1.Regulatory effect of Jiedu Huayu granules on liver injury in mice with acute liver failure and its mechanism
Chengyu YA ; Tingshuai WANG ; Huiping YAN ; Yi WANG ; Qingrui ZHAO ; Shenglan ZENG ; Weiyu CHEN ; Rongzhen ZHANG
Journal of Clinical Hepatology 2026;42(1):143-150
ObjectiveTo investigate the mechanism of action of Jiedu Huayu granules in improving liver injury in mice with acute liver failure (ALF) by observing its effect on a mouse model of ALF after prophylactic administration, and to provide a basis for clinical medication. MethodsA total of 60 specific pathogen-free male C57BL/6J mice were divided into normal group, model group, Jiedu Huayu granules group (JDHY group), and farnesoid X receptor (FXR) agonist (GW4064) group using a random number table, with 15 mice in each group. The model of ALF was induced by a single intraperitoneal injection of D-galactosamine combined with lipopolysaccharide. The mice in the JDHY group were given prophylactic administration of 0.3 g/mL drug solution of Jiedu Huayu granules by gavage for 3 days before modeling, those in the normal group and the model group were given 0.9% NaCl solution by gavage, and those in the GW4064 group were given intraperitoneal injection of GW4064 for 3 consecutive days before modeling. The mice were sacrificed after modeling, and serum and liver tissue samples were collected. A veterinary automatic biochemical analyzer was used to measure the serum levels of total bilirubin (TBil), total bile acids (TBA), gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in mice from each group; HE staining was used to observe liver pathological changes; RT-PCR was used to measure the mRNA expression levels of FXR, fibroblast growth factor 15 (FGF15), fibroblast growth factor receptor 4 (FGFR4), small heterodimer partner (SHP), and bile salt export pump (BSEP) in mice, and Western blot was used to measure the protein expression levels of FXR, FGF15, FGFR4, SHP, and BSEP. A one-way analysis of variance was used for comparison between groups, and the Dunett method was used for further comparison between two groups. ResultsCompared with the normal group, the model group had significant increases in the serum levels of TBil, ALT, AST, TBA, and GGT (all P<0.01), and compared with the model group, the JDHY group and the GW4064 group had significant reductions in the serum levels of TBil, ALT, AST, TBA, and GGT (all P <0.01). HE staining showed that compared with the model group, the JDHY group and the GW4064 group had milder pathological injury, a reduction in the area of hepatocyte necrosis, and alleviation of cellular swelling and edema. Compared with the normal group, the model group had significant reductions in the mRNA and protein expression levels of FXR, FGF15, FGFR4, SHP, and BSEP in liver tissue (all P <0.01), and compared with the model group, the JDHY group and the GW4064 group had significant increases in the mRNA and protein expression levels of FXR, FGF15, FGFR4, SHP, and BSEP in liver tissue (all P <0.05). ConclusionJiedu Huayu granules may alleviate liver injury in mice with ALF through the FXR/SHP axis.
2.Zhuluan Decoction Ameliorates Premature Ovarian Insufficiency by Inhibiting Excessive Autophagy of KGN Through Regulation of PI3K/Akt/mTOR Pathway
Yao CHEN ; Sainan TIAN ; Jing ZENG ; Xingxing YI ; Wen'e LIU ; Lei LEI ; Li TANG
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(8):89-98
ObjectiveTo elucidate the underlying mechanism through which Zhuluan decoction suppresses excessive autophagy in human ovarian granulosa cells (KGN) and ameliorates premature ovarian insufficiency (POI) via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. MethodsThe optimal concentration of cyclophosphamide for inducing a POI model in KGN cells was identified via the cell counting kit-8 (CCK-8) assay. Subsequently, the impacts of varying concentrations of Zhuluan decoction-containing serum on the viability of the KGN cell model were assessed. After the optimal drug concentration was determined, KGN cells were categorized into the following groups: blank control (20% blank serum), model (20% blank serum + 5 μmol·L-1 cyclophosphamide), Zhuluan decoction-containing serum (20% Zhuluan decoction-containing serum + 5 μmol·L-1 cyclophosphamide), autophagy inhibitor (20% blank serum + 5 μmol·L-1 cyclophosphamide + 20 μmol·L-1 chloroquine phosphate), autophagy inhibitor + Zhuluan decoction-containing serum (20% Zhuluan decoction-containing serum + 5 μmol·L-1 cyclophosphamide + 20 μmol·L-1 chloroquine phosphate), and estradiol valerate (20% estradiol valerate-containing serum + 5 μmol·L-1 cyclophosphamide). Following 48 hours of incubation, flow cytometry was utilized to measure the apoptosis rate of KGN cells in each group. Western blotting was employed to quantify the protein levels of PI3K, phosphorylated (p)-Akt, Akt, p-mTOR, and mTOR, along with the expression levels of autophagy-related proteins such as Beclin1, autophagy-related 5 homolog (ATG5), and microtubule-associated protein 1 light chain 3 (LC3), in each group. Additionally, monodansylcadaverine (MDC) staining was performed to evaluate the extent of autophagy in each group. ResultsIncubation of KGN cells with 5 μmol·L-1 cyclophosphamide for 48 h successfully established a POI model, marked by a significant inhibition of KGN cell proliferation. Notably, the inhibitory effect of cyclophosphamide on KGN cell proliferation exhibited a positive correlation with its concentration. Zhuluan decoction-containing serum at 20% and 30% promoted cell proliferation and mitigated the inhibitory effect of cyclophosphamide on KGN cell proliferation, with comparable therapeutic efficacy observed at both concentrations. Compared with the blank control group, the model group displayed an elevated apoptosis rate (P<0.01), reduced protein levels of PI3K, p-Akt, and p-mTOR (P<0.01), increased protein levels of Beclin1, LC3, and ATG5 (P<0.01), no significant alterations in the protein levels of Akt and mTOR, and an enhanced MDC autophagy fluorescence intensity (P<0.01). In comparison to that the model group, the apoptosis rates in the blank control group, model group, Zhuluan decoction-containing serum group, autophagy inhibitor group, autophagy inhibitor + Zhuluan decoction-containing serum group, and estradiol valerate group all reduced (P<0.05, P<0.01), with the most pronounced reduction observed in the autophagy inhibitor + Zhuluan decoction-containing serum group. The protein levels of PI3K, p-Akt, and p-mTOR were higher in other groups than in the model group (P<0.05, P<0.01), being the highest in the autophagy inhibitor + Zhuluan decoctio-containing serum group (P<0.01). The protein levels of Beclin1 and ATG5 were lower in other groups than in the model group (P<0.05, P<0.01). The expression level of LC3 declined in the Zhuluan decoction-containing serum group and the estradiol valerate group (P<0.05, P<0.01), while it decreased without statistical significance in the autophagy inhibitor group and the autophagy inhibitor + Zhuluan decoction-containing serum group. ConclusionZhuluan decoction may activate the PI3K/Akt/mTOR pathway to inhibit excessive autophagy and counteract the detrimental effects of cyclophosphamide on the KGN cell model, thus managing POI.
3.Zhuluan Decoction Ameliorates Premature Ovarian Insufficiency by Inhibiting Excessive Autophagy of KGN Through Regulation of PI3K/Akt/mTOR Pathway
Yao CHEN ; Sainan TIAN ; Jing ZENG ; Xingxing YI ; Wen'e LIU ; Lei LEI ; Li TANG
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(8):89-98
ObjectiveTo elucidate the underlying mechanism through which Zhuluan decoction suppresses excessive autophagy in human ovarian granulosa cells (KGN) and ameliorates premature ovarian insufficiency (POI) via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway. MethodsThe optimal concentration of cyclophosphamide for inducing a POI model in KGN cells was identified via the cell counting kit-8 (CCK-8) assay. Subsequently, the impacts of varying concentrations of Zhuluan decoction-containing serum on the viability of the KGN cell model were assessed. After the optimal drug concentration was determined, KGN cells were categorized into the following groups: blank control (20% blank serum), model (20% blank serum + 5 μmol·L-1 cyclophosphamide), Zhuluan decoction-containing serum (20% Zhuluan decoction-containing serum + 5 μmol·L-1 cyclophosphamide), autophagy inhibitor (20% blank serum + 5 μmol·L-1 cyclophosphamide + 20 μmol·L-1 chloroquine phosphate), autophagy inhibitor + Zhuluan decoction-containing serum (20% Zhuluan decoction-containing serum + 5 μmol·L-1 cyclophosphamide + 20 μmol·L-1 chloroquine phosphate), and estradiol valerate (20% estradiol valerate-containing serum + 5 μmol·L-1 cyclophosphamide). Following 48 hours of incubation, flow cytometry was utilized to measure the apoptosis rate of KGN cells in each group. Western blotting was employed to quantify the protein levels of PI3K, phosphorylated (p)-Akt, Akt, p-mTOR, and mTOR, along with the expression levels of autophagy-related proteins such as Beclin1, autophagy-related 