1.Analyzing Differences in Volatile Components of Citri Reticulatae Pericarpium Before and After Being Stir-fried with Halloysitum Rubrum Based on HS-GC-MS and Intelligent Sensory Technology
Li XIN ; Jiawen WEN ; Wenhui GONG ; Beibei ZHAO ; Shihao YAN ; Huashi CHEN ; Haiping LE ; Jinlian ZHANG ; Yanhua XUE
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):157-162
ObjectiveTo analyze the differences in color, odor and volatile components of Citri Reticulatae Pericarpium(CRP) before and after being stir-fried with Halloysitum Rubrum, and to explore the material basis of enhancing the effect of strengthening spleen after processing and the scientific connotation of decoction pieces processed with Halloysitum Rubrum as the auxiliary material. MethodsThe volatile components of the samples before and after processing were identified and relatively quantified by headspace gas chromatography-mass spectrometry(HS-GC-MS), and the volatile components were analyzed by principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA). According to the principle of variable importance in the projection(VIP) value>1.5, volatile differential components before and after processing were screened. And combined with intelligent sensory technologies such as colorimeter and electronic nose, the chroma and odor information of CRP before and after being stir-fried with Halloysitum Rubrum were identified. Pearson correlation analysis was used to explore the correlation between volatile differential components and chroma values. ResultsA total of 112 volatile components were identified from CRP and CRP stir-fried with Halloysitum Rubrum, of which 84 were from CRP and 97 were from CRP stir-fried with Halloysitum Rubrum. And 7 differential components were selected, including α-pinene, β-myrcene, linalool, sabinene, ocimene isomer mixture, A-ocimene, and δ-elemene. After being processed with Halloysitum Rubrum, the brightness value(L*), yellow-blue value(b*) and total chromatic value(E*ab) of CRP were decreased(P<0.01), and red-green value(a*) was increased(P<0.01), the response values of S4, S5, S10 and S13 sensors were significantly increased(P<0.05), and the response values of S3 and S8 sensors were significantly decreased(P<0.05). Correlation analysis showed that α-pinene and β-myrcene were negatively correlated with L* and E*ab, but positively correlated with a*. Sabinene was positively correlated with L* and E*ab. Linalool was positively correlated with L* and E*ab, and negatively correlated with a*. The ocimene isomer mixture was positively correlated with the L*. ConclusionAfter being processed with Halloysitum Rubrum, the appearance color, odor and volatile components of CRP change significantly, and α-pinene, β-myrcene, sabinene, linalool and A-ocimene are the characteristic volatile components before and after processing, which can provide references for the quality evaluation and clinical application of CRP and its processed products.
2.Textual Research on Key Information of Famous Classical Formula Jiegengtang
Yang LEI ; Yuli LI ; Xiaoming XIE ; Zhen LIU ; Shanghua ZHANG ; Tieru CAI ; Ying TAN ; Weiqiang ZHOU ; Zhaoxu YI ; Yun TANG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):182-190
Jiegengtang is a basic formula for treating sore throat and cough. By means of bibliometrics, this study conducted a textual research and analysis on the key information such as formula origin, decocting methods, and clinical application of Jiegengtang. After the research, it can be seen that Jiegengtang is firstly contained in Treatise on Febrile and Miscellaneous Disease, which is also known as Ganjietang, and it has been inherited and innovated by medical practitioners of various dynasties in later times. The origins of Chinese medicines in this formula is basically clear, Jiegeng is the dried roots of Platycodon grandiflorum, Gancao is the dried roots and rhizomes of Glycyrrhiza uralensis, the two medicines are selected raw products. The dosage is 27.60 g of Glycyrrhizae Radix et Rhizoma and 13.80 g of Platycodonis Radix, decocted with 600 mL of water to 200 mL, taken warmly after meals, twice a day, 100 mL for each time. In ancient times, Jiegengtang was mainly used for treating Shaoyin-heat invasion syndrome, with cough and sore throat as its core symptoms. In modern clinical practice, Jiegengtang is mainly used for respiratory diseases such as pharyngitis, esophagitis, tonsillitis and lung abscess, especially for pharyngitis and lung abscess with remarkable efficacy. This paper can provide literature reference basis for the modern clinical application and new drug development of Jiegengtang.
3.Textual Research on Historical Evolution and Key Information of Classical Famous Formula of Da Qinjiaotang
Na LI ; Jianying BAI ; Fuping LI ; Xiufen ZHANG ; Di LU ; Yishuo BAI ; Cuixiang WANG ; Kun SU
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):201-211
Da Qinjiaotang is the 54th formula of the 100 formulas in the Catalogue of Ancient Classical Formulas (the first batch) ,and it originated from the Collection of Writings on the Mechanism of Disease, Suitability of Qi, and Safeguarding of Life Discussed in Plain Questions. Da Qinjiaotang is composed of Gentiana macrophylla, Glycyrrhizae Radix et Rhizoma, Ligusticum chuanxiong, Angelica sinensis, Paeonia lactiflora, Asari Radix et Rhizoma, Notopterygium incisum, Saposhnikoviae Radix, Scutellariae Radix, Gypsum, Angelica dahurica, Atractylodis Macrocephalae Rhizoma, Rehmanniae Radix, Rehmanniae Radix Praeparata, Poria, and Angelicae Pubescentis Radix. It is a classical formula for treating strokes. Da Qinjiaotang is widely used in modern clinical practices for treating ischemic stroke, peripheral facial paralysis, cervical spondylosis, rheumatic arthritis, neurodermatitis, and other multisystem diseases. Therefore, following the Principles of Textual Research on the Key Information of Ancient Classical Famous Formulas, the authors collected the ancient Chinese medical literature of Da Qinjiaotang by the method of bibliometrics and screened out 177 valid data, involving 100 ancient books of traditional Chinese medicine. Based on the historical evolution, composition, dosage, method of preparation, and preparation of the original medicinal materials of Da Qinjiaotang, a systematic study was carried out. It was found that among the 175 records of the main diseases and syndromes, stroke (144) was the most, accounting for 82.29% of the total diseases and syndromes. Later generations mostly followed the practice of LIU Wansu in using Da Qinjiaotang to treat stroke caused by "weak blood and inability to nourish tendon", featuring "hands and feet cannot move, stiff tongue hinders speaking", as well as other symptoms, such as slant of the mouth, hemiplegia, numbness of the limbs, paroxysmal pain, and acerbic syncope. The treatment scope was expanded, covering tendon dryness, clonic convulsion, spasm syndrome, and arthralgia syndrome. At the same time, it was found that there was a controversy between "internal wind" and "external wind" in the treatment of stroke by Da Qinjiaotang. LIU Wansu thought that stroke was caused by internal factors, created the theory of "hot stroke", and used Da Qinjiaotang to treat "internal wind". Many doctors in later generations focused on treating the "external wind" of "internal deficiency and evil". There were 76 valid data on the composition of drugs, 59 of which had doses for each drug. It was suggested to use the modern conversion dosage of the original formula, with 41.30 g per dose. The drug should be boiled in 600 mL water until 300 mL, decocted once, and taken in a warm state after removing the dregs anytime. Through the analysis and study of the ancient books about Da Qinjiaotang, the paper clarified its historical evolution and confirmed its key information, so as to provide the ancient literature evidence for the research and development of the classical famous formula Daqinjiaotan and its better clinical application.
