1.Mechanism of Electroacupuncture Alleviating Inflammatory Pain in Rats by Regulating ErbB Subtypes in the Spinal Dorsal Horn
Yuxin WU ; Shuxin TIAN ; Zhengyi LYU ; Dingru JI ; Xingzhen LI ; Yue DONG ; Binyu ZHAO ; Yi LIANG ; Jianqiao FANG
Journal of Traditional Chinese Medicine 2026;67(1):69-78
ObjectiveTo observe the changes in the levels of different subtypes of epidermal growth factor receptor (ErbB), namely ErbB1, ErbB2, ErbB3, and ErbB4, in the spinal dorsal horn of inflammatory pain model rats, and to explore their mechanism of mediating hyperalgesia as well as the intervention mechanism of electroacupuncture at "Zusanli (ST 36)" and "Kunlun (BL 60)". MethodsThe study was divided into five parts. In experiment 1, 14 Sprague Dawley (SD) rats were randomly divided into control and inflammatory pain group (7 rats each group) to observe the pain behavior and the protein expression of different ErbB receptor subtypes in the spinal dorsal horn. In experiment 2, 30 rats were randomly divided into control group 1, inflammatory pain group 1, and low-, medium-, and high-concentration TX1-85-1 groups, with 6 rats in each group, to observe the effect of inhibiting spinal ErbB3 on inflammatory pain. In experiment 3, 12 rats were randomly divided into control virus group and ErbB3 knockdown virus group, with 6 rats in each group, to observe the effect of knocking down ErbB3 in the spinal dorsal horn on inflammatory pain. In experiment 4, 44 rats were randomly divided into control group 2, inflammatory pain group 2, electroacupuncture group, and sham electroacupuncture group, with 11 rats in each group, to observe the effect of electroacupuncture. In experiment 5, 40 rats were randomly divided into control group 3, inflammatory pain group 3, electroacupuncture group 1, and electroacupuncture + NRG1 group, with 10 rats in each group, to observe the effect of activating ErbB3 on electroacupuncture. A rat model of inflammatory pain was established by subcutaneous injection of 100 μl of complete Freund's adjuvant into the sole of the unilateral hind foot of SD rats. Rats in the low-, medium-, and high-concentration TX1-85-1 groups were intrathecally injected with ErbB3 inhibitor TX1-85-1 on day 5 to day 7 after modeling. Rats in the ErbB3 knockdown virus group were injected with ErbB3 knockdown virus packaged with adenovirus vector-based short hairpin RNA (shRNA) into the spinal dorsal horn in situ 3 weeks before modeling. Rats in each electroacupuncture group received electroacupuncture at bilateral "Zusanli (ST 36)" and "Kunlun (BL 60)" from day 1 to day 7 after modeling, with dense-sparse waves at a frequency of 2 Hz/100 Hz and a current of 0.5-1.5 mA for 30 minutes once a day. Rats in the electroacupuncture + NRG1 group were intrathecally injected with ErbB3 ligand recombinant human neuregulin-1 (NRG1) after electroacupuncture intervention from day 5 to day 7 after modeling. The mechanical withdrawal threshold and thermal withdrawal latency of rats were measured on day 1, 3, 5, and 7 after modeling to evaluate behavior, and Western Blot was used to detect the protein and phosphorylation levels of each ErbB subtype in the spinal dorsal horn. ResultsCompared with the control group, rats in the inflammatory pain group showed decreased mechanical withdrawal threshold and thermal withdrawal latency of rats, and increased expression of phosphorylated ErbB3 (p-ErbB3) protein in the spinal dorsal horn on days 1, 3, 5, and 7 after modeling (P<0.01). On day 5 and day 7 after modeling, compared with the inflammatory pain group 1, the mecha-nical withdrawal threshold and thermal withdrawal latency of rats in the medium- and high-concentration TX1-85-1 groups increased, and the expression of p-ErbB3 protein decreased (P<0.05). On day 1, 3, 5, and 7 after modeling, compared with the control virus group, the mechanical withdrawal threshold and thermal withdrawal latency of rats in the ErbB3 knockdown virus group increased (P<0.05). On day 5 and day 7 after modeling, compared with the inflammatory pain group 2 and the sham electroacupuncture group, the mechanical withdrawal threshold and thermal withdrawal latency of rats in the electroacupuncture group increased, and the expression of p-ErbB3 protein decreased (P<0.05). On day 5 and day 7 after modeling, compared with the electroacupuncture + NRG1 group, the mechanical withdrawal threshold and thermal withdrawal latency of rats in the electroacupuncture group 1 increased (P<0.05). ConclusionThe p-ErbB3 in the spinal dorsal horn involved in hyperalgesia in rats with inflammatory pain, and electroacupuncture at "Zusanli (ST 36)" and "Kunlun (BL 60)" can alleviate inflammatory pain by inhibiting the expression of p-ErbB3 protein in the spinal dorsal horn of rats.
