1.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.
2.Effect of Huanglian Jiedutang in Regulating Ferroptosis in Mice with Atherosclerosis Based on Nrf2/GPX4 Signaling Pathway
Zhaohui GONG ; Li GAO ; Huiqi ZHAI ; Jinzi YU ; Qingmin CHU ; Chuanjin LUO ; Lijin QING ; Wei WU ; Rong LI
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(3):22-28
ObjectiveTo study the mechanism of Huanglian Jiedutang (HLJDT) in treating mice with atherosclerosis (AS) by improving ferroptosis. MethodsA total of 10 SPF C57BL/6J mice were selected as a normal group, and 50 ApoE-/- mice were randomly divided into five groups: model group, low-dose group of HLJDT, medium-dose group of HLJDT, high-dose group of HLJDT, and atorvastatin (ATV) group. ApoE-/- mice were fed a high-fat diet for eight weeks to establish the AS model, and at the 9th week, they were given normal saline, low, medium, and high doses of HLJDT (3.9, 7.8, 15.6 g·kg-1·d-1), and atorvastatin calcium tablets (0.01 g·kg-1·d-1), respectively, for a total of eight weeks. The formation of aortic plaque in mice was observed by gross oil red O staining and Masson staining. The levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), and high-density lipoprotein cholesterol (HDL-C) in blood fat were measured by the automatic biochemical analyzer, and the mitochondrial structure of the aorta was observed by transmission electron microscopy. The content of serum superoxide dismutase (SOD) in serum was detected by enzyme-linked immunosorbent assay (ELISA). The content of reduced glutathione (GSH) in serum was detected by the microplate method, and that of malondialdehyde (MDA) in serum was detected by the TBA method. The protein expression of nuclear factor E2-associated factor 2 (Nrf2)/glutathione peroxidase 4 (GPX4) signaling pathway was detected by Western blot. ResultsCompared with those of the normal group, the contents of TC, LDL-C, TG, HDL-C, and MDA in the serum and the aortic vascular plaque deposition of the model group were significantly increased (P<0.01), while the expression levels of SOD and GSH in serum, as well as Nrf2, solute carrier family 7 member 11 (SLC7A11), and GPX4 in aorta were significantly decreased (P<0.01). Mice in the model group appeared mitochondrial fragmentation and vacuolation in the aorta, volume atrophy, mitochondrial crista reduction, or a loose and disorganized form. Compared with those in the model group, the aortic vascular plaque deposition was significantly decreased in the low-dose, medium-dose, and high-dose groups of HLJDT and ATV group, and the contents of serum TC, LDL-C, TG, and MDA in serum were significantly decreased (P<0.05, P<0.01). The contents of serum SOD and GSH and the expression levels of Nrf2, SLC7A11, and GPX4 in the aorta were increased (P<0.05, P<0.01), and the symptoms of aortic mitochondrial vacuolation were alleviated. The number of cristae was increased, and they were ordered neatly. ConclusionHLJDT can reduce aortic vascular plaque deposition, decrease blood lipid and MDA expression, increase SOD and GSH expression, and ameliorate the pathological changes of ferroptosis, the mechanism of which is related to the Nrf2/GPX4 signaling pathway.
