1.Mechanism of Huayu Jiedu Prescription in Preventing and Treating Cerebral Ischemia Injury by Regulating NETosis After Acute Cerebral Infarction with Blood Stasis and Toxin Syndrome
Wuchaonan LIU ; Dingxiang LI ; Le YANG ; Jing LIU ; Shengping LUO ; Fang LEI ; Hanlin LEI ; Yihui DENG
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(4):50-59
ObjectiveTo investigate the material basis of the pathogenesis of cerebral ischemic injury with blood stasis and toxin syndrome and to explore the protective effects of Huayu Jiedu prescription (HYJDP) on neutrophil extracellular trap-related cell death (NETosis) in cerebral ischemic injury following acute cerebral infarction. MethodsSeventy-two Sprague-Dawley (SD) rats were randomly divided into six groups (n=12 per group): sham operation (Sham) group, blood stasis and toxin model (Model) group, low-, medium-, and high-dose HYJDP groups (HYJDP-L, HYJDP-M, and HYJDP-H; 9, 18, and 36 g·kg-1, respectively), and butylphthalide (NBP) group (0.06 g·kg-1). Except for the Sham group, rats in all other groups were subjected to carrageenan/dry yeast combined with a modified intraluminal filament method to establish a focal cerebral ischemia model of the middle cerebral artery with blood stasis and toxin syndrome. Neurological function was evaluated at 24 h after modeling using the Zea-Longa neurological deficit score. Cerebral infarction rate was assessed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Pathological morphology of brain tissue was observed using hematoxylin-eosin (HE) staining. Enzyme-linked immunosorbent assay (ELISA) was used to determine serum levels of interleukin-8 (IL-8), myeloperoxidase-DNA complexes (MPO-DNA), and citrullinated histone H3 (CitH3). Protein expression of phosphorylated phosphatidylinositol 3-kinase (p-PI3K), protein kinase B (p-Akt), mammalian target of rapamycin (p-mTOR), sequestosome 1 (p62), and CitH3 in brain tissue was detected by Western blot. Immunofluorescence (IF) was used to detect the expression of neutrophil-specific marker Ly6G, CitH3, and neuron-specific nuclear protein (NeuN) in brain tissue. ResultsCompared with the Sham group, neurological deficit scores and cerebral infarction rates in the model group were significantly increased (P<0.01 for both). HE staining showed varying degrees of neuronal degeneration and necrosis, characterized by blurred neuronal structures, nuclear pyknosis and fragmentation, cytoplasmic dissolution into a vacuolated reticular pattern, and mild glial cell proliferation. ELISA results showed that serum levels of IL-8, MPO-DNA, and CitH3 were significantly increased (P<0.01). Western blot analysis demonstrated decreased expression of p-PI3K, p-Akt, p-mTOR, and p62, while CitH3 expression was significantly increased (P<0.01). IF results showed an increased number of NETs+ cells and a significant decrease in NeuN+ cells (P<0.01). Compared with the Model group, neurological deficit scores in the HYJDP-H group were significantly decreased (P<0.05), and cerebral infarction rates in the HYJDP-H and NBP groups were significantly reduced (P<0.01). HE staining showed that brain tissue damage was markedly alleviated in the HYJDP-H group. ELISA results showed that levels of IL-8, MPO-DNA, and CitH3 were significantly decreased in the HYJDP-M, HYJDP-H, and NBP groups (P<0.01). Western blot analysis showed that expression of p-PI3K, p-Akt, p-mTOR, and p62 was significantly increased in the HYJDP-H and NBP groups, while CitH3 expression was significantly reduced in all drug intervention groups (P<0.01). IF results showed that the number of NETs+ cells was significantly decreased and the number of NeuN⁺ cells was significantly increased in all drug intervention groups (P<0.01). ConclusionNETs may be the material basis of the pathogenesis of cerebral ischemic injury characterized by blood stasis and toxin. HYJDP can regulate the PI3K/Akt/mTOR signaling pathway, reduce the release of pro-inflammatory mediators and NETosis-related products, alleviate cerebral ischemic injury caused by autophagy-dependent NETosis, and thereby exert a neuroprotective effect.