4.Treatment of IgA Nephropathy by Tonifying Kidney and Invigorating Spleen as Well as Detoxifying and Relieving Sore-throat Based on PIgR-CR1-mediated Mucosal-renal Axis
Fan LI ; Hongan WANG ; He NAN ; Mingyu HE ; Chengji CUI ; Yinping WANG ; Yutong LIU ; Shoulin ZHANG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):237-244
Immunoglobulin A nephropathy (IgAN) is the primary glomerulonephritis with the highest incidence rate in the world. It is also the main cause of end-stage renal disease (ESRD) in China, which has brought heavy economic burden to the society and patient families. Traditional Chinese medicine (TCM) has certain advantages in treating IgAN. In TCM, IgAN is classified into consumptive disease, hematuria, and edema categories, with the location in the kidney and involving the lung, liver, and spleen. Professor Ren Jixue, a master of TCM, believes that kidney deficiency and spleen deficiency are the root causes of IgAN, and the throat is the source of the disease. He proposed the theory of throat-kidney correlation and used the method of tonifying kidney and invigorating spleen as well as detoxifying and relieving sore-throat to treat IgAN, achieving significant therapeutic effects. Studies have shown that IgAN is closely related to mucosal immune defense. IgAN patients often experience recurrent and gradually worsening symptoms due to mucosal infections, and polymeric Ig receptor (PIgR) is an important component of mucosal defense function. The lack of PIgR leads to the accumulation of IgA molecules in the mucosal lamina propria, and the molecules enter the bloodstream in large quantities and ultimately deposit in the kidneys, causing kidney damage. Complement regulatory protein complement receptor type 1 (CR1) exists on red blood cells and glomeruli and has the function of inhibiting the activation and differentiation of B cells, clearing immune complexes, and inhibiting excessive activation of the complement system. Therefore, regulating the immune defense function through the mucosal-renal axis mediated by PIgR-CR1 will be an important target for preventing and treating IgAN. Based on the theory of throat-kidney correlation, this article explores the effects and molecular mechanisms of tonifying kidney and invigorating spleen as well as detoxifying and relieving sore-throat in preventing and treating IgAN by regulating the mucosal-kidney axis mediated by PIgR-CR1. It provides effective theoretical support and a scientific basis for TCM prevention and treatment of IgAN based on the theory of throat-kidney correlation.
5.Exploration of Traditional Chinese and Western Medicine in Prevention and Treatment of DKD Based on Mitochondrial Autophagy Mediated by PINK1/Parkin Signaling Pathway: A Review
Runsheng LIU ; Xiaodong ZHANG ; Zhaoqing LI ; Jing WANG ; Jinglu ZHANG ; Lixia JIN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):302-313
Diabetic kidney disease (DKD) is one of the more common chronic kidney diseases,and its causes are complex. DKD is very easy to progress to end-stage renal disease,and the current therapeutic effect still needs to be improved. As an important excretive organ of the human body, the kidney has physiological functions such as discharging metabolic waste, regulating fluid balance, and maintaining the stability of the body's internal environment. These highly complex biochemical processes all depend on the energy support provided by mitochondria. Mitochondrial dysfunction is a key factor causing kidney injury, and the imbalance of mitochondrial homeostasis is an important link leading to mitochondrial dysfunction. The occurrence and development of DKD are often accompanied by the imbalance of mitochondrial homeostasis in renal cells. Mitochondrial autophagy, as a means of regulating mitochondrial homeostasis, is very important for the prevention and treatment of DKD. The PTEN-induced putative kinase 1 (PINK1)/Parkin pathway is one of the most classical pathways to regulate mitochondrial autophagy. Recent studies have found that some drugs can regulate the PINK1/Parkin signaling pathway to target mitochondrial homeostasis and exert renoprotective effects. In particular, traditional Chinese medicine has a significant effect on early and middle stage DKD by regulating PINK1/Parkin pathway-mediated mitochondrial autophagy. This article discussed the mechanism of PINK1/Parkin pathway in mitochondrial autophagy and DKD and reviewed the effect of PINK1/Parkin pathway-mediated mitochondrial autophagy on DKD. At the same time, it explored the therapeutic effect of traditional Chinese and western medicine on DKD mediated by PINK1/Parkin-mediated mitochondrial autophagy, aiming to broaden the ideas of traditional Chinese and western medicine for the prevention and treatment of DKD from the perspective of PINK1/Parkin regulating mitochondrial autophagy.