2.Staged Efficacy of Qijia Rougan Prescription Combined with Entecavir for Chronic Hepatitis B-related Hepatic Fibrosis with Qi Deficiency and Collateral Stasis Syndrome Based on "Zhu Ke Jiao" Theory
Baixue LI ; Xin WANG ; Jibin LIU ; Li WEN ; Cen JIANG ; Wenjun WU ; Dong WANG ; Shuwan LIU ; Huabao LIU ; Yongli ZHENG ; Liang HUANG ; Yue SU ; Song ZHANG ; Yanan SHANG ; Hang ZHOU ; Quansheng FENG
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(9):180-188
ObjectiveThis paper aims to investigate and evaluate the staged efficacy and safety of the representative empirical prescription of the “Zhu Ke Jiao” theory, Qijia Rougan prescription, combined with entecavir in the treatment of hepatic fibrosis in chronic hepatitis B. MethodsA multicenter randomized controlled clinical study was conducted, and 101 patients diagnosed with chronic hepatitis B-related hepatic fibrosis (CHB-HF) who met the diagnosis and inclusion criteria were randomly assigned to an observation group (Qijia Rougan prescription + entecavir) and a control group (entecavir). The treatment duration was 24 weeks. Liver stiffness measurement (LSM), fibrosis-4 index (FIB-4), portal vein diameter, hepatitis B serology, biochemical indicators, hepatic fibrosis markers in serum [hyaluronic acid (HA), laminin (LN), procollagen Ⅲ peptide (PⅢP), and type Ⅳ collagen (Ⅳ-C)], and traditional Chinese medicine syndrome scores were used as efficacy evaluation indicators. Efficacy assessments and explorations of different staged subgroups of Qijia Rougan prescription were conducted according to LSM values based on the Metavir pathological staging standard. ResultsA total of 98 cases were included for statistical analysis, with 49 cases in the observation group and 49 in the control group. The general data of the patients in both groups were comparable. Compared with the same group before treatment, the observation group showed a significant reduction in LSM and FIB-4 (P<0.01), as well as notable improvements in LN, Ⅳ-C, and various TCM syndrome scores (P<0.05, P<0.01). When compared to the control group after treatment, the observation group demonstrated significant improvements in LSM, FIB-4, and various TCM syndrome score indicators (P<0.05, P<0.01), indicating that the observation group performed better than the control group. Subgroup analysis of the regression of hepatic fibrosis stages showed that compared to the same group before treatment, the observation group had better improvement in regression of stages F2 and F3 (P<0.05). When compared to the control group after treatment, the observation group exhibited superior improvement in regression of stage F3 (P<0.05). No adverse events occurred in either group during the treatment period. ConclusionCompared with entecavir alone, the combination of Qijia Rougan prescription and entecavir significantly improves the degree of hepatic fibrosis and clinical TCM symptoms in patients. The optimal intervention period is primarily during stage F3, which is a potential “interception” point of the “Zhu Ke Jiao” theory.