2.Perioperative immune dynamics and clinical outcomes in patients undergoing on-pump cardiac surgery
Zhiyuan CHENG ; Xinyi LIAO ; Juan WU ; Ping YANG ; Tingting WANG ; Qinjuan WU ; Wentong MENG ; Zongcheng TANG ; Jiayi SUN ; Jia TAN ; Jing LIN ; Dan LUO ; Hao WANG ; Chaonan LIU ; Jiyue XIONG ; Liqin LING ; Jing ZHOU ; Lei DU
Chinese Journal of Blood Transfusion 2026;39(1):31-43
Objective: To characterize perioperative dynamic changes in immune-cell phenotypes and inflammatory cytokines in patients undergoing CPB (cardiopulmonary bypass) cardiac surgery, and to explore their associations with postoperative outcomes. Methods: In this prospective cohort study, 120 adult patients who underwent elective cardiac surgery under CPB at West China Hospital from May 2022 to March 2023 were enrolled. Perioperative immune-cell phenotypes and concentrations of 40 inflammation-related cytokines were measured. The primary outcomes were the sequential organ failure assessment (SOFA) score at 24 h after surgery and ΔSOFA (the peak SOFA score within 48 h after surgery minus the preoperative SOFA score). Secondary outcomes included major adverse cardiovascular events (MACE), acute kidney injury (AKI), respiratory failure, severe liver injury, and infection. Results: The mean age of enrolled patients was 57±10 years. Of these, 52% (62/120) were male and 90% (108/120) underwent valve surgery. During the rewarming to the end of CPB, neutrophil counts rapidly increased (7.39×10
/L vs preoperative 3.07×10
/L, P<0.001), with significant upregulation of CD11b (7.30×10
/L vs preoperative 3.05×10
/L, P<0.001) and CD54 (7.15×10
/L vs preoperative 2.99×10
/L, P<0.001). Lymphocyte counts increased at the end of CPB (1.75×10
/L vs preoperative 1.12×10
/L, P<0.001) but decreased significantly at 24 h after surgery (0.59×10
/L vs preoperative 1.12×10
/L, P<0.001). Plasma analysis showed that multiple pro-inflammatory cytokines increased during CPB and remained elevated up to 24 h after surgery; five chemokines and the anti-inflammatory cytokine IL-10 peaked at the end of CPB. The SOFA score increased from 1 (1, 2) preoperatively to 7 (5, 10) at 24 h after surgery, with a ΔSOFA of 6 (4, 8). Within 30 days after surgery, 48 patients (40.0%) developed AKI, 17 (14.2%) developed infection, 4 (3.3%) developed severe liver injury, 3 (2.5%) developed respiratory failure, and 3 (2.5%) experienced MACE. During the 2-year follow-up, 8 patients (6.7%) experienced MACE and 5 (4.2%) died. Conclusion: Multi-organ dysfunction is common after cardiac surgery under CPB (median ΔSOFA, 6), accompanied by perioperative activation of multiple immune-cell subsets and upregulation of pro-inflammatory, anti-inflammatory, and chemotactic mediators. This study provides data-driven evidence and research clues for further investigation of the associations between CPB-related immune perturbations and postoperative organ dysfunction and clinical outcomes.
3.Early warning of influenza epidemic based on CUSUM and EWMA models in Daxing District, Beijing
Hong LEI ; Qiuling LI ; Qi LIU ; Meichen LIU ; Enhuan DU ; Jinfeng TANG ; Zhiping LI ; Yadi GAN ; Lijie ZHANG
Journal of Public Health and Preventive Medicine 2026;37(1):13-17
Objective To explore the effectiveness of the cumulative sum (CUSUM) and the exponentially weighted moving average (EWMA) for early warning of influenza epidemic using two datasets of reported influenza cases and influenza-like illness (ILI) cases. Methods Using the reported cases of influenza and ILI in Daxing District, Beijing, from week 23 of 2018 to week 22 of 2024 as data sets, the CUSUM and EWMA models were established, respectively. The positive rate of influenza etiology was used as the “gold standard”, and the Youden index was used as the evaluation index to compare the early warning effect of the two models under different data sets and different parameters. Results In CUSUM, the optimal Youden indices of the reported influenza cases set and the ILI cases set were 0.751 and 0.635, respectively. In EWMA, the optimal Youden indices of the reported influenza cases set and the ILI cases set were 0.544 and 0.464, respectively. The optimal EWMA and CUSUM models could both issue early warning signals in advance of the “gold standard”. Conclusion In the influenza epidemic early warning in Daxing District, Beijing, the CUSUM model established with the reported cases of influenza can achieve good early warning effects, but the model parameters need to be dynamically adjusted according to the local epidemic characteristics.
4.Investigation of somatization symptoms and related factors in adolescents during frequent earthquakes in Hefei
Yu ZHUANG ; Pei TANG ; Yinghan TIAN ; Peng YAO ; Lei XIA ; Huanzhong LIU
Acta Universitatis Medicinalis Anhui 2026;61(1):141-145
ObjectiveTo investigate somatization symptoms in adolescents during frequent earthquakes in Hefei, and to explore their correlation with earthquake experiences. MethodsA cross-sectional survey was used to select 324 adolescents in Hefei as the survey objects. The self-rating scale of somatization symptoms (SSS) and the fatigue intensity scale (FIS) were used to evaluate the somatization symptoms and fatigue degree of middle school students, and multivariate Logistic regression analysis was used to explore the related factors of somatization symptoms and fatigue among middle school students. ResultsA total of 324 adolescents were included, and the overall detection rate of somatization symptoms was 6.5%, and the detection rate of moderate or above fatigue was 20.1%. The results of regression analysis showed that adolescents who were concerned about the earthquake for a longer time (≥1 h) had a higher risk of somatization symptoms (OR=5.430, 95%CI: 1.547-19.058), and adolescents who received pre-earthquake training had a lower degree of fatigue (OR=0.535, 95%CI: 0.292-0.981) (P<0.05). ConclusionDuring the frequent earthquakes, adolescents have more somatization symptoms and fatigue. Therefore, it is crucial to enhance health education, reduce the emphasis on event-related reports, and implement earthquake prevention and disaster reduction training to improve the physical and mental health of adolescents.
5.Effect of Runmu Dihuang Decoction on Perimenopausal Dry Eye in Rats with Liver-kidney Yin Deficiency Syndrome Based on SIRT3/HIF-1α/NF-κB Signaling Pathway
Sainan TIAN ; Wei MA ; Yao CHEN ; Yu CAO ; Guicheng LIU ; Pei LIU ; Junxian LEI ; Qinghua PENG ; Jun PENG
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(7):201-210
ObjectiveTo investigate the mechanisms of Runmu Dihuang decoction (RMDHD) in treating perimenopausal dry eye with liver-kidney Yin deficiency syndrome based on the silent information regulator 3 (SIRT3)/hypoxia-inducible factor-1α (HIF-1α)/nuclear factor-κB (NF-κB) signaling pathway. MethodsSixty female Sprague-Dawley rats were randomly divided into six groups (n=10 per group): Sham operation group, model group, sodium hyaluronate eye drop group, and low-, medium-, and high-dose RMDHD groups (5.625, 11.25, 22.50 g·kg-1). Except for the sham operation group, all rats underwent bilateral ovariectomy and were administered 0.1% benzalkonium chloride eye drops combined with long-term chronic irritation to establish a perimenopausal dry eye model with liver-kidney Yin deficiency syndrome. Drug administration began in the 11th week after modeling and continued for 21 days. General conditions, screen-grip test scores, tear secretion volume, tear film breakup time (TFBUT), and corneal fluorescein staining were recorded. Serum levels of reactive oxygen species (ROS), follicle-stimulating hormone (FSH), estradiol (E2), and progesterone (PROG) were measured by enzyme-linked immunosorbent assay (ELISA). Pathological changes in the lacrimal glands, corneas, and uteri were observed using hematoxylin-eosin (HE) staining. Protein expression levels of SIRT3, HIF-1α, phosphorylated NF-κB p65 (p-NF-κB p65), and total NF-κB p65 in the lacrimal glands were detected by Western blot. The expression of inflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the lacrimal glands was assessed by immunohistochemistry (IHC). ResultsAfter model establishment, no significant differences were observed among the groups except the sham operation group. Compared with the sham operation group, the other groups exhibited slowed movement, dull responses, increased irritability, reduced body weight, elevated rectal temperature, decreased screen-grip test scores, reduced tear secretion, and significantly shortened TFBUT (P<0.05). After treatment, compared with the model group, the sodium hyaluronate eye drop group and all RMDHD groups showed improved general conditions, significantly