6.Influence of self-esteem, alexithymia and alienation on non-suicidal self-injury behaviors in adolescents
Chunxing XU ; Hongzheng LI ; Yongcai MENG ; Yuqing ZHANG ; Meng LI ; Yaomu ZHANG ; Yaqin WANG
Sichuan Mental Health 2025;38(1):65-70
BackgroundIn recent years, the incidence of non-suicidal self-injury (NSSI) behaviors among adolescents has been increasing annually. Self-esteem and alexithymia are strongly associated with NSSI behaviors, and alienation is closely linked to both self-esteem and alexithymia. However, there is limited research on the relationship between alienation and NSSI behaviors among adolescents in China. ObjectiveTo analyze the relationship between alienation and NSSI behaviors among adolescents, and to explore the factors influencing NSSI behaviors in this population, so as to provide insights for the prevention and treatment of NSSI behaviors in adolescents. MethodsAdolescents admitted to the Department of Psychiatry and Psychology at the 923rd Hospital of the Joint Logistics Support Force of the Chinese People's Liberation Army from September 1, 2021 to March 1, 2023, who met the diagnostic criteria for NSSI in the Diagnostic and Statistical Manual of Mental Disorders, fifth edition (DSM-5), were selected as the study group (n=60). Concurrently, middle school students from Nanning were recruited as the control group (n=60). Participants were assessed using Adolescent Self Harm Scale (ASHS), Rosenberg Self-Esteem Scale (RSES), Toronto Alexithymia Scale (TAS) and Adolescent Students′ Alienation Scale (ASAS). Pearson correlation analysis was employed to examine the relationships between scale scores in the study group, and Logistic regression analysis was used to identify the influencing factors of NSSI behaviors among adolescents. ResultsThe RSES score of the study group was significantly lower than that of the control group (t=-7.033, P<0.01). The TAS and ASAS scores of the study group were significantly higher than those of the control group (t=5.591, 8.124, P<0.01). The ASHS score was negatively correlated with RSES score (r=-0.410, P<0.01) and positively correlated with ASAS score (r=0.555, P<0.01). The RSES scores of the study group were negatively correlated with TAS and ASAS scores (r=-0.317, -0.590, P<0.05 or 0.01). Logistic regression analysis showed that being female (OR=0.714, 95% CI: 0.042~0.709) was a protective factor for NSSI behaviors among adolescents, while high alienation (OR=1.028, 95% CI: 1.013~1.043) and residing in rural areas (OR=6.692, 95% CI: 2.038~21.967) were risk factors for NSSI behaviors among adolescents. ConclusionAlienation was positively correlated with NSSI behaviors in adolescents. Female adolescents had a lower risk of NSSI behaviors, while those with higher levels of alienation or residing in rural areas were more prone to NSSI behaviors. [Funded by Self-financed Scientific Research Project of the Health Commission of Guangxi Zhuang Autonomous Region (number, Z20210656); Self-financed Scientific Research Project of the Health Commission of Guangxi Zhuang Autonomous Region (number, Z-A20231057)]
7.Effect of Shenxiong Huanglian Jiedu Decoction on Neuronal Damage and Aβ Clearance in Mice Model of Alzheimer's Disease
Jing LIU ; Kang CHEN ; Yushun ZHOU ; Zhezuo ZHANG ; Guran YU ; Hao LI
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):43-52
ObjectiveTo investigate the effects of Shenxiong Huanglian Jiedu decoction on the clearance of amyloid β-protein (Aβ) and neuronal damage in the mouse model of Alzheimer's disease (AD). MethodsA total of 36 SPF-grade 2-month-old C57BL/6J mice were used in this study, and the modeling was performed by bilateral hippocampal injection of Aβ oligomers in C57BL/6J mice. The experiment was conducted with a blank group, a sham operation group, a model group, low- and high-dose (3.27,6.54 g·kg-1, respectively) Shenxiong Huanglian Jiedu decoction groups, and a positive control (donepezil hydrochloride, 0.65 mg·kg-1) group. At the end of the drug intervention, the learning and memory abilities and the activities of mice were evaluated by the Morris water maze and open field tests. Brain histopathology was examined by hematoxylin-eosin and Nissl staining. Additionally, in vivo imaging was employed to measure the metabolism of fluorescent Aβ in the cerebrospinal fluid, and staining of ionized calcium-binding adapter molecule-1 (Iba-1) was employed to assess microglial activation in the hippocampal tissue. Additionally, neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) levels in the brain tissue and serum were determined by the immunofluorescence assay and enzyme-linked immunosorbent assay. Western blot was conducted to determine the expression of inflammation and pathway-related proteins in the hippocampal tissue. ResultsCompared with the blank group and the sham operation group, the escape latency of the mice in the model group was prolonged, the platform residence time was shortened, the hippocampal tissue showed pathological manifestations such as neuronal pyknosis, Nissl body dissolution, and microglia activation. The metabolic rate of fluorescent Aβ through cerebrospinal fluid was slowed down, and the expression levels of BDNF, NT-3, and interleukin-10 (IL-10) in the hippocampus were significantly decreased (P<0.01). The expression levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and phosphorylated nuclear transcription factor-κB (p-NF-κB p65) in hippocampus were significantly increased (P<0.05, P<0.01). Compared with the model group, the escape latency of mice in the low and high dose groups of Chinese medicine and donepezil group was shortened, and the platform residence time was prolonged. Neuronal karyopyknosis, Nissl body dissolution and microglia activation in hippocampus were improved. Fluorescence Aβ was metabolized faster by cerebrospinal fluid. The expression of BDNF and NT-3 in hippocampus was increased (P<0.01), and the expression of TLR4, MyD88 and p-NF-κB p65 was significantly decreased (P<0.05, P<0.01). The expression of TNF-α in the hippocampus of the high-dose group was significantly decreased (P<0.05), and the expression of IL-10 was significantly increased (P<0.05). The expression of TNF-α, IL-6 and IL-1β in the hippocampus of the donepezil group was significantly decreased (P<0.05, P<0.01). ConclusionShenxiong Huanglian Jiedu decoction may mitigate neuronal damage and enhance cerebrospinal fluid flow in the mouse model of AD, thereby promoting the clearance of Aβ and improving the learning and memory abilities. These beneficial effects are likely mediated through the inhibition of microglial activation, reduction of inflammation, and modulation of the TLR4/MyD88/NF-κB signaling pathway.
8.Effect of Zishen Tongguan Formula on "Gut-prostate" Axis of Rats with Chronic Non-bacterial Prostatitis Based on 16S rDNA Sequencing
Xiran LI ; Mengjiao CHEN ; Kaiping ZOU ; Chenguang ZHAO ; Xingbin DAI ; Xiaoqing ZHANG ; Shun LIU
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):63-71
ObjectiveBased on the theory of "gut-prostate" axis, this study explored the effects and mechanisms of Zishen Tongguan formula and Cinnamomi Cortex in the formula in treating rats with chronic non-bacterial prostatitis(CNP) by detecting the levels of inflammatory factors, and the composition and structure of intestinal flora in CNP rats. MethodsEight out of 42 SD rats were randomly selected as the normal group, and the remaining rats were injected with carrageenan to prepare the CNP model. After successful modeling, 32 rats were randomly divided into the model group, Ningmitai capsule group(0.50 g·kg-1), Zishen Tongguan formula group(2.00 g·kg-1), and the Phellodendri Chinensis Cortex-Anemarrhenae Rhizoma pair group(PCC-AR group, 2.00 g·kg-1), with 8 rats in each group. The administered groups were given the corresponding medicinal solution by gavage, and the normal and model groups were intragastrically administered with an equal volume of normal saline, once a day for 14 consecutive days. The prostate tissues of rats were collected and subjected to hematoxylin-eosin(HE) staining and Masson staining to observe the pathological changes of the tissues in each group. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of related inflammatory factors in rat serum, and 16S rDNA sequencing was used to analyze the abundance and diversity changes of gut microbiota before and after administration, and species difference analysis was performed. ResultsAll the administered groups could alleviate the inflammatory symptoms of CNP rats, increase the expression levels of anti-inflammatory factors and decrease the expression levels of pro-inflammatory factors, with the most sIgnificant effect observed in the Zishen Tongguan formula group. Compared with the normal group, the expression levels of interleukin(IL)-8, hypersensitive C-reactive protein(hs-CRP), immunoglobulin(Ig)M, secretory IgA (sIgA), and inducible nitric oxide synthase(iNOS) were sIgnificantly increased in the model group(P<0.01). Compared with the model group, the expression levels of the above inflammatory factors in all administered groups were significantly reduced(P<0.01). When compared with the PCC-AR group, the Zishen Tongguan formula group showed a significant decrease in transforming growth factor(TGF)-β1 expression level(P<0.05) and a significant increase in IgM expression level(P<0.01). The results of gut microbiota analysis showed that, compared with the PCC-AR group, at the order level, the Zishen Tongguan formula group significantly reduced the relative abundance of conditional pathogens such as Bacteroidales, Acidaminococcales, Rhodospirillales, Clostridiales, and Elusimicrobiales(P<0.01). And at the genus level, the Zishen Tongguan formula group significantly decreased the relative abundance of pathogenic microbiota such as Lachnospira and Bacteroides(P<0.01) and significantly increased the relative abundances of beneficial microbiota such as Ruminococcus and Lactobacillus(P<0.01). ConclusionZishen Tongguan formula can reduce the level of harmful intestinal bacteria, increase the level of beneficial intestinal bacteria, down-regulate the expression of serum inflammatory factors, and the small amount of Cinnamomi Cortex in the formula may play a key role in the treatment of CNP with this formula.