5 homolog (ATG5), and microtubule-associated protein 1 light chain 3 (LC3), in each group. Additionally, monodansylcadaverine (MDC) staining was performed to evaluate the extent of autophagy in each group. ResultsIncubation of KGN cells with 5 μmol·L-1 cyclophosphamide for 48 h successfully established a POI model, marked by a significant inhibition of KGN cell proliferation. Notably, the inhibitory effect of cyclophosphamide on KGN cell proliferation exhibited a positive correlation with its concentration. Zhuluan decoction-containing serum at 20% and 30% promoted cell proliferation and mitigated the inhibitory effect of cyclophosphamide on KGN cell proliferation, with comparable therapeutic efficacy observed at both concentrations. Compared with the blank control group, the model group displayed an elevated apoptosis rate (P<0.01), reduced protein levels of PI3K, p-Akt, and p-mTOR (P<0.01), increased protein levels of Beclin1, LC3, and ATG5 (P<0.01), no significant alterations in the protein levels of Akt and mTOR, and an enhanced MDC autophagy fluorescence intensity (P<0.01). In comparison to that the model group, the apoptosis rates in the blank control group, model group, Zhuluan decoction-containing serum group, autophagy inhibitor group, autophagy inhibitor + Zhuluan decoction-containing serum group, and estradiol valerate group all reduced (P<0.05, P<0.01), with the most pronounced reduction observed in the autophagy inhibitor + Zhuluan decoction-containing serum group. The protein levels of PI3K, p-Akt, and p-mTOR were higher in other groups than in the model group (P<0.05, P<0.01), being the highest in the autophagy inhibitor + Zhuluan decoctio-containing serum group (P<0.01). The protein levels of Beclin1 and ATG5 were lower in other groups than in the model group (P<0.05, P<0.01). The expression level of LC3 declined in the Zhuluan decoction-containing serum group and the estradiol valerate group (P<0.05, P<0.01), while it decreased without statistical significance in the autophagy inhibitor group and the autophagy inhibitor + Zhuluan decoction-containing serum group. ConclusionZhuluan decoction may activate the PI3K/Akt/mTOR pathway to inhibit excessive autophagy and counteract the detrimental effects of cyclophosphamide on the KGN cell model, thus managing POI.
4.Concept, design and clinical application of minimally invasive liver transplantation through laparoscopic combined upper midline incision
Shuhong YI ; Hui TANG ; Kaining ZENG ; Xiao FENG ; Binsheng FU ; Qing YANG ; Jia YAO ; Yang YANG ; Guihua CHEN
Organ Transplantation 2025;16(1):67-73
Objective To explore the technical process and clinical application of laparoscopic combined upper midline incision minimally invasive liver transplantation. Methods A retrospective analysis was conducted on 30 cases of laparoscopic combined upper midline incision minimally invasive liver transplantation. The cases were divided into cirrhosis group (15 cases) and liver failure group (15 cases) based on the primary disease. The surgical and postoperative conditions of the two groups were compared. Results All patients successfully underwent laparoscopic "clockwise" liver resection, with no cases of passive conversion to open surgery or intolerance to pneumoperitoneum. In 6 cases, the right lobe was relatively large, and the right hepatic ligaments could not be completely mobilized. One case required an additional reverse "L" incision during open surgery. All patients successfully completed the liver transplantation, with no major intraoperative bleeding, cardiovascular events, or other occurrences in the 30 patients. The model for end-stage liver disease (MELD) score in the cirrhosis group was lower than that in the liver failure group (P<0.001). There were no statistically significant differences between the two groups in terms of age, surgical time, blood loss, anhepatic phase, or cold ischemia time (all P>0.05). During the perioperative period, there was 1 case of hepatic artery embolism, 1 case of portal vein anastomotic stenosis, no complications of hepatic vein and inferior vena cava, and 3 cases of biliary anastomotic stenosis, all of which occurred in the liver failure group. Conclusions In strictly selected cases, the minimally invasive liver transplantation technique combining laparoscopic hepatectomy with upper midline incision for graft implantation has the advantages of smaller incisions, less bleeding, relatively easier operation, and faster postoperative recovery, which is worthy of clinical promotion and application.