3.Correlation between liver fibrosis degree and carotid plaque in patients with lean metabolic dysfunction-associated fatty liver disease
Shuai ZHANG ; Shoulu JIN ; Wanqing LI ; Xijing SHI ; Hao LIANG ; Hao DONG ; Dailong LU ; Ying ZHU ; Xiaoxing XIANG ; Jun LIU
Journal of Clinical Hepatology 2026;42(2):319-325
ObjectiveTo investigate the association between noninvasive liver fibrosis markers and carotid plaque (CP) in patients with lean metabolic dysfunction-associated fatty liver disease (MAFLD), and to provide a basis for screening high-risk populations. MethodsA total of 957 patients with lean MAFLD who underwent physical examination in Subei People’s Hospital from January 2021 to June 2023 was enrolled as the observation cohort, with the presence or absence of CP as the outcome, and fibrosis-4 (FIB-4) index and nonalcoholic fatty liver disease fibrosis score (NFS) were used to assess liver fibrosis degree. The independent-samples t test was used for comparison of normally distributed continuous data between two groups, and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups; the chi-square test was used for comparison of categorical data between two groups. The multivariate logistic regression analysis, the restricted cubic spline analysis, the receiver operating characteristic curve, and the mediation effect analysis were used to investigate the association between liver fibrosis degree and CP. ResultsThe prevalence rate of CP was 36.6% in the lean MAFLD population. Compared with the non-CP group(n=607), the CP group (n=350) had a significantly higher proportion of male patients, a significantly higher proportion of patients with smoking/diabetes/hypertension, and significantly higher levels of age, creatinine, blood urea nitrogen, triglycerides, fasting blood glucose, aspartate aminotransferase, aspartate aminotransferase/alanine aminotransferase ratio, NFS, and FIB-4 index, as well as significantly lower levels of platelet count and albumin (all P<0.05). The multivariate logistic regression analysis showed that after adjustment for confounding factors, FIB-4 index (odds ratio[OR]=2.979, 95% confidence interval[CI]:2.141 — 4.219, P<0.001) and NFS (OR=1.747, 95%CI: 1.499 — 2.046, P<0.001) were positively correlated with CP. Both FIB-4 index and NFS had a good value in predicting CP. Hypertension had a significant indirect effect on the prevalence rate of CP through its impact on liver fibrosis markers, and its mediating effect accounted for 39.5% — 40.8% of the total effect (P<0.001). ConclusionIn patients with lean MAFLD, NFS and FIB-4 index are significantly positively correlated with the prevalence rate of CP, and they can be used as potential epidemiological predictive indicators. Liver fibrosis markers may play a mediating role in the association between hypertension and CP. Interventions targeting hypertension and liver fibrosis markers may help to prevent and delay the progression of CP.
4.Ectopic expression of hemoglobin subunits enhances the in vitro cytotoxicity of CAR-T cells against tumor cells under hypoxic conditions
YANG Jianxun1,2 ; ZHENG Rui3 ; LIANG Sixin3 ; PAN Jie4 ; LI Yanlong5 ; ZHAI Chenxi5 ; ZHAO Xiaojuan2 ; WANG Pengju3 ; DONG Hao4 ; YAN Bo2 ; SUN Zhihong1 ; YANG Angang3
Chinese Journal of Cancer Biotherapy 2026;33(3):233-242
[摘 要] 目的:探讨异位表达血红蛋白亚基(HBA/HBB)对缺氧条件下嵌合抗原受体T细胞(CAR-T细胞)功能障碍的改善作用及其对肿瘤细胞的杀伤效应。方法:全基因合成技术合成靶向HER2的CAR序列,构建共表达HBA或HBB的CAR慢病毒载体,包装慢病毒后感染人原代T淋巴细胞,制备异位表达HBA/HBB的CAR-T细胞,命名为HBA CAR-T和HBB CAR-T。采用缺氧探针检测小鼠实体瘤缺氧状态。通过流式细胞术检测瘤内CAR-T细胞占比、异位表达血红蛋白亚基的CAR-T细胞阳性率及CAR-T细胞的活性氧、凋亡水平。WB法检测HBA CAR-T和HBB CAR-T内相关血红蛋白亚基表达情况,采用细胞计数板计数检测细胞增殖水平,通过萤光素酶报告基因法检测CAR-T细胞对肿瘤细胞的杀伤能力,qPCR检测CAR-T细胞中缺氧诱导因子-1α(HIF-1α)表达水平,利用MitoXpress Intra试剂盒检测CAR-T细胞内氧气含量。结果:不同细胞构建的实体瘤模型均存在明显缺氧情况,且CAR-T细胞浸润水平与缺氧程度呈显著负相关(P < 0.000 1)。HBA CAR-T与HBB CAR-T构建成功(阳性率 > 60%),相应血红蛋白亚基可稳定表达。缺氧环境下HBA CAR-T和HBB CAR-T的ROS水平、凋亡水平显著下降,增殖、对肿瘤细胞的体外杀伤能力显著强于传统CAR-T细胞(均P < 0.05)。HBA CAR-T与HBB CAR-T内HIF-1α表达降低(均P < 0.001),且缺氧程度显著降低(均P < 0.001)。结论:异位表达血红蛋白亚基可改善缺氧条件下CAR-T细胞功能障碍并增强其对肿瘤细胞的体外杀伤作用。