9.Zishen Tiaogan Prescription Treats Diminished Ovarian Reserve in Rats via Keap1/Nrf2/HO-1 Signaling Pathway
Zhongtong LI ; Yaping ZHANG ; Chen YOU ; Qingqing LI ; Yingjie WANG ; Siwen OU ; Taomei XUE ; Chuqi ZHANG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):72-80
ObjectiveTo observe the effect of Zishen Tiaogan prescription on the oxidative stress injury in the rat model of diminished ovarian reserve (DOR) and explore the role of the Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. MethodsForty-eight female SD rats were randomly assigned into a normal group (n=12) and a modeling group (n=36). The rats in the modeling group received subcutaneous injection of galactose (350 mg·kg-1) combined with immobilization stress daily. After 28 days of modeling, 6 rats in the normal group and 6 rats in the modeling group were sacrificed to examine the modeling results. The successfully modeled rats were assigned into model, estradiol valerate (0.09 mg·kg-1), and low-, medium-, and high-dose (6.39, 12.78, 25.56 g·kg-1, respectively) Zishen Tiaogan prescription groups. The intervention lasted for 4 weeks with 6 animals per group. Hematoxylin-eosin staining was used to observe the estrous cycle and the pathological changes in the ovarian tissue. The ovarian index was calculated. Enzyme-linked immunosorbent assay was employed to measure the serum levels of sex hormones and oxidative stress-related indexes. Western blot and real-time PCR were employed to determine the protein and mRNA levels, respectively, of Nrf2, Keap1 and HO-1 in the ovarian tissue. The positive expression of superoxide dismutase 2 (SOD2) in the ovarian tissue was detected by immunohistochemistry (IHC). ResultsCompared with the normal group, the model group showed reduced follicles in the ovary, loose arrangement of the follicle granule layer, declined levels of anti-Mullerian hormone (AMH) and estradiol (E2) in the serum, elevated levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) (P<0.01), lowered levels of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) (P<0.01), and increased accumulation of malondialdehyde (MDA) (P<0.01). In addition, the modeling led to up-regulated protein and mRNA levels of Keap1 (P<0.01), the expression of Nrf2 and HO-1 protein was significantly decreased (P<0.01), the mRNA expression of Nrf2 was significantly decreased (P<0.05), the mRNA expression of HO-1 was significantly decreased (P<0.01), in the ovarian tissue. Compared with model group, the estradiol valerate and low-, medium-, and high-dose Zishen Tiaogan prescription groups showed increases in the ovarian index (P<0.01) and serum E2 and AMH levels (P<0.01), declined levels of FSH and LH (P<0.01), increased follicles in the ovary, elevated levels of SOD, CAT, and GSH, and reduced accumulation of MDA (P<0.05, P<0.01). Furthermore, these groups showcased down-regulated protein and mRNA levels of Keap1 (P<0.01), the expression of Nrf2 protein was significantly increased (P<0.01), the expression level of HO-1 protein was increased (P<0.05,P<0.01), and increased positive expression of SOD2 (P<0.01). ConclusionZishen Tiaogan prescription can regulate the serum levels of hormones, down-regulate the expression of Keap1, up-regulate the expression of Nrf2, HO-1, and SOD2, enhance the antioxidant capacity, and reduce the peroxidation damage in the ovarian tissue to improve the ovarian reserve function in the rat model of DOR. High-dose Zishen Tiaogan prescription demonstrated the best effect and the mechanism is associated with the regulation of the Keap1/Nrf2/HO-1 pathway.
10.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.

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