5.Research progress of meibomian gland dysfunction-related dry eye
Jianbo ZHONG ; Guoqiang ZENG ; Yi ZHANG ; Xiaoyan DOU ; Wanmei TANG ; Kunling CHEN ; Li CAI
International Eye Science 2025;25(2):259-263
In recent years, with the endless emergence of meibomian gland dysfunction(MGD)diagnostic equipment, rich treatment methods, and in-depth clinical and basic research on MGD at home and abroad, the understanding of MGD has entered a new stage. MGD-related dry eye is considered to be the main cause of lipid abnormal dry eye, and its occurrence and development is a chronic and multi-factorial pathological process. This article reviews the pathogenesis, imaging analysis and clinical treatment progress of MGD-related dry eye, in order to provide scientific evidence and ideas for clinical diagnosis and therapy of MGD-related dry eye.
6.Treating premature ejaculation combined with anxiety and depression based on the "four-dimensional integration" of the "holism of body and spirit" theory
Yi WEI ; Zhiming HONG ; Junfeng QIU ; Zilong CHEN ; Hao KUANG ; Yangling ZENG ; Quan WANG ; Wenbin ZHOU
Journal of Beijing University of Traditional Chinese Medicine 2025;48(3):418-423
Premature ejaculation refers to a sexual dysfunction in which men experience a short intravaginal ejaculation latency and a lack of control over ejaculation during sexual activity. The onset of this condition is often accompanied by anxiety and depression, which can seriously affect the quality of the patient′s sexual life and the relationship between partners. Based on the "integration of body and spirit" theory in traditional Chinese medicine, our team believes that this condition is a comorbidity of physical and spiritual factors. We propose that the core pathogenesis of this disease lies in the "loss of form and essence, impairment of spirit, and depression of the mind, "while the primary treatment principle involves "nourishing form and regulating spirit." As a result, a new diagnosis and treatment approach of "four-dimensional integration" is summarized in this study. The disease is treated through the four dimensions of shape, body, spirit, and emotion. Traditional Chinese medicine is used to adjust the shape in cases where the physical form is damaged. For individuals with depression of heart and liver qi, the treatment focuses on soothing the heart and smoothing liver qi, and the modified Wangyou Powder and Xuanzhi Decoction is used. In cases where the heart and kidney function are compromised, the treatment involves nourishing both the heart and kidney while restoring interaction between the heart and the kidney, and modified Jihuo Yansi Elixir is used. To reduce the sensitivity of the glans penis, the patient′s body is washed with a traditional Chinese medicine formula, and a delicate fumigation formula is decocted for external washing. For those who are not in tune with their god, psychological counseling can be used to regulate their spirit and advocate "self-partner" and psychotherapy. If there are issues with intimacy, partners should focus on cooperating during foreplay, sexual intercourse, and post-coital interactions. Overall, the treatment aims to harmonize the body and spirit, addressing both physical and psychological factors through a comprehensive, multi-dimensional approach. This method provides new perspectives and ideas for the clinical diagnosis and treatment of this condition.
7.Mechanism of Guben Jiannao Liquid on Alzheimer's disease by regulating autophagy based on LKB1/AMPK/mTOR pathway.