5.Association of liver fibrosis markers and inflammation markers with the risk of gallstones in patients with metabolic dysfunction-associated fatty liver disease
Shuai ZHANG ; Shoulu JIN ; Wanqing LI ; Xijing SHI ; Hao LIANG ; Hao DONG ; Dailong LU ; Ying ZHU ; Xiaoxing XIANG ; Jun LIU
Journal of Clinical Hepatology 2026;42(3):579-585
ObjectiveTo investigate the association of liver fibrosis scores and inflammation markers with gallstones in patients with metabolic dysfunction-associated fatty liver disease (MAFLD), as well as the mediating role of liver fibrosis scores in the relationship between inflammation markers and gallstones. MethodsA total of 14 567 patients who received physical examination and were diagnosed with MAFLD in Subei People’s Hospital from January 2014 to June 2023 were enrolled in this study, and according to the results of abdominal color Doppler ultrasound, they were divided into gallstone group with 1 724 patients and non-gallstone group with 12 843 patients. Related clinical data were collected from all patients, including demographic data, medical history, family history, physical examination, Color Doppler ultrasound, and biochemical parameters. The biomarkers associated with metabolic disorders and insulin resistance included triglyceride-glucose index (TyG), TyG-body mass index (BMI) index, atherogenic index of plasma (AIP), and non-high-density lipoprotein cholesterol-to-high-density lipoprotein cholesterol ratio (NHHR); the biomarkers associated with inflammation and nutritional status included neutrophil-to-lymphocyte ratio (NLR), neutrophil percentage-to-albumin ratio (NPAR), and monocyte-to-lymphocyte ratio (MLR); the biomarkers for assessing liver fibrosis degree and liver function included albumin-bilirubin (ALBI) score, NAFLD fibrosis score (NFS), fibrosis-4 (FIB-4) index, and aspartate aminotransferase-to-platelet ratio index (APRI). The independent-samples t test was used for comparison of normally distributed continuous data between two groups, while the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups; the chi-square test was used for comparison of categorical data between two groups. Multivariate Logistic regression analysis, restricted cubic spline analysis, and mediating effect analysis were used to assess the association of liver fibrosis markers and inflammation markers with the risk of gallstones. ResultsThe prevalence rate of gallstones was 11.8% among the MAFLD patients. There were significant differences between the gallstone group and the non-gallstone group in sex, age, smoking history, diabetes, hypertension, lymphocytes, platelets, glucose, albumin, serum uric acid, alanine aminotransferase, aspartate aminotransferase, red blood cell, NLR, NPAR, MLR, NFS, FIB-4 index, and ALBI score (all P<0.05). The multivariate Logistic regression analysis showed that NLR (odds ratio [OR]=1.091, 95% confidence interval [CI]: 1.028 — 1.160, P<0.05), NPAR (OR=1.073, 95%CI: 1.042 — 1.105, P<0.05), MLR (OR=1.142, 95%CI: 1.057 — 1.232, P<0.05), NFS (OR=1.239, 95%CI: 1.190 — 1.291, P<0.05), and FIB-4 index (OR=1.326, 95%CI: 1.241 — 1.417, P<0.05) were influencing factors for the prevalence rate of gallstones. The restricted cubic spline analysis showed a significant non-linear association between NFS/FIB-4 index and the risk of gallstone (non-linear P<0.05). The mediating effect analysis further showed that the association of NLR, MLR, and NPAR with gallstones was partially mediated by NFS or FIB-4 index, with a mediating effect accounting for 36.79%、28.09%、29.67% and 18.31%、17.70、11.57%, respectively. ConclusionNFS and FIB-4 index have a non-linear association with the prevalence rate of gallstones in MAFLD patients, and they also mediate the association of NLR, NPAR, and MLR with the risk of gallstone.
6.Research Advances in Traditional Chinese Medicine Regulation of Pyroptosis for Lung Cancer Prevention and Treatment
Qiongqiong GUO ; Meihao XUE ; Xuchao DONG ; Ping TIAN ; Rong HU ; Longxin XU ; Juan LI ; Jianqing LIANG ; Jintian LI
Medical Journal of Peking Union Medical College Hospital 2026;17(3):716-725
Lung cancer remains one of the leading causes of cancer-related morbidity and mortality worldwide, and its treatment continues to face major challenges such as therapeutic resistance and tumor recurrence. Pyroptosis, a newly characterized form of programmed cell death, induces tumor cell death through gasdermin-mediated membrane pore formation and is accompanied by the release of inflammatory mediators, thereby playing complex roles in lung cancer initiation, progression, and modulation of the tumor microenvironment. Active components and herbal formulas derived from traditional Chinese medicine can modulate pyroptosis-related signaling pathways through multi-target mechanisms, showing potential advantages in inducing lung cancer cell death, inhibiting proliferation and migration, and reversing chemoresistance. This review systematically summarizes relevant studies from domestic and international sources, focusing on the molecular mechanisms of pyroptosis, its roles in lung cancer development and tumor microenvironment remodeling, and the current research progress on traditional Chinese medicine-based interventions targeting pyroptosis, with the aim of providing references for the prevention and treatment of lung cancer using traditional Chinese medicine.
7.Molecular Characterization Network of Dampness-heat Syndrome in Patients with Chronic Hepatitis B Complicated by Glucose Metabolism Disorder Based on Shadowless Scleral Imaging and Metabolomics Technology
Caiying HE ; Hang ZHOU ; Yanqi CHI ; Baixue LI ; Liang HUANG ; Zhu CHEN ; Dafeng LIU ; Dong WANG
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(14):271-285
ObjectiveThis paper aims to conduct the feature analysis and correlation analysis on the ocular collateral features and differential metabolites in patients with chronic hepatitis B (CHB) complicated by glucose metabolism disorder (GMD),particularly those with the damp-heat syndrome type,by integrating shadowless scleral imaging and metabolomics technologies. MethodsA total of 313 patients were recruited from the Hepatology and Endocrinology Outpatient Departments of Public Health Clinical Center of Chengdu according to the inclusion/exclusion criteria,and they were divided into a CHB group and a CHB complicated by GMD groups (damp-heat syndrome group and non-damp-heat syndrome group). All patients underwent high-definition ocular image acquisition and feature extraction using an intelligent analysis system for shadowless scleral imaging to analyze the differences in the counting of morphological feature scores of ocular collaterals among groups. By using a digital sampling method,24 patients from each group were randomly selected,along with 20 healthy volunteers,for untargeted metabolomic analysis of peripheral serum. Differential metabolites were identified,statistically analyzed,and subjected to potential biomarker analysis and pathway enrichment. Spearman method was performed to conduct the correlation analysis on the differential ocular collateral features and differential metabolites,followed by correlation network construction. ResultsCompared with those in the CHB group,patients with CHB complicated by GMD showed significant changes in ocular collateral feature scores such as "hillock","blood vessels",and "pale dusky coloration" (P<0.05). In comparison with those in the healthy group,metabolites including N-acetylglucosamine,acetylhomoserine,and myo-inositol (AUC>0.7) were identified as potential biomarkers for the disease. Compared with those in the CHB complicated by GMD group with non-damp-heat syndrome,patients with damp-heat syndrome exhibited significant changes in feature scores of "plaques","yellow coloration","spleen",and "gallbladder" (P<0.05). In comparison with those in the healthy group,metabolites such as O2′-4a-cyclic tetrahydrobiopterin,theobromine,xanthurenic acid,and L-glutamic acid 5-phosphate (AUC>0.7) were identified as potential biomarkers for the damp-heat syndrome type. The Spearman correlation analysis reveals weak to moderate linear correlations between the differential scleral collateral features and metabolites. By constructing a "disease-syndrome" network of ocular diagnosis and metabolites,"xanthurenic acid-gallbladder" and "theobromine-plaque/yellow coloration" were identified as specific molecular-phenotypic correlated biomarker clusters for CHB complicated by GMD with dampness-heat syndrome. ConclusionPatients with CHB complicated by GMD demonstrate differential ocular diagnostic features and serum metabolites corresponding to disease states and dampness-heat syndrome. These objective biomarkers can guide both clinical syndrome differentiation and medication. The macro-micro integration based on ocular feature clusters and potential metabolic biomarkers offers an innovative approach to a combined traditional Chinese and Western medicine diagnosis and treatment model for this disease.
8.Qiangjing Tablets Regulate CDK4-E2F Signaling Pathway to Delay Aging of Leydig Cells and Testicular Tissue in Rats
Xiucheng LAN ; Meijing WANG ; Jingyi ZHANG ; Junjun LI ; Liang DONG ; Xujun YU ; Fang YANG ; Degui CHANG
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(14):328-336
ObjectiveTo reveal the molecular mechanism by which the traditional Chinese medicine compound prescription Qiangjing tablets regulate the aging of the testicular tissue and Leydig cells in rats through the cyclin-dependent kinase 4 (CDK4)-early 2 factor (E2F) signaling pathway. MethodsFor the cell experiment, 2-month-old SPF-grade SD male rats were selected and randomly assigned into a blank control group (administrated with an equal volume of 0.9% sodium chloride injection) and a Qiangjing tablets group (20 rats in each group) according to body weight. The Leydig cell model of aging was established by treatment of TM3 cells with 100 μmol·L-1 H2O2, and the modeling performance was evaluated based on the levels of p16 and p21 determined by Western blot. The antioxidant NAC (1 mmol·L-1) was used as the positive control for eliminating reactive oxygen species (ROS). Cells were intervened with Qiangjing tablets-containing serum at low (2.5%), medium (5%), and high (10%) concentrations. The testosterone level in the cell supernatant was determined by enzyme-linked immunosorbent assay (ELISA), and the protein levels of CDK4, E2F1, and E2F2 were analyzed by Western blot. In the animal experiment, 19-month-old naturally aging rats were used as the model group, and 2-month-old rats as the young control group. The positive control group was subcutaneously injected with 5.21 mg·kg-1·d-1 testosterone propionate. Qiangjing tablets were administered by gavage at low, medium, and high doses of 0.72, 1.44, 2.88 g·kg-1·d-1, respectively. The general conditions of rats were observed, and the protein levels of CDK4, E2F1, and E2F2 in the testicular tissue were determined by Western blot. ResultsIn the cell experiment, compared with the blank control group, the model group showed upregulated expression of CDK4 and E2F1 (P<0.05) and slightly downregulated expression of E2F2. Compared with that in the model group, the expression of CDK4 was upregulated in the NAC group and the low-dose Qiangjing tablets group (P<0.05), slightly upregulated in the medium-dose Qiangjing tablets group, and downregulated in the high-dose Qiangjing tablets group (P<0.05). The NAC group showed downregulated expression of E2F1 (P<0.05) and E2F2, and the low-, medium-, and high-dose Qiangjing tablets groups showed downregulated expression of both E2F1 and E2F2 (P<0.05). Compared with that in the NAC group, the expression of CDK4 was upregulated in the low-dose Qiangjing tablets group and downregulated in the medium-dose and high dose (P<0.05) groups. The expression of E2F1 was down-regulated in all the three dose groups, with statistically significance in the high dose group (P<0.05), and that of E2F2 were downregulated in all the three dose groups (P<0.05). In the animal experiment, compared with the young control group, the model group exhibited downregulated expression of CDK4 (P<0.05) and slightly upregulated expression of E2F1 and E2F2. Compared with that in the model group, the expression of CDK4 decreased in the testosterone propionate group and the low-dose Qiangjing tablets group (P<0.05) but increased in the medium-dose (P<0.05) and high-dose groups. In addition, the expression of E2F1 decreased (P<0.05), and that of E2F2 was slightly elevated. Compared with that in the NAC group, CDK4 expression was elevated in the Qiangjing tablets groups, with statistical significance in the medium- and high-dose groups (P<0.05). Similarly, the E2F1 expression was also upregulated in the Qiangjing tablets groups, with statistical significance in the medium-dose group (P<0.05). The expression of E2F2 was downregulated in all the Qiangjing tablets groups. ConclusionQiangjing tablets delay the aging process of Leydig cells and testicular tissue by up-regulating the expression of CDK4 and lowering the levels of E2F1 and E2F2.