3.Textual Research on Historic Evolution and Ancient and Modern Application of Classic Prescription Huangqintang
Yuxin LI ; Lyuyuan LIANG ; Jialei CAO ; Tongyi HUANG ; Hejia WAN ; Bingqi WEI ; Mengting ZHAO ; Xiaoyang TIAN ; Bingxiang MA ; Weili DANG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(3):184-191
Huangqintang, with its accurate efficacy, is a classic formula specialized in treating dysentery recommended and promoted by medical experts from successive generations, and it was included in the Catalogue of Ancient Classic Prescriptions (the Second Batch, Han Chinese medicine prescriptions) published by the National Administration of Traditional Chinses Medicine (TCM) in 2023. The method of bibliometrics was applied in this study to conduct textual research on the classic formula Huangqintang and provide a literature reference for the development of modern preparations of Huangqintang. A total of 2 026 pieces of ancient literature were searched with "Huangqintang" as the key word, and 23 pieces of effective data were selected, involving 15 ancient TCM books. The historic evolution, composition, dosage, origin, processing methods, preparation and decocting methods, efficiency, and application of Huangqintang were carefully reviewed. The results showed that Huangqintang was first recorded in the Treatise on Febrile Diseases written by ZHANG Zhongjing. It has the effect of clearing heat, stopping dysentery, regulating the middle, and downbearing counterflow and has become one of the classic formulas widely used in clinical practice. Because of its accurate efficacy, medical experts from later generations have modified it from its original composition. Though many prescriptions have different names, it is the manifestation of physicians' inheritance and development of the thought of ZHANG Zhongjing. Ancient literature showed this prescription had wide indications yet centered on digestive system diseases such as dysentery and abdominal pain. Modern applications of Huangqintang involve digestive, respiratory, ophthalmology and otolaryngology, gynecological, skin, musculoskeletal system, and connective tissue, and this prescription has great potential in treating ulcerative colitis, diarrhea, acute enteritis, and damp-heat dysentery. Through a systematic textual excavation and review of the ancient literature about Huangqintang, the paper has confirmed its key information, so as to provide a scientific basis for the clinical application and new drug development of classic formulas.
4.Textual Research on Key Information of Classic Formula Houpo Qiwutang and Its Ancient and Modern Applications
Jinlong ZHANG ; Wei CHEN ; Ruobing LI ; Baikun YIN ; Yaodong GU ; Jun LEI ; Xicheng JIANG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(3):213-222
Houpo Qiwutang originated from the Synopsis of the Golden Chamber, and it consists of seven medicines: Magnoliae Officinalis Cortex, Rhei Radix et Rhizoma, Aurantii Fructus Immaturus, Cinnamomi Ramulus, Zingiberis Rhizoma Recens, Glycyrrhizae Radix et Rhizoma, and Jujubae Fructus. It is a basic formula for the treatment of abdominal fullness. Through the bibliometric method, the historical history, drug base, preparation and dosage, decoction method, and ancient and modern applications of Houpu Qiwu Tang were analyzed by means of textual research. The research finds that Houpu Qiwu Tang has been passed down through the generations in an orderly manner with fewer changes. The drug base of this formula is basically clear, and the base of Magnoliae Officinalis Cortex, Rhei Radix et Rhizoma, Cinnamomi Ramulus, Zingiberis Rhizoma Recens, and Jujubae Fructus is consistent with the 2020 edition of Chinese Pharmacopoeia. The mainstream base of Aurantii Fructus Immaturus is the dried young fruit of Citrus aurantium of Rutaceae family, and the historical mainstream base of Glycyrrhizae Radix et Rhizoma is the dried root of Glycyrrhiza uralensis of Leguminosae family. The modern dosage of this formula is 110.40 g of Magnoliae Officinalis Cortex, 41.40 g of Rhei Radix et Rhizoma, 69 g of Aurantii Fructus Immaturus, 27.60 g of Cinnamomi Ramulus, 69 g of Zingiberis Rhizoma Recens, 41.40 g of Glycyrrhizae Radix et Rhizoma, and 30 g of Jujubae Fructus. In addition, the decoction method is to add 2 000 mL of water with the above seven flavors of the medicine, boil it to 800 mL, and then take 160 mL in a warm state each time. The amount of the medicine taken for each time is 22.08 g of Magnoliae Officinalis Cortex, 8.28 g of Rhei Radix et Rhizoma, 13.80 g of Aurantii Fructus Immaturus, 5.52 g of Cinnamomi Ramulus, 13.80 g of Zingiberis Rhizoma Recens, 8.28 g of Glycyrrhizae Radix et Rhizoma, and 6 g of Jujubae Fructus. The modern application of this formula involves the digestive system, respiratory system, and urinary system. It is more advantageous in digestive system diseases such as early postoperative inflammatory bowel obstruction, functional dyspepsia, gastric pain, functional abdominal distension, and gastric reflux esophagitis. By comprehensively examining the key information of Houpu Qiwu Tang, this paper aims to provide literature support for the development and clinical application of this formula.
5.Exploration of the Application of Fengfu (GV 16) Acupoint in BIAN Que Heart Book (《扁鹊心书》)
Yawei ZHAO ; Haoying LI ; Lintong WEN ; Hefei WANG ; Wei WANG ; Hongyu WU ; Shijiang SUN
Journal of Traditional Chinese Medicine 2025;66(1):98-101
By examining the records related to the Fengfu (GV 16) acupoint in BIAN Que Heart Book (《扁鹊心书》) compiled by the Song Dynasty physician DOU Cai, this study analyzed various aspects, including the differentiation of conditions treated with Fengfu (GV 16) acupoint, the theoretical foundation for selection of Fengfu (GV 16) acupoint, the application of needling manipulation, and the sensation of obtaining qi during acupuncture. The findings suggest that DOU Cai's approach to utilizing Fengfu (GV 16) acupoint differs from traditional methods, particularly emphasizing the effectiveness of achieving a sensation of heat and numbness. His unique techniques include transverse insertion at Fengfu (GV 16) acupoint and penetrated insertion to Fengchi (GB 20) and Yifeng (TE 17) acupoints. The records of Fengfu (GV 16) acupoint in BIAN Que Heart Book provide a valuable reference for its modern clinical application and further development.
6.Investigation and analysis of drug use and pharmaceutical care in tight medical alliance in Wanzhou District of Chongqing
Suxin WAN ; Qiuyan SUN ; Caibing XU ; Li SHEN ; Hongmei GONG ; Wei FANG
China Pharmacy 2025;36(1):19-23
OBJECTIVE To investigate the use of drugs and the development of pharmaceutical care in the tight medical alliance (shorted for “medical alliance”) of Wanzhou District of Chongqing, and provide reference for the further construction of the medical alliance. METHODS A survey form was designed and distributed to 21 constituent units (5 leading units and 16 member units) of 5 medical alliances in Wanzhou District of Chongqing. The statistical analysis was conducted in aspects of basic drug allocation and use, pharmaceutical personnel team construction, the development of pharmaceutical care, and rational use of antibiotics. RESULTS Among the 21 constituent units, 4 leading units and 14 member units achieved the target for the proportion of essential drug procurement varieties, with a total compliance rate of 85.71%; 4 leading units and 13 member units achieved the target for the proportion of national essential drug allocation and usage amount, with a total compliance rate of 80.95%. The proportions of personnel with doctoral degrees in the 5 leading units and 16 member units were 1.71% and 0 respectively, and the proportions of personnel with senior professional titles were 8.56% and 1.63%, respectively. A total of 5 pharmacy or pharmaceutical combined outpatient clinics were set up in the 21 medical alliance units, and 5 clinical pharmacy information service platforms were established; all 5 leading units were able to regularly carry out clinical pharmacy projects, while only 4 out of 16 member units had conducted medical order review and evaluation. The proportions of irrational use of antibiotics in outpatient prescriptions and inpatient medical records of the 16 member units (4.81%, 5.21%) were significantly higher than those of the 5 leading units (2.80%, 4.00%). CONCLUSIONS The allocation and usage of national essential drugs in 21 constituent units from Wanzhou District of Chongqing are both in good standing. However, the data on the allocation of pharmaceutical professionals and the number, qualifications, and job titles of clinical pharmacists in member units are generally low. Moreover, the pharmaceutical service projects and service quality in member units need to be further improved.