increased tear secretion (P<0.05), prolonged TFBUT (P<0.05), and elevated screen-grip test scores (P<0.05). Serum ROS and FSH levels were significantly decreased, while E2 and PROG levels were significantly increased (P<0.05). Pathological damage to the cornea, lacrimal glands, and uterus was ameliorated. In addition, protein expression levels of SIRT3 and HIF-1α in the lacrimal glands were significantly upregulated (P<0.05), whereas the expression of p-NF-κB p65, IL-1β, and TNF-α was significantly downregulated (P<0.05). ConclusionRMDHD increases tear secretion and TFBUT, improves lacrimal gland and corneal injury, and alleviates dry eye symptoms in a perimenopausal dry eye rat model with liver-kidney Yin deficiency syndrome. The underlying mechanism may be related to regulation of the SIRT3/HIF-1α/NF-κB signaling pathway, inhibition of oxidative stress and inflammatory responses, and reduction of ocular surface tissue damage.
6.Exploration on Scientific Connotation of "Removing Calyx" of Inulae Flos Based on Processing Experience of Old Medicine Workers
Qiao ZHOU ; Zhenni QU ; Menghan LIU ; Lei ZHANG ; Benzheng SU ; Yanpeng DAI ; Dianhua SHI
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(7):260-266
ObjectiveTo conduct a comparative quality analysis of Inulae Flos, fuzz of Inulae Flos and calyx of Inulae Flos, elucidating the scientific connotation of the "removing calyx" process in the traditional processing of Inulae Flos. MethodsInulae Flos decoction pieces were collected, and the fuzz and calyx of Inulae Flos were prepared according to the experiences of old medicine workers. Subsequently, according to the methods under the "Inulae Flos" item in the 2025 edition of the Pharmacopoeia of the People's Republic of China, the appearance characteristics and thin-layer chromatography(TLC) identification of these samples were tested, and the moisture content, total ash content, extract content were also measured. The characteristic fingerprint patterns of Inulae Flos and fuzz of Inulae Flos were established by high-performance liquid chromatography(HPLC), followed by similarity evaluation, principal component analysis(PCA), and partial least squares-discriminant analysis(PLS-DA). The contents of cryptochlorogenic acid, caffeic acid, 1,3-O-dicaffeoylqunic acid, 1,5-O-dicaffeoylqunic acid, and 1-O-acetyl britannilactone were determined to compare the quality differences of Inulae Flos, fuzz of Inulae Flos, and calyx of Inulae Flos. ResultsThe moisture content of Inulae Flos, fuzz of Inulae Flos, and calyx of Inulae Flos was all<10%. The determination results of total ash content were as follows:Calyx of Inulae Flos>Inulae Flos>fuzz of Inulae Flos, and the determination results of alcohol-soluble extract content were as follows:Fuzz of Inulae Flos>Inulae Flos>calyx of Inulae Flos. HPLC fingerprint patterns of Inulae Flos and fuzz of Inulae Flos were established, and 22 common peaks were identified. The similarity analysis and PCA showed that the overall quality of Inulae Flos and fuzz of Inulae Flos was similar, while the overall quality of calyx of Inulae Flos differed significantly from that of Inulae Flos and fuzz of Inulae Flos. PLS-DA results showed that Inulae Flos, fuzz of Inulae Flos, and calyx of Inulae Flos clustered into distinct groups, indicating significant differences among them. Cryptochlorogenic acid and caffeic acid had relatively high contents in calyx of Inulae Flos, the contents of 1,3-O-dicaffeoylqunic acid and 1,5-O-dicaffeoylqunic acid in Inulae Flos and fuzz of Inulae Flos were higher than those in calyx of Inulae Flos. The order of 1-O-acetyl britannilactone content was determined as follows:fuzz of Inulae Flos>Inulae Flos>calyx of Inulae Flos. ConclusionThe scientific nature of "Removing Calyx" process in the cleansing of Inulae Flos by old medicine workers is demonstrated by the resulting fuzz of Inulae Flos decoction pieces exhibiting enhanced cleanliness and higher content of the index component 1-O-acetyl britannilactone. This study provides a reference basis for further improving and enhancing the processing method and quality control standards of Inulae Flos.