9.Qiangjing Tablets Alleviate Oxidative Stress Damage in Varicocele by Regulating Keap1/Nrf2 Signaling Pathway
Liang DONG ; Fang YANG ; Jingyi ZHANG ; Xinyi TANG ; Yulin LI ; Xujun YU ; Degui CHANG
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(14):347-359
ObjectiveTo explore the mechanism by which Qiangjing tablets (QJT) alleviate the spermatogenic function damage caused by varicocele (VC) based on the Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway-mediated oxidative stress. MethodsTen Sprague-Dawley (SD) rats were randomly assigned into a control group and a model group. Pathological examination confirmed the stability of the model. Thirty-six SD rats were randomized into control, model, low-dose (0.23 g·kg-1) QJT, medium-dose (0.46 g·kg-1) QJT, high-dose (0.92 g·kg-1) QJT, and mazhilin (61.7 mg·kg-1) groups, with 6 rats in each group. A rat model of experimental left varicocele (ELV) was established by partially ligating the left renal vein to simulate the human nutcracker syndrome. The rats were administrated with corresponding agents once a day for 28 consecutive days. The in vitro testicular culture model of rats was established through the Transwell chamber method and intervened with QJT-containing sera (2.3, 4.6, and 9.2 g·kg-1). Microscopic observation was carried out for the morphology of the left kidney. A micrometer was used to measure the diameter of the left spermatic vein (LSV). The body weights of rats were recorded weekly, and the epididymis and testis weights were measured. The pathological changes of the testicular tissue was observed via hematoxylin-eosin (HE) staining. The levels of testosterone (T) in the cell culture supernatant and reactive oxygen species (ROS) in the rat testicular tissue were measured by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was employed to determine the ROS content. Immunohistochemical staining was conducted to analyze Keap1, Nrf2, 3β-hydroxysteroid dehydrogenase (3β-Hsd), GATA-binding protein-4 (Gata-4), and proto-oncogene receptor tyrosine kinase (C-kit). The ultrastructure of the tissue was observed by transmission electron microscopy (TEM). Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The expression of Keap1, Nrf2, glutathione S-transferase α2 (Gsta2), glutathione S-transferase μ1 (Gstm1), heme oxygenase-1 (HO-1), quinone oxidoreductase 1 (Nqo1), and thioredoxin reductase 1 (Txnrd1) was quantified by Real-time quantitative polymerase chain reaction(Real-time PCR) and Western blot. ResultsCompared with the control group, the ROS content and the percentage of apoptotic cells in the model group were significantly increased (P<0.01), the T concentration was significantly decreased (P<0.01), the mRNA and protein expressions of Keap1 were significantly increased (P<0.01), and the mRNA and protein expressions of Nrf2, Gsta2, Gstm1, HO-1, Nqo1 and Txnrd1 were significantly decreased (P<0.05). Compared with the model group, the ROS content and the percentage of apoptotic cells in each dose group of the Qiangjing Tablets were significantly reduced (P<0.05), and the mRNA and protein expressions of Keap1 were significantly decreased (P<0.05), while the mRNA and protein expressions of Nrf2, Gsta2, Gstm1, HO-1, Nqo1 and Txnrd1 were significantly increased (P<0.05). ConclusionQJT improves sperm motility in the rat model of VC by modulating the Keap1/Nrf2 signaling pathway and reducing oxidative stress injury.
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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