Jing-Fan ZHANG ; Qing-Hua LONG ; Chu-Hua ZENG ; Yi-Min CHEN ; Zhe-Yao XIE ; Yuan-Qin CAI ; Xi WANG
China Journal of Chinese Materia Medica 2025;50(2):293-300
This study explores the mechanism of Guben Jiannao Liquid on Alzheimer's disease(AD) by regulating autophagy based on the liver kinase B1(LKB1)/adenosine monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR) pathway. Male SD rats were randomly divided into the blank group, model group, low-dose and high-dose groups of Guben Jiannao Liquid, and rapamycin group, with 10 rats in each group. Except for the blank group, all other groups of rats were injected bilaterally in the hippocampus with β-amyloid(Aβ)_(1-42) to establish the AD model. The low-dose(6.21 g·kg~(-1)) and high-dose(12.42 g·kg~(-1)) groups of Guben Jiannao Liquid and rapamycin group(1 mg·kg~(-1)) were given the corresponding drugs by gavage, and the blank and model groups were given an equal volume of saline by gavage for four weeks. Morris water maze was used to test the learning and memory ability of rats in each group; hematoxylin-eosin(HE) and Nissl staining were used to observe the morphological and quantitative changes of neurons and Nissl bodies in the CA1 region of rat hippocampus; immunohistochemistry was utilized to detect Aβ-positive cell expression in the CA1 region of rat hippocampus; transmission electron microscopy was employed to observe ultrastructural changes in rat hippocampal tissue, and Western blot was used to examine the protein expression levels of LKB1, p-AMPK/AMPK, p-mTOR/mTOR, Beclin1, p62, and LC3-Ⅱ in the hippocampal tissue of the rats. The results showed that compared with those in the blank group, rats in the model group had elevated evasion latency and decreased number of platform transversal and residence time in the platform quadrant. The number of neurons in the hippocampal area was reduced, and the morphology was impaired. The average integral optical density value of Aβ-positive cells was elevated; the expression levels of LKB1, p-AMPK/AMPK, Beclin1, and LC3-Ⅱ were decreased, and the expression levels of p-mTOR/mTOR and p62 were increased. Compared with those in the model group, rats in the low-dose and high-dose groups of Guben Jiannao Liquid had shorter evasion latency, higher number of platform transversal, longer residence time in the platform quadrant, increased number of neurons, decreased expression of Aβ-positive cells and average integral optical density values, and increased number of autophagic lysosomes in hippocampal tissue. The expression levels of LKB1, Beclin1, and LC3-Ⅱ were elevated in the hippocampus of rats in the low-dose group of Guben Jiannao Liquid. The expression levels of LKB1, p-AMPK/AMPK, Beclin1, and LC3-Ⅱ were elevated in the hippocampal tissue of rats in the high-dose group of Guben Jiannao Liquid, and the expression levels of p-mTOR/mTOR and p62 were decreased. The findings suggest that Guben Jiannao Liquid can improve cognitive impairment in AD rats, and its mechanism of action may be related to the activation of the LKB1/AMPK/mTOR signaling pathway and the up-regulation of autophagy level.
Animals
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Alzheimer Disease/physiopathology*
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Male
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TOR Serine-Threonine Kinases/genetics*
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Autophagy/drug effects*
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Rats, Sprague-Dawley
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Protein Serine-Threonine Kinases/genetics*
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AMP-Activated Protein Kinases/genetics*
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Rats
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Drugs, Chinese Herbal/administration & dosage*
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Signal Transduction/drug effects*
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AMP-Activated Protein Kinase Kinases
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Humans
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Hippocampus/metabolism*
8.Role of miR-140-5p/BCL2L1 in apoptosis and autophagy of HFOB1.19 and effect of Bushen Jianpi Huoxue Decoction.
Tong-Ying CHEN ; Sai FU ; Xiao-Yun LI ; Shu-Hua LIU ; Yi-Fu YANG ; Dong-Sheng YANG ; Yun-Jie ZENG ; Yang-Bo LI ; Dan LUO ; Hong-Xing HUANG ; Lei WAN
China Journal of Chinese Materia Medica 2025;50(3):583-589
Osteoporosis(OP) is a senile bone disease characterized by an imbalance between bone remodeling and bone formation. Targeting pathogenesis of kidney deficiency, spleen deficiency, and blood stasis, Bushen Jianpi Huoxue Decoction has a significant effect on the treatment of OP by tonifying kidney, invigorating spleen, and activating blood circulation. MicroRNA(miRNA) and the anti-apoptotic protein B-cell lymphoma-2-like protein 1(BCL2L1) are closely related to bone cell metabolism. Therefore, in this study, the binding of miR-140-5p to BCL2L1 was detected by dual luciferase assay and polymerase chain reaction(PCR). After silencing or overexpressing miR-140-5p, the apoptosis, autophagy, and osteogenic function of human fetal osteoblast cell line 1.19(HFOB1.19) were observed by flow cytometry and Western blot. Bushen Jianpi Huoxue Decoction-containing serum was prepared by intragastric administration of Bushen Jianpi Huoxue Decoction in rats. Different concentrations of Bushen Jianpi Huoxue Decoction-containing serum were used to treat HFOB1.19 with or without miR-140-5p mimic. The expression of osteogenic proteins in each group was observed, and the role of miR-140-5p/BCL2L1 in apoptosis and autophagy of HFOB1.19 was studied, along with the effect of Bushen Jianpi Huoxue Decoction on these processes. As indicated by the dual luciferase assay, miR-140-5p bound to BCL2L1. Flow cytometry and Western blot showed that miR-140-5p promoted apoptosis and inhibited autophagy in HFOB1.19. After intervention with high, medium, and low doses of Bushen Jianpi Huoxue Decoction-medicated serum, compared with the miR-140-5p NC group, the expression of osteocalcin(OCN), osteopontin(OPN), Runt-related transcription factor 2(RUNX2), and transforming growth factor beta 1(TGF-β1) decreased in the miR-140-5p mimic group, while the expression of bone morphogenetic protein 2(BMP2) showed no significant difference under high-dose intervention. Therefore, miR-140-5p/BCL2L1 can promote apoptosis and inhibit autophagy in HFOB1.19. Bushen Jianpi Huoxue Decoction can affect the osteogenic effect of miR-140-5p through BMP2.
MicroRNAs/metabolism*
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Autophagy/drug effects*
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Apoptosis/drug effects*
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Humans
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Drugs, Chinese Herbal/administration & dosage*
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Animals
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Cell Line
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bcl-X Protein/metabolism*
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Osteoblasts/metabolism*
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Rats
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Osteoporosis/physiopathology*
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Male
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Rats, Sprague-Dawley
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Osteogenesis/drug effects*
9.Traditional Chinese medicine dry powder inhalers: research status and development ideas and methods.
Yu-Wen MA ; Yi-Chen ZENG ; Hao-Ran WANG ; Guang-Fu LIU ; Jun JIANG ; Yu-Song ZENG ; Bai-Xiu ZHAO ; Jin FANG
China Journal of Chinese Materia Medica 2025;50(3):620-631
As an innovative dosage form, traditional Chinese medicine(TCM) dry powder inhalers have emerged as a focal point in the research and development of new preparations due to its high efficiency, safety, and bioavailability. This paper systematically reviewed the relevant literature and patents associated with TCM dry powder inhalers to analyze the origins and the current research and development status. Furthermore, this paper probed into the research and development ideas of TCM dry powder inhalers regarding clinical positioning, prescription screening, and druggability. Additionally, the paper thoroughly analyzed the technical barriers in druggability studies and elaborated on corresponding research techniques and coping measures. Furthermore, it emphasized the need for improved regulations and policies governing TCM dry powder inhalers, advocated for strengthened oversight, and called for the establishment of a scientific quality evaluation system. Measures such as promoting production-education-research collaboration, enhancing personnel training, and fostering international exchanges were proposed to provide a scientific and systematic reference for the future research, development, and application of TCM dry powder inhalers, thereby facilitating the rapid modernization of TCM.
Humans
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Dry Powder Inhalers/trends*
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Drugs, Chinese Herbal/chemistry*
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Medicine, Chinese Traditional/instrumentation*
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Administration, Inhalation
10.Buyang Huanwu Decoction promotes angiogenesis after oxygen-glucose deprivation/reoxygenation injury of bEnd.3 cells by regulating YAP1/HIF-1α signaling pathway via caveolin-1.
Bo-Wei CHEN ; Yin OUYANG ; Fan-Zuo ZENG ; Ying-Fei LIU ; Feng-Ming TIAN ; Ya-Qian XU ; Jian YI ; Bai-Yan LIU
China Journal of Chinese Materia Medica 2025;50(14):3847-3856
This study aims to explore the mechanism of Buyang Huanwu Decoction(BHD) in promoting angiogenesis after oxygen-glucose deprivation/reoxygenation(OGD/R) of mouse brain microvascular endothelial cell line(brain-derived Endothelial cells.3, bEnd.3) based on the caveolin-1(Cav1)/Yes-associated protein 1(YAP1)/hypoxia-inducible factor-1α(HIF-1α) signaling pathway. Ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was used to analyze the blood components of BHD. The cell counting kit-8(CCK-8) method was used to detect the optimal intervention concentration of drug-containing serum of BHD after OGD/R injury of bEnd.3. The lentiviral transfection method was used to construct a Cav1 silent stable strain, and Western blot and polymerase chain reaction(PCR) methods were used to verify the silencing efficiency. The control bEnd.3 cells were divided into a normal group(sh-NC control group), an OGD/R model + blank serum group(sh-NC OGD/R group), and an OGD/R model + drug-containing serum group(sh-NC BHD group). Cav1 silent cells were divided into an OGD/R model + blank serum group(sh-Cav1 OGD/R group) and an OGD/R model + drug-containing serum group(sh-Cav1 BHD group). The cell survival rate was detected by the CCK-8 method. The cell migration ability was detected by a cell migration assay. The lumen formation ability was detected by an angiogenesis assay. The apoptosis rate was detected by flow cytometry, and the expression of YAP1/HIF-1α signaling pathway-related proteins in each group was detected by Western blot. Finally, co-immunoprecipitation was used to verify the interaction between YAP1 and HIF-1α. The results showed astragaloside Ⅳ, formononetin, ferulic acid, and albiflorin in BHD can all enter the blood. The drug-containing serum of BHD at a mass fraction of 10% may be the optimal intervention concentration for OGD/R-induced injury of bEnd.3 cells. Compared with the sh-NC control group, the sh-NC OGD/R group showed significantly decreased cell survival rate, cell migration rate, mesh number, node number, and lumen length, significantly increased cell apoptotic rate, significantly lowered phosphorylation level of YAP1 at S127 site, and significantly elevated nuclear displacement level of YAP1 and protein expression of HIF-1α, vascular endothelial growth factor(VEGF), and vascular endothelial growth factor receptor 2(VEGFR2). Compared with the same type of OGD/R group, the sh-NC BHD group and sh-Cav1 BHD group had significantly increased cell survival rate, cell migration rate, mesh number, node number, and lumen length, a significantly decreased cell apoptotic rate, a further decreased phosphorylation level of YAP1 at S127 site, and significantly increased nuclear displacement level of YAP1 and protein expression of HIF-1α, VEGF, and VEGFR2. Compared with the sh-NC OGD/R group, the sh-Cav1 OGD/R group exhibited significantly decreased cell survival rate, cell migration rate, mesh number, node number, and lumen length, a significantly increased cell apoptotic rate, a significantly increased phosphorylation level of YAP1 at S127 site, and significantly decreased nuclear displacement level of YAP1 and protein expression of HIF-1α, VEGF, and VEGFR2. Compared with the sh-NC BHD group, the sh-Cav1 BHD group showed significantly decreased cell survival rate, cell migration rate, mesh number, node number, and lumen length, a significantly increased cell apoptotic rate, a significantly increased phosphorylation level of YAP1 at the S127 site, and significantly decreased nuclear displacement level of YAP1 and protein expression of HIF-1α, VEGF, and VEGFR2. YAP1 protein was present in the protein complex precipitated by the HIF-1α antibody, and HIF-1α protein was also present in the protein complex precipitated by the YAP1 antibody. The results confirmed that the drug-containing serum of BHD can increase the activity of YAP1/HIF-1α pathway in bEnd.3 cells damaged by OGD/R through Cav1 and promote angiogenesis in vitro.
Drugs, Chinese Herbal/pharmacology*
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Animals
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Mice
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Signal Transduction/drug effects*
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Glucose/metabolism*
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Caveolin 1/genetics*
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Hypoxia-Inducible Factor 1, alpha Subunit/genetics*
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YAP-Signaling Proteins
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Oxygen/metabolism*
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Endothelial Cells/metabolism*
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Cell Line
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Adaptor Proteins, Signal Transducing/genetics*
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Neovascularization, Physiologic/drug effects*
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Cell Hypoxia/drug effects*
;
Angiogenesis


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