7.Optimization of simmering technology of Rheum palmatum from Menghe Medical School and the changes of chemical components after processing
Jianglin XUE ; Yuxin LIU ; Pei ZHONG ; Chanming LIU ; Tulin LU ; Lin LI ; Xiaojing YAN ; Yueqin ZHU ; Feng HUA ; Wei HUANG
China Pharmacy 2025;36(1):44-50
OBJECTIVE To optimize the simmering technology of Rheum palmatum from Menghe Medical School and compare the difference of chemical components before and after processing. METHODS Using appearance score, the contents of gallic acid, 5-hydroxymethylfurfural (5-HMF), sennoside A+sennoside B, combined anthraquinone and free anthraquinone as indexes, analytic hierarchy process (AHP)-entropy weight method was used to calculate the comprehensive score of evaluation indicators; the orthogonal experiment was designed to optimize the processing technology of simmering R. palmatum with fire temperature, simmering time, paper layer number and paper wrapping time as factors; validation test was conducted. The changes in the contents of five anthraquinones (aloe-emodin, rhein, emodin, chrysophanol, physcion), five anthraquinone glycosides (barbaloin, rheinoside, rhubarb glycoside, emodin glycoside, and emodin methyl ether glycoside), two sennosides (sennoside A, sennoside B), gallic acid and 5-HMF were compared between simmered R. palmatum prepared by optimized technology and R. palmatum. RESULTS The optimal processing conditions of R. palmatum was as follows: each 80 g R. palmatum was wrapped with a layer of wet paper for 0.5 h, simmered on high heat for 20 min and then simmered at 140 ℃, the total simmering time was 2.5 h. The average comprehensive score of 3 validation tests was 94.10 (RSD<1.0%). After simmering, the contents of five anthraquinones and two sennosides were decreased significantly, while those of 5 free anthraquinones and gallic acid were increased to different extents; a new component 5-HMF was formed. CONCLUSIONS This study successfully optimizes the simmering technology of R. palmatum. There is a significant difference in the chemical components before and after processing, which can explain that simmering technology slows down the relase of R. palmatum and beneficiate it.
8.Effect and mechanism of compatibility of Astragali Radix-Puerariae Lobatae Radix on ferroptosis in T2DM insulin resistance rats
Shuang WEI ; Feng HAO ; Wenchun ZHANG ; Zhangyang ZHAO ; Ji LI ; Dongwei HAN ; Huan XING
China Pharmacy 2025;36(1):57-63
OBJECTIVE To explore the effect and potential mechanism of the compatibility of Astragali Radix-Puerariae Lobatae Radix on ferroptosis of liver cells in type 2 diabetes mellitus (T2DM) insulin resistance (IR) rats. METHODS Sixty male SD rats were randomly divided into control group (12 rats) and modeling group (48 rats). The modeling group was fed with a high- fat diet for 4 consecutive weeks and then given a one-time tail vein injection of 1% streptozotocin to establish T2DM IR model. The model rats were randomly divided into model group, the compatibility of Astragali Radix-Puerariae Lobatae Radix group [QG group, 4.05 g/(kg·d), intragastric administration], ferroptosis inhibitor ferrostatin-1 group [Fer-1 group, 5 mg/kg by intraperitoneal injection, once every other day], the compatibility of Astragali Radix-Puerariae Lobatae Radix+ferroptosis inducer erastin group [QG+erastin group, 4.05 g/(kg·d) by intragastric administration+erastin 10 mg/(kg·d), intraperitoneal injection]. After 4 weeks of intervention, serum fasting blood glucose (FBG) and fasting insulin (FINS) were measured in each group of rats, and homeostasis model assessment of insulin resistance (HOMA-IR) and the natural logarithm of insulin action index(IAI) were calculated; the serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), aspartate transaminase (AST) and alanine transaminase (ALT), Fe2+ and Fe content, glutathione (GSH), malondialdehyde (MDA) and superoxide dismutase (SOD) levels, NADP+/NADPH ratio and reactive oxygen species (ROS) were determined. The pathological morphology of its liver tissue was observed; the protein expressions of glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1), long-chain acyl-CoA synthetase 3 (ACSL3), ACSL4, ferritin mitochondrial (FTMT), and cystine/glutamate anti-porter (xCT) in the liver tissue of rats were detected. RESULTS Compared with control group, the liver cells in the model group of rats showed disordered arrangement, swelling, deepened nuclear staining, and more infiltration of inflammatory cells, as well as a large number of hepatocyte vacuoles and steatosis; FBG (after medication), the levels of TC, TG, LDL-C, AST, ALT, FINS, MDA and ROS, HOMA-IR, Fe2+ and Fe content, NADP+/NADPH ratio and protein expression of ACSL4 were significantly increased or up-regulated, while the levels of HDL-C, GSH and SOD, IAI, protein expressions of GPX4, FTH1, ACSL3, FTMT and xCT were significantly reduced or down-regulated (P<0.01). Compared with the model group, both QG group and Fer-1 group showed varying degrees of improvement in pathological damage of liver tissue and the levels of the above indicators, the differences in the changes of most indicators were statistically significant (P<0.01 or P<0.05). Compared with QG group, the improvement of the above indexes of QG+erastin group had been reversed significantly (P<0.01). CONCLUSIONS The compatibility decoction of Astragali Radix-Puerariae Lobatae Radix can reduce the level of FBG in T2DM IR rats, and alleviate IR degree, ion overload and pathological damage of liver tissue. The above effects are related to the inhibition of ferroptosis.
9.Effect and Mechanism of Wulingsan Decoction in Protecting Blood Brain Barrier and Ameliorating Cerebral Edema after Intracerebral Hemorrhage in Mice
Damei TAO ; Huihong LI ; Xiaoqing ZHENG ; Yunfei DENG ; Wei WEI ; Xiehua XUE
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(4):1-9
ObjectiveTo study the effect of Wulingsan on cerebral edema after intracerebral hemorrhage (ICH) in mice and explore the treatment mechanism. MethodsThe mouse model of ICH was established by injection of collagenase into the caudate nucleus. Mice were randomly assigned into the following groups: sham, ICH, intervention before modeling with low-dose and high-dose (3.69, 11.07 g·kg-1, respectively) Wulingsan, and intervention after modeling with high-dose Wulingsan. The modified neurological severity score (mNSS) was recorded, and the small animal MRI T2 sequential scanning was performed to measure the volume of cerebral hemorrhage after the modeling of ICH in each group. The Y-maze test, open field test, and Morris water maze test were conducted to evaluate the neurological behaviors of mice in each group. Hematoxylin-eosin staining was employed to observe the pathological changes in the brain tissue. Immunohistochemistry was employed to observe the expression of aquaporin 4 (AQP4), neuronal nuclei (NeuN), and glial fibrillary acidic protein (GFAP) in the brain tissue. Western blot was employed to determine the protein levels of AQP4, Claudin-5, and zonula occludens-1 (ZO-1) in the hematoma area. ResultsCompared with the sham group, the ICH group showed increases in the mNSS, the cerebral hemorrhage volume, and the escape latency in the Morris water maze test (P<0.01), decreases in the times of touching the platform and times of entering the quadrant where the platform was located in the Morris water maze test, and reductions in the spontaneous alternation rate in the Y-maze test and the ratio of distance of center travel to total travel distance in the open field test (P<0.01). Moreover, pathological changes such as cell disarrangement, cell space enlargement, and cell swelling were observed in the ICH group. Immunohistochemistry results showed that the ICH group had higher proportions of AQP4- and GFAP-positive cells and lower proportion of NeuN-positive cells than the sham group (P<0.01). Compared with the sham group, the ICH group showed an up-regulated protein level of AQP4 and down-regulated protein levels of Claudin-5 and ZO-1 (P<0.01). Compared with the ICH group, the intervention with Wulingsan decreased the mNSS, the volume of cerebral hemorrhage, and the escape latency in the Morris water maze test (P<0.05, P<0.01), while increasing the times of touching the platform and times of entering the quadrant where the platform was located in the Morris water maze test, the spontaneous alternation rate in the Y-maze test, and the ratio of distance of center travel to total travel distance in the open field test (P<0.05, P<0.01). Furthermore, the intervention with Wulingsan alleviated the pathological changes in the brain tissue after ICH, decreased the proportion of AQP4- and GFAP-positive cells (P<0.01), increased the proportion of NeuN-positive cells (P<0.01), down-regulated the protein level of AQP4 (P<0.01), and up-regulated the protein levels of Claudin-5 and ZO-1 (P<0.01). ConclusionThe intervention with Wulingsan could reduce the neural function score and the cerebral hemorrhage volume, up-regulate the expression of Claudin-5 and ZO-1, and down-regulate the expression of AQP4 to ameliorate the neurological function defect and cerebral edema after ICH, thereby protecting the brain.
