1.Herbal Textual Research and Modern Research Progress of Ostreae Concha
Hongyi ZHANG ; Bin WANG ; Jiawen LIU ; Yuan HU ; Lin CHEN ; Youping LIU ; Hongping CHEN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(3):223-234
By consulting relevant literature of ancient herbal books and processing specifications, this paper made a systematic research and analysis of Ostreae Concha, including the name, producing area, harvesting, quality, historical evolution of processing, relevant processing specifications, modern processing technology, and changes in chemical composition and pharmacological effects before and after processing, in order to provide documentary evidence for the research on processing technology and the establishment of quality standards. According to the textual research, it is known that Ostreae Concha has a long history of being used in medicine, and there have been many aliases and local names in each historical period. Shennong's Classic of the Materia Medica(Shennong Bencaojing) began to use Muli as the correct name, which has continued to use to today, and there were also aliases such as Muge, Zuogu Muli and Haoke. Ostreae Concha has a wide range of localities and irregular harvesting periods. The ancients believed that its left shell was of superior quality, but this has not been seen in modern. And there were many kinds of processing methods of Ostreae Concha, such as grinding, roasting, calcining, frying, simmering, quenching and so on, and the calcining was still in use. The different editions of Chinese Pharmacopoeia from 1963 to 2020 contain only calcined Ostreae Concha, and the local processing specifications mainly include three kinds of processed products(calcined products, salt-soaked products and vinegar-soaked products). Modern processing research mainly focuses on process optimization, changes in chemical composition and pharmacological effects, and the research methods are relatively single. Overall, there are currently issues such as inconsistent processing standards, unclear process parameters and imperfect quality standards, which are not conducive to the quality control and standardized clinical use of Ostreae Concha. Therefore, it is necessary to further investigate the pharmacological substance basis of Ostreae Concha and its processed products in order to elucidate the processing mechanism, standardize the processing technology and improve the quality standard.
2.Effect and mechanism of Qingxue xiaozhi jiangtang formula on insulin resistance in rats with type 2 diabetes mellitus
Yuxin HONG ; Lei ZHANG ; Mingxue ZHOU ; Sinai LI ; Li LIN ; Meng ZHANG ; Zixuan GUO ; Weihong LIU
China Pharmacy 2025;36(1):24-29
OBJECTIVE To investigate the improvement effect and potential mechanism of Qingxue xiaozhi jiangtang formula on insulin resistance (IR) in type 2 diabetes mellitus (T2DM) rats. METHODS T2DM rat model was established by intraperitoneal injection of 30 mg/kg streptozotocin combined with high-fat and high-sugar diet. The rats were randomly divided into normal control group, model group, Qingxue xiaozhi jiangtang formula low-dose and high-dose groups (6.525, 13.05 g/kg, calculated by raw material) and metformin group (positive control, 0.18 g/kg), with 8 rats in each group. Administration groups were given relevant medicine intragastrically; normal control group and model group were given constant volume of normal saline intragastrically, once a day, for consecutive 6 weeks. Body mass and fasting blood glucose (FBG) were determined, and oral glucose tolerance test was conducted. Serum fasting insulin (FINS) level was measured to calculate the insulin resistance index (HOMA-IR) and insulin sensitivity index (ISI). Additionally, the level of serum lipids, liver function, oxidative stress indicators and inflammatory factors were assessed. The phosphorylation levels of kinase R-like endoplasmic reticulum kinase (PERK) and forkhead box O1 (FOXO1) protein in liver tissue of rats were determined. RESULTS Compared with model group, the body weight, ISI, the levels of high-density lipoprotein cholesterol and superoxide dismutase were increased significantly in Qingxue xiaozhi jiangtang formula high-dose group and metformin group (P<0.05); FBG, blood glucose level at 120 minutes of glucose loading, area under the curve of glucose, FINS, HOMA-IR, low-density lipoprotein cholesterol, total cholesterol, triglyceride, alanine transaminase, aspartate transaminase, alkaline phosphatase, malondialdehyde, interleukin-6, tumor necrosis factor-α, and C-reactive protein levels were significantly reduced (P< Δ0.05); the pathological damage of liver tissue had significantlyimproved, and the phosphorylation levels of PERK and FOXO1 proteins in liver tissue were significantly decreased (P<0.05). CONCLUSIONS Qingxue xiaozhi jiangtang formula can regulate glucose and lipid metabolism, inflammation factor and oxidative stress levels, and alleviate insulin resistance in T2DM rats. Its mechanism of action may be related to the inhibition of the PERK/FOXO1 signaling pathway.
3.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.
4.Huoluo Xiaolingdan Suppresses Triple-negative Breast Cancer in Mice by Regulating TCF1+ CD8+ Stem Cell-like T Cells Infiltration
Bo LUO ; Qu ZHANG ; Yujie SUN ; Lin LIU ; Lan ZENG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(4):108-115
ObjectiveTo investigate the inhibitory effect of Huoluo Xiaolingdan on triple negative breast cancer (TNBC) in mice through its regulation of TCF1+CD8+ stem cell-like T cells infiltration. MethodsA mouse model of TNBC was established and the mice were randomly divided into the model group, low-dose (3.9 g·kg-1), medium-dose (7.8 g·kg-1) and high-dose (15.6 g·kg-1) Huoluo Xiaolingdan groups, and anti-PD-1 antibody treatment group. Each group was given a dose of 0.01 mL·g-1, while the model group and the anti-PD-1 treatment group were also given an equivalent volume of normal saline. The drug was administered for 21 days. In the anti-PD-1 antibody group, mice were intraperitoneally injected with 100 μg of mouse anti-PD-1 antibody twice a week, for a total of five injections. The tumor volume, survival time and tumor mass were measured at different time points. Hematoxylin-eosin (HE) staining was used to observe the histological changes of the tumor. The expression of CD8+T cells and TCF1+CD8+ stem-like T cells in tumor tissues was detected by immunohistochemistry and immunofluorescence staining. Flow cytometry was used to detect the difference of immune cell subsets in tumors and the expression difference of TCF1+CD8+ stem cell-like T cells in tumors and peripheral blood. The expression level of PD-L1 in tumor tissues was detected by Western blot. ResultsCompared with model group, the tumor volume and mass of in low-, medium- and high-dose Huoluo Xiaolingdan groups and anti-PD-1 group were decreased (P<0.05, P<0.01). The median survival time of mice in low-, medium- and high-dose Huoluo Xiaolingdan groups and anti-PD-1 group was as follows: 27.00 days (95%CI, 0.45-2.65), 31.00 days (95%CI, 0.32-1.89), 34.00 days (95%CI, 0.40-2.33), and 35.00 days (95%CI, 0.42-2.47). All of them were higher than that of the model group [24.50 days (95%CI, 0.37-10.5)]. Flow cytometry showed that compared with the model group, the proportion and number of infiltrating CD8+ T cells in tumor were increased in low-, medium- and high-dose Huoluo Xiaolingdan groups and anti-PD-1 group (P<0.05, P<0.01), while the proportion of tumor regulatory T cells (Treg) and M2 macrophages decreased (P<0.05). Compared with the model group, the proportion of IFN-γ+CD8+ T and GrzB+CD8+ T cells in tumors in low-, medium- and high-dose Huoluo Xiaolingdan groups and anti-PD-1 group was increased (P<0.01), and the proportion of TCF1+CD8+ T cells in tumor and peripheral blood was also increased. Immunofluorescence staining further showed that the number of TCF1+CD8+ T cells in tumor tissues increased in low-, medium- and high-dose Huoluo Xiaolingdan groups. Western blot analysis showed no significant decrease in the PD-L1 protein expression in tumor tissues between the Huoluo Xiaolingdan groups and the model group. ConclusionHuoluo Xiaolingdan can inhibit TNBC in mice by increasing tumor infiltration of TCF1+CD8+ stem-like T cells, enhancing CD8+ T cell activity, and regulating immune cell subgroups such as M2 macrophages and Treg cells to enhance anti-tumor immunity. This study provides a theoretical basis for the clinical application of Huoluo Xiaolingdan in breast cancer treatment and combination therapy.
5.Expert Consensus on Clinical Application of Qinbaohong Zhike Oral Liquid in Treatment of Acute Bronchitis and Acute Attack of Chronic Bronchitis
Jian LIU ; Hongchun ZHANG ; Chengxiang WANG ; Hongsheng CUI ; Xia CUI ; Shunan ZHANG ; Daowen YANG ; Cuiling FENG ; Yubo GUO ; Zengtao SUN ; Huiyong ZHANG ; Guangxi LI ; Qing MIAO ; Sumei WANG ; Liqing SHI ; Hongjun YANG ; Ting LIU ; Fangbo ZHANG ; Sheng CHEN ; Wei CHEN ; Hai WANG ; Lin LIN ; Nini QU ; Lei WU ; Dengshan WU ; Yafeng LIU ; Wenyan ZHANG ; Yueying ZHANG ; Yongfen FAN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(4):182-188
The Expert Consensus on Clinical Application of Qinbaohong Zhike Oral Liquid in Treatment of Acute Bronchitis and Acute Attack of Chronic Bronchitis (GS/CACM 337-2023) was released by the China Association of Chinese Medicine on December 13th, 2023. This expert consensus was developed by experts in methodology, pharmacy, and Chinese medicine in strict accordance with the development requirements of the China Association of Chinese Medicine (CACM) and based on the latest medical evidence and the clinical medication experience of well-known experts in the fields of respiratory medicine (pulmonary diseases) and pediatrics. This expert consensus defines the application of Qinbaohong Zhike oral liquid in the treatment of cough and excessive sputum caused by phlegm-heat obstructing lung, acute bronchitis, and acute attack of chronic bronchitis from the aspects of applicable populations, efficacy evaluation, usage, dosage, drug combination, and safety. It is expected to guide the rational drug use in medical and health institutions, give full play to the unique value of Qinbaohong Zhike oral liquid, and vigorously promote the inheritance and innovation of Chinese patent medicines.
6.Insights on Peripheral Blood Biomarkers for Parkinson’s Disease
Yu-Meng LI ; Jing-Kai LIU ; Zi-Xuan CHEN ; Yu-Lin DENG
Progress in Biochemistry and Biophysics 2025;52(1):72-87
Parkinson’s disease (PD) is a common neurodegenerative disorder with profound impact on patients’ quality of life and long-term health, and early detection and intervention are particularly critical. In recent years, the search for precise and reliable biomarkers has become one of the key strategies to effectively address the clinical challenges of PD. In this paper, we systematically evaluated potential biomarkers, including proteins, metabolites, epigenetic markers, and exosomes, in the peripheral blood of PD patients. Protein markers are one of the main directions of biomarker research in PD. In particular, α‑synuclein and its phosphorylated form play a key role in the pathological process of PD. It has been shown that aggregation of α-synuclein may be associated with pathologic protein deposition in PD and may be a potential marker for early diagnosis of PD. In terms of metabolites, uric acid, as a metabolite, plays an important role in oxidative stress and neuroprotection in PD. It has been found that changes in uric acid levels may be associated with the onset and progression of PD, showing its potential as an early diagnostic marker. Epigenetic markers, such as DNA methylation modifications and miRNAs, have also attracted much attention in Parkinson’s disease research. Changes in these markers may affect the expression of PD-related genes and have an important impact on the onset and progression of the disease, providing new research perspectives for the early diagnosis of PD. In addition, exosomes, as a potential biomarker carrier for PD, are able to carry a variety of biomolecules involved in intercellular communication and pathological regulation. Studies have shown that exosomes may play an important role in the pathogenesis of PD, and their detection in blood may provide a new breakthrough for early diagnosis. It has been shown that exosomes may play an important role in the pathogenesis of PD, and their detection in blood may provide new breakthroughs in early diagnosis. In summary, through in-depth evaluation of biomarkers in the peripheral blood of PD patients, this paper demonstrates the important potential of these markers in the early diagnosis of PD and in the study of pathological mechanisms. Future studies will continue to explore the clinical application value of these biomarkers to promote the early detection of PD and individualized treatment strategies.
7.Mechanism of Zuogui Jiangtang Jieyu Prescription Against Damage to Hippocampal Synaptic Microenvironment via Suppressing GluR2/Parkin Signal-mediated Mitophagy in Rats with Diabetes-related Depression
Jian LIU ; Lin LIU ; Xiaoyuan LIN ; Wei LI ; Yuhong WANG ; Hui YANG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(1):104-112
ObjectiveTo reveal the mechanism of Zuogui Jiangtang Jieyu prescription against damage to hippocampal synaptic microenvironment via suppressing glutamate receptor 2 (GluR2)/Parkin signal-mediated mitophagy in rats with diabetes-related depression (DD). MethodsEighty male SD rats underwent adaptive feeding for 5 days before the study. Ten rats were randomly assigned to the normal group. The model of DD rats was established with the rest by 2-week high-fat diet + streptozotocin (STZ) tail intravenous injection + 28 days of chronic unpredictable mild stress (CUMS) combined with isolation. The rats were randomly divided into a normal group, a model group, a GluR2 blocker group (5 μg·kg-1), a GluR2 agonist group (10 μg·kg-1), a metformin + fluoxetine group (0.18 g·kg-1 metformin + 1.8 mg·kg-1 fluoxetine), and high- and low-dose Zuogui Jiangtang Jieyu prescription groups (20.52 and 10.26 g·kg-1, respectively). The rats in the GluR2 blocker group and the GluR2 agonist group were continuously injected with CNQX and Cl-HIBO in the dentate gyrus of the hippocampus once a week starting from stress modeling, respