo evaluate the therapeutic effect of Huazhuo SanJie Chubi presciption (HSCD) on chronic gouty arthritis (CGA) rats with low-grade inflammation and to explore the underlying mechanism with a focus on macrophage polarization. MethodsThe 41 male 6-week-old SD rats were randomly allocated, using the random number table, to a normal group (n=8) and a model group (n =33). CGA with low-grade inflammation was induced in the model group by daily gavage of potassium oxonate (250 mg·kg-1·d-1) and hypoxanthine (300 mg·kg-1·d-1), combined with intra-articular injection of a monosodium urate (MSU) crystal suspension (50 μL, 25 g·L-¹) into the left ankle twice weekly. After 4 weeks of modeling, 3 rats were randomly selected from each group for model validation. The remaining successfully modeled rats were randomly divided into a model group, an HSCD group (10.35 g·kg-1·d-1, gavage once daily), an M1 polarization agonist group (L-methionine sulfoximine, 300 mg·kg-1, subcutaneous injection every other day), an M1 polarization agonist + HSCD group, an M2 polarization inhibitor group (PD0325901, 10 mg·kg-1·d-1, gavage once daily), and M2 polarization inhibitor + HSCD group. The corresponding drug or drug combination was administered according to group assignment, whereas rats in the normal and model groups received 0.5% carboxymethyl cellulose sodium (CMC-Na) vehicle (10.35 g·kg-1·d-1, gavage once daily). All interventions were continued for four weeks. During the intervention period, except for the normal group, potassium oxonate (250 mg·kg⁻¹) and hypoxanthine (300 mg·kg-1) were co-administered by gavage every other day to maintain the model. At the end of treatment, serum uric acid (SUA), ankle joint diameter and joint swelling index were measured. The levels of high-sensitivity C-reactive protein (hs-CRP), interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), chemokine C-C motif ligand 2 (CCL2), S100 calcium-binding protein A8/A9 (S100A8/A9), interleukin-10 (IL-10) and arginase-1 (Arg-1) in serum and joint fluid were determined by enzyme-linked immunosorbent assay (ELISA). High-frequency ultrasound was used to assess MSU deposition in the ankle joint. Hematoxylin-eosin (HE) staining was performed to evaluate synovial histopathological changes. Quantitative Real-time PCR and immunofluorescence were used to detect the mRNA and protein expression of the M1 macrophage polarization markers inducible nitric oxide synthase (iNOS) and the M2 macrophage polarization marker scavenger receptor cysteine-rich type 1 protein M130 (CD163) in synovial tissue. ResultsCompared with the normal group, the model group showed significantly elevated SUA level and joint swelling index, and increased levels of pro-inflammatory cytokines, CCL2, and S100A8/A9 in both serum and joint fluid (P<0.05), accompanied by MSU deposition and synovial inflammation in the ankle joint. The mRNA and protein expression levels of macrophage polarization M1/M2 markers iNOS and CD163 in synovial tissues were also significantly up-regulated (P<0.05). Compared with model group, rats in HSCD group had significantly lower SUA levels, attenuated joint swelling, reduced serum levels of pro-inflammatory cytokines, and decreased levels of CCL2 and S100A8/A9 in both serum and joint fluid, accompanied with alleviated MSU deposition and synovial inflammation (P<0.05). HSCD markedly downregulated the mRNA and protein expression of M1 marker iNOS (P<0.05), whereas it had no significant effect on the expression of M2 marker CD163. Compared with the M1 polarization agonist group, the M1 polarization agonist + HSCD group showed significantly reduced joint swelling, lower serum levels of pro-inflammatory cytokines, and decreased levels of CCL2 and S100A8/A9 in joint fluid (P<0.05). In addition, synovial inflammatory cell infiltration and angiogenesis were attenuated, and iNOS mRNA and protein expression levels were significantly reduced (P<0.05). Compared with the M2 polarization inhibitor group, the M2 polarization inhibitor + HSCD group exhibited reduced joint swelling, decreased levels of CCL2 and S100A8/A9 in joint fluid and ameliorated synovial inflammation (P<0.05), whereas the levels of anti-inflammatory mediators (IL-10, Arg-1) and CD163 mRNA and protein expression were not significantly increased. ConclusionHSCD alleviates low-grade inflammation in CGA rats, at least in part, by inhibiting macrophage polarization toward the M1 phenotype.
6.Modified Morrow procedure for the treatment of hypertrophic obstructive cardiomyopathy: A single-center retrospective study in 318 patients
Jie LI ; Fan WENG ; Nan CHEN ; Yongxin SUN ; Changfa GUO ; Chunsheng WANG ; Yi LIN ; Wenjun DING
Chinese Journal of Clinical Thoracic and Cardiovascular Surgery 2026;33(03):431-437
Objective To summarize the clinical efficacy of modified Morrow surgery in the treatment of hypertrophic obstructive cardiomyopathy. Methods A retrospective analysis was conducted on the clinical data of patients with hypertrophic obstructive cardiomyopathy treated with modified Morrow surgery at Zhongshan Hospital Affiliated to Fudan University from 2020 to 2023. Results A total of 318 patients were enrolled, including 156 males and 162 females, with an average age of (55.6±13.1) years. Preoperative echocardiography showed a mean interventricular septal thickness of (18.1±3.8) mm, peak left ventricular outflow tract pressure difference of (86.4±24.9) mm Hg. The surgery time was (162.3±51.0) min, extracorporeal circulation time was (80.9±31.0) min, and aortic occlusion time was (44.8±20.8) min. After the surgery, transesophageal echocardiography showed that the interventricular septal thickness was (11.0±1.8) mm and left ventricular outflow tract peak pressure difference was (9.4±5.1) mm Hg. The incidence rate of postoperative complete left bundle branch block was 45.3%, Ⅲ° atrioventricular block was 3.8%, and postoperative newly developed atrial fibrillation was 3.1%. The postoperative hospital stay was (6.6±4.9) days, and one perioperative death occurred, with a mortality rate of 0.3%. The follow-up time was (10.3±9.4) months, during which the transthoracic echocardiography revealed a ventricular septal thickness of (12.9±2.9) mm and a peak left ventricular outflow tract pressure difference of (13.9±10.0) mm Hg. Conclusion The modified Morrow procedure for the treatment of hypertrophic obstructive cardiomyopathy is safe and effective, with good results in the short and medium term.
7.Expert Consensus on Clinical Application of Qinbaohong Zhike Oral Liquid in Treatment of Acute Bronchitis and Acute Attack of Chronic Bronchitis
Jian LIU ; Hongchun ZHANG ; Chengxiang WANG ; Hongsheng CUI ; Xia CUI ; Shunan ZHANG ; Daowen YANG ; Cuiling FENG ; Yubo GUO ; Zengtao SUN ; Huiyong ZHANG ; Guangxi LI ; Qing MIAO ; Sumei WANG ; Liqing SHI ; Hongjun YANG ; Ting LIU ; Fangbo ZHANG ; Sheng CHEN ; Wei CHEN ; Hai WANG ; Lin LIN ; Nini QU ; Lei WU ; Dengshan WU ; Yafeng LIU ; Wenyan ZHANG ; Yueying ZHANG ; Yongfen FAN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(4):182-188
The Expert Consensus on Clinical Application of Qinbaohong Zhike Oral Liquid in Treatment of Acute Bronchitis and Acute Attack of Chronic Bronchitis (GS/CACM 337-2023) was released by the China Association of Chinese Medicine on December 13th, 2023. This expert consensus was developed by experts in methodology, pharmacy, and Chinese medicine in strict accordance with the development requirements of the China Association of Chinese Medicine (CACM) and based on the latest medical evidence and the clinical medication experience of well-known experts in the fields of respiratory medicine (pulmonary diseases) and pediatrics. This expert consensus defines the application of Qinbaohong Zhike oral liquid in the treatment of cough and excessive sputum caused by phlegm-heat obstructing lung, acute bronchitis, and acute attack of chronic bronchitis from the aspects of applicable populations, efficacy evaluation, usage, dosage, drug combination, and safety. It is expected to guide the rational drug use in medical and health institutions, give full play to the unique value of Qinbaohong Zhike oral liquid, and vigorously promote the inheritance and innovation of Chinese patent medicines.
8.Optimization and Mechanism Exploration of Tusizi Prescription for Ovarian Reserve Function Based on Uniform Design Method
Yuan LI ; Hanqian DU ; Jiashan LI ; Li GUO ; Zehui LI ; Na LIN ; Ying XU
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):53-62
ObjectiveTo optimize Tusizi prescription for ovarian reserve function based on the uniform design method combined with in vitro experiments and explore the underlying mechanisms of this prescription. MethodsThe uniform design method was adopted to design a 5-factor 11-level experiment on the water extract of Tusizi prescription. The cell-counting kit-8 (CCK-8) assay was employed to measure the viability of human ovarian granulosa cells (KGN cells) treated with Tusizi prescription extracts 1-11, and multivariate regression analysis was performed to determine the optimal herb ratio in this prescription. The potential targets of active ingredients in the prescription were retrieved from traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP) and encyclopedia of traditional Chinese medicine (ETCM). The common targets shared by Tusizi prescription and diminished ovarian reserve (DOR) were selected and imported into search tool for the retrieval of interacting genes/proteins (STRING) to construct a protein-protein interaction (PPI) network and into gene function annotation database (DAVID) for gene ontology (GO) analysis. The CCK-8 assay was used to measure the viability of ovarian germline stem cells treated with hyperoside. The CCK-8 assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, terminal-deoxynucleoitidyl transferase mediated nick-end labeling (TUNEL), and enzyme-linked immunosorbent assay (ELISA) were employed to examine the proliferation, apoptosis, and estradiol (E2) secretion of KGN cells treated with the water extract 11 of Tusizi prescription (Cuscutae Semen-Lycii Fructus-Dioscoreae Rhizoma-Poria-Nelumbinis Semen 4∶4∶2∶1∶1) and the optimal prescription screened by uniform design. On this basis, the optimal prescription composition for maximizing the effect on ovarian reserve function was determined and preliminary insights into the underlying mechanisms of this prescription were gained. ResultsA total of 147 common targets were obtained from 278 targets of Tusizi prescription and 1 721 targets of DOR. GO analysis revealed 194 biological processes, primarily involving cellular responses to exogenous compound stimuli, negative regulation of apoptotic process, and positive regulation of cell proliferation. It identified 84 cellular components, including cell membrane, mitochondria, and neuronal cell body, as well as 144 molecular functions such as enzyme binding, estrogen response element binding, and nuclear estrogen receptor binding. The multivariate regression analysis revealed that when Tusizi prescription was composed of Cuscutae Semen, Lycii Fructus, Dioscoreae Rhizoma, Poria, and Nelumbinis Semen in a ratio of 27∶30∶17∶12∶14, the water extract of Tusizi prescription had the best effect of enhancing the viability of KGN cells. CCK-8 results showed that compared with the normal group, the hyperoside group demonstrated increased viability of ovarian germline stem cells (P<0.01). The CCK-8, EdU, and ELISA results showed that compared with the normal group, the optimal prescription screened by uniform design and the water extract 11 of Tusizi prescription increased the proliferation and reduced the apoptosis of KGN cells (P<0.05, P<0.01). ELISA results showed that compared with the normal group, the water extract 11 of Tusizi prescription promoted the E2 secretion of KGN cells (P<0.05), while the optimal prescription screened by uniform design had no significant effect on the E2 secretion. ConclusionBoth the water extract 11 of Tusizi prescription (Cuscutae Semen-Lycii Fructus-Dioscoreae Rhizoma-Poria-Nelumbinis Semen 4∶4∶2∶1∶1) and the optimal prescription screened by uniform design (Cuscutae Semen-Lycii Fructus-Dioscoreae Rhizoma-Poria-Nelumbinis Semen 27∶30∶17∶12∶14) can improve the ovarian reserve function, and the former has better effect. Tusizi prescription can modulate biological processes (such as cell proliferation and apoptosis) and molecular functions (such as enzyme binding and estrogen response element binding) through active components like hyperoside to promote the proliferation and E2 secretion and inhibit the apoptosis of KGN cells, thereby protecting the ovarian reserve function.
9.Optimization and Mechanism Exploration of Tusizi Prescription for Ovarian Reserve Function Based on Uniform Design Method
Yuan LI ; Hanqian DU ; Jiashan LI ; Li GUO ; Zehui LI ; Na LIN ; Ying XU
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):53-62
ObjectiveTo optimize Tusizi prescription for ovarian reserve function based on the uniform design method combined with in vitro experiments and explore the underlying mechanisms of this prescription. MethodsThe uniform design method was adopted to design a 5-factor 11-level experiment on the water extract of Tusizi prescription. The cell-counting kit-8 (CCK-8) assay was employed to measure the viability of human ovarian granulosa cells (KGN cells) treated with Tusizi prescription extracts 1-11, and multivariate regression analysis was performed to determine the optimal herb ratio in this prescription. The potential targets of active ingredients in the prescription were retrieved from traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP) and encyclopedia of traditional Chinese medicine (ETCM). The common targets shared by Tusizi prescription and diminished ovarian reserve (DOR) were selected and imported into search tool for the retrieval of interacting genes/proteins (STRING) to construct a protein-protein interaction (PPI) network and into gene function annotation database (DAVID) for gene ontology (GO) analysis. The CCK-8 assay was used to measure the viability of ovarian germline stem cells treated with hyperoside. The CCK-8 assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, terminal-deoxynucleoitidyl transferase mediated nick-end labeling (TUNEL), and enzyme-linked immunosorbent assay (ELISA) were employed to examine the proliferation, apoptosis, and estradiol (E2) secretion of KGN cells treated with the water extract 11 of Tusizi prescription (Cuscutae Semen-Lycii Fructus-Dioscoreae Rhizoma-Poria-Nelumbinis Semen 4∶4∶2∶1∶1) and the optimal prescription screened by uniform design. On this basis, the optimal prescription composition for maximizing the effect on ovarian reserve function was determined and preliminary insights into the underlying mechanisms of this prescription were gained. ResultsA total of 147 common targets were obtained from 278 targets of Tusizi prescription and 1 721 targets of DOR. GO analysis revealed 194 biological processes, primarily involving cellular responses to exogenous compound stimuli, negative regulation of apoptotic process, and positive regulation of cell proliferation. It identified 84 cellular components, including cell membrane, mitochondria, and neuronal cell body, as well as 144 molecular functions such as enzyme binding, estrogen response element binding, and nuclear estrogen receptor binding. The multivariate regression analysis revealed that when Tusizi prescription was composed of Cuscutae Semen, Lycii Fructus, Dioscoreae Rhizoma, Poria, and Nelumbinis Semen in a ratio of 27∶30∶17∶12∶14, the water extract of Tusizi prescription had the best effect of enhancing the viability of KGN cells. CCK-8 results showed that compared with the normal group, the hyperoside group demonstrated increased viability of ovarian germline stem cells (P<0.01). The CCK-8, EdU, and ELISA results showed that compared with the normal group, the optimal prescription screened by uniform design and the water extract 11 of Tusizi prescription increased the proliferation and reduced the apoptosis of KGN cells (P<0.05, P<0.01). ELISA results showed that compared with the normal group, the water extract 11 of Tusizi prescription promoted the E2 secretion of KGN cells (P<0.05), while the optimal prescription screened by uniform design had no significant effect on the E2 secretion. ConclusionBoth the water extract 11 of Tusizi prescription (Cuscutae Semen-Lycii Fructus-Dioscoreae Rhizoma-Poria-Nelumbinis Semen 4∶4∶2∶1∶1) and the optimal prescription screened by uniform design (Cuscutae Semen-Lycii Fructus-Dioscoreae Rhizoma-Poria-Nelumbinis Semen 27∶30∶17∶12∶14) can improve the ovarian reserve function, and the former has better effect. Tusizi prescription can modulate biological processes (such as cell proliferation and apoptosis) and molecular functions (such as enzyme binding and estrogen response element binding) through active components like hyperoside to promote the proliferation and E2 secretion and inhibit the apoptosis of KGN cells, thereby protecting the ovarian reserve function.
10.Effect of Serum Containing Zhenwutang on Apoptosis of Myocardial Mast Cells and Mitochondrial Autophagy
Wei TANG ; Meiqun ZHENG ; Xiaolin WANG ; Zhiyong CHEN ; Chi CHE ; Zongqiong LU ; Jiashuai GUO ; Xiaomei ZOU ; Lili XU ; Lin LI
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(3):11-21
ObjectiveTo explore the effect of serum containing Zhenwutang on myocardial mast cell apoptosis induced by angiotensin Ⅱ (AngⅡ) and the mechanism of the correlation between apoptosis and mitochondrial autophagy. MethodsIn this experiment, AngⅡ and serum containing Zhenwutang with different concentrations were used to interfere with H9C2 cardiomyocytes for 24 h, and the survival rate of H9C2 cardiomyocytes was detected by cell counting kit-8 (CCK-8) to screen the optimal concentration for the experiment. Enzyme-linked immunosorbent assay (ELISA) was used to detect the content of B-type natriuretic peptide (BNP) in cell culture supernatant, and immunofluorescence was used to detect the cell surface area to verify the construction of the myocardial mast cell model. Subsequently, the experiment was divided into a blank group (20% blank serum), a model group (20% blank serum + 5×10-5 mol·L-1 AngⅡ), low-, medium-, and high-dose (5%, 10% and 20%) serum containing Zhenwutang groups, an autophagy inhibitor group (1×10-4 mol·L-1 3-MA), and autophagy inducer group (1×10-7 mol·L-1 rapamycin). The apoptosis level of H9C2 cells and the changes of mitochondrial membrane potential were detected by flow cytometry. The lysosomal probe (Lyso Tracker) and mitochondrial probe (Mito Tracker) co-localization was employed to detect autophagy. Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) was used to detect Caspase-3, Caspase-9, B-cell lymphoma 2 (Bcl-2), Bcl-2-related X protein (Bax), and cytochrome C (Cyt C) in apoptosis-related pathways and the relative mRNA expression of ubiquitin ligase (Parkin), phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1), and p62 protein in mitochondrial autophagy-related pathways. Western blot was used to detect cleaved Caspase-3, cleaved Caspase-9, Bax, Bcl-2, and Cyt C in apoptosis-related pathways, phosphorylated ubiquitin ligase (p-Parkin), phosphorylated PTEN-induced kinase 1 (p-PINK1), p62, and Bcl-2 homology domain protein Beclin1 in mitochondrial autophagy-related pathways, and the change of microtubule-associated protein 1 light chain 3 (LC3) Ⅱ/Ⅰ ratio. ResultsCCK-8 showed that when the concentration of AngⅡ was 5×10-5 mol·L-1, the cell activity was the lowest, and there was no cytotoxicity. At this concentration, the surface area of cardiomyocytes was significantly increased (P<0.01), and the content of BNP in the supernatant of culture medium was significantly increased (P<0.05). Therefore, AngⅡ with a concentration of 5×10-5 mol·L-1 was selected for the subsequent modeling of myocardial mast cells. Compared with the blank group, the model group and the autophagy inhibitor 3-MA group had a significantly increased apoptosis rate (P<0.01) and significantly decreased mitochondrial membrane potential (P<0.01). The results of immunofluorescence co-localization showed that compared with the blank group, the model group had a significantly decreased number of red and green fluorescence spots. The results of Real-time PCR showed that compared with that in the blank group, the relative mRNA expression of Bax, Caspase-3, Caspase-9, Cyt C, and p62 in the model group was significantly up-regulated (P<0.01), while the relative mRNA expression of Bcl-2, Parkin, and PINK1 was significantly down-regulated (P<0.01). In addition, the relative protein expression of Bax, cleaved Caspase-3, cleaved Caspase-9, Cyt C, and p62 was significantly up-regulated (P<0.01). The LC3Ⅱ/Ⅰ was significantly decreased, and the relative protein expression of Bcl-2, p-Parkin, p-PINK1, and Beclin1 was significantly down-regulated (P<0.01). Compared with the model group, the serum containing Zhenwutang groups and the autophagy inducer group had significantly decreased apoptosis rate (P<0.01), and the decrease ratio of mitochondrial membrane potential is significantly lowered (P<0.01) in a dose-dependent manner. Additionally, both red and green fluorescence spots became more in these groups. In the 3-MA group, the number of red and green fluorescence spots decreased significantly. The relative mRNA expression of Bax, Caspase-3, Caspase-9, Cyt C, and p62 was significantly down-regulated (P<0.05, P<0.01), while that of Bcl-2, Parkin, and PINK1 was significantly up-regulated (P<0.01). In the serum containing Zhenwutang groups, the relative protein expression levels of Bax, cleaved Caspase-3, cleaved Caspase-9, Cyt C, and p62 were significantly down-regulated (P<0.05,P<0.01). The LC3Ⅱ/Ⅰ was significantly increased, and the relative protein expression levels of Bcl-2, p-Parkin, p-PINK1, and Beclin1 were significantly up-regulated (P<0.01). ConclusionThe serum containing Zhenwutang can reduce the apoptosis of myocardial mast cells and increase mitochondrial autophagy. This is related to the inhibition of intracellular Bax/Bcl-2/Caspase-3 apoptosis pathway and regulation of Parkin/PINK1 mitochondrial autophagy pathway.

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