10.Mechanism of Herbal Cake-separated Moxibustion in Improving Neuroimmune Inflammation in Rats with Chronic Fatigue Syndrome by Interfering TLR4/MyD88/NF-κB Pathway
Chuntao ZHAI ; Yawei HOU ; Linjuan SHI ; Yixiao WANG ; Wei LI ; Yuefeng TIAN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(4):140-149
ObjectiveTo explore the mechanism of herbal cake-separated moxibustion using the classical formula Xiaoyaosan in alleviating neuroimmune inflammatory responses in chronic fatigue syndrome (CFS) rats, based on the regulation of the toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear transcription factor-κB (NF-κB) signaling pathway. MethodsFifty SPF-grade SD rats aged 6-8 weeks were randomly divided into five groups: Normal group, model group, sham herbal cake moxibustion group, Chinese medicine intragastric administration group, and herbal cake-separated moxibustion group, with 10 rats in each group. Except for the normal group, all other groups underwent a 21-day modeling process, followed by behavioral testing. The herbal cake-separated moxibustion and sham herbal cake moxibustion groups received interventions at the Shenque (CV8), Guanyuan (CV4), Zusanli (ST36), and Qimen (LR14) acupoints. The Chinese medicine intragastric administration group was treated with a Xiaoyaosan suspension via gavage. Behavioral tests were conducted after 10 days of continuous intervention. Serum levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α), as well as hippocampal levels of IL-1β, IL-6, TNF-α, and NF-κB, were detected by enzyme-linked immunosorbent assay (ELISA). Morphological changes in the hippocampus were observed using hematoxylin-eosin (HE) staining. Real-time quantitative polymerase chain reaction (Real-time PCR) was used to detect mRNA expression levels of TLR4, MyD88, and NF-κB in the hippocampus. Western blot analysis was performed to detect the relative expression levels of TLR4, MyD88, NF-κB, and p65 proteins in the hippocampus. ResultsCompared with the normal group, the model group showed a significant decrease in upright times during the open field test (P<0.01), as well as significant reductions in total movement distance, resting time, and center region duration (P<0.01). In the tail suspension test, immobility time increased (P<0.01), and struggle times decreased (P<0.01). Serum and hippocampal levels of IL-1β, IL-6, and TNF-α, as well as hippocampal NF-κB levels and TLR4, MyD88, and NF-κB mRNA expression, were significantly elevated (P<0.01). After treatment, compared with the model group, the total movement distance and upright times in the open field test were significantly increased in all treatment groups (P<0.01), while resting time and center region duration were notably prolonged (P<0.05, P<0.01). Immobility time in the tail suspension test was significantly shortened (P<0.01), and struggle times significantly increased (P<0.05). Serum and hippocampal levels of IL-1β, IL-6, TNF-α, hippocampal NF-κB levels, and TLR4 and NF-κB mRNA expression were significantly reduced (P<0.05, P<0.01). Compared with the sham herbal cake moxibustion group, the herbal cake-separated moxibustion group showed a significant extension in center region duration during the open field test (P<0.05) and a significant increase in upright times (P<0.01). In the tail suspension test, immobility time was reduced (P<0.01), and struggle times increased (P<0.01). Serum TNF-α levels in the Chinese medicine intragastric administration group were significantly reduced (P<0.01), while serum IL-6 levels, as well as hippocampal levels of IL-1β, TNF-α, NF-κB, and TLR4, MyD88, and NF-κB mRNA expression, were significantly decreased in both the Chinese medicine intragastric administration group and the herbal cake-separated moxibustion group (P<0.05, P<0.01). Compared with the Chinese medicine intragastric administration group, the herbal cake-separated moxibustion group exhibited significantly increased upright times in the open field test (P<0.01). In the tail suspension test, immobility time was reduced (P<0.01), and struggle times increased (P<0.01). Serum IL-1β, hippocampal TNF-α levels, and TLR4, MyD88, and NF-κB mRNA expression were significantly decreased (P<0.05, P<0.01). ConclusionHerbal cake-separated moxibustion effectively improves fatigue and memory function in CFS rats, regulates neuroimmune inflammatory responses, and its mechanism may be related to the modulation of the TLR4/MyD88/NF-κB signaling pathway.

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