7.A retrospective analysis of clinical outcomes: a retroauricular transmeatal approach for condylar fractures
ZHANG Jiaxiang ; GAO Yu ; Sadam Ahmed Elayah ; LIU Lei ; CHEN Jinlong
Journal of Prevention and Treatment for Stomatological Diseases 2026;34(3):246-254
Objective:
To evaluate the safety and efficacy of the retroauricular transmeatal approach in the treatment of condylar head and neck fractures, and to provide a reference for clinical practice.
Methods:
This study has been reviewed and approved by the institutional medical ethics committee and has obtained informed consent from the patients. A retrospective analysis was conducted on the clinical data of patients with condylar head and neck fractures treated via the retroauricular transmeatal approach between March and October 2024. Postoperative follow-up was performed for at least 3 months, including clinical and radiographical evaluations. The assessed parameters included facial nerve function, temporal region numbness, hematoma, infection, salivary fistula, mouth opening, malocclusion, mandibular movement function, temporomandibular joint (TMJ) pain and clicking, external auditory canal (EAC) stenosis, hearing function impairment, surgical scar concealment, postoperative reduction and fixation outcomes.
Results:
A total of 16 patients with condylar fractures were treated via the retroauricular transmeatal approach, including 10 unilateral and 6 bilateral cases. Specifically, 18 sides were condylar head fractures, and 4 sides were condylar neck fractures. All patients achieved a House-Brackmann Grade I, indicating normal facial nerve function postoperatively. On postoperative day 1, 3 sides experienced temporal numbness in the temporal region on the surgical side, with spontaneous resolution in all cases. All patients recovered after surgery without hematoma, infection, or salivary fistula. Limited mouth opening was improved (14 cases with restricted mouth opening preoperatively vs. 3 cases postoperatively). No malocclusion occurred in any patient. All patients achieved satisfactory recovery of postoperative mandibular movement function, manifested as restored range of motion without pain. No TMJ clicking was observed within 3 months after surgery. One keloid-prone patient developed progressive EAC stenosis (2 mm lumen constriction) accompanied by conductive hearing impairment, which recovered after dilation therapy. All patients were satisfied with the aesthetic outcomes. Radiographically parametric assessment demonstrated satisfactory fracture reduction and fixation; with no loosening of titanium plates or screws.
Conclusion
The retroauricular transmeatal approach effectively reduces the risk of facial nerve injury and salivary fistulas formation with providing concealed scarring and high patient satisfaction. However, caution should be exercised regarding EAC stenosis, especially for keloid-prone patients.
8.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.
9.Occupational fatigue and influencing factors of live-line power distribution workers
Ruijian PAN ; Conghan LIU ; Xin LU ; Chu CHEN ; Min LI ; Lei LIU
Journal of Environmental and Occupational Medicine 2026;43(2):196-200
Background Fatigue among distribution network live-line workers in complex operational environments has become increasingly severe and requires widespread attention. Objective To investigate the positive rates of fatigue and associated influencing factors of live-line power distribution workers, and to make a reasonable strategy to reduce the fatigue of front-line workers. Methods Power supply companies in Guangdong, Guangxi, and Yunnan provinces were selected by cluster sampling in 2023, and all front-line live-line workers in the selected companies were recruited. The questionnaire used in this study consisted of two parts: one was the Fatigue Scale-14 (FS-14) for investigating fatigue status and the other was for associated influencing factors. A FS-14 score greater than 3 points was defined as fatigue.
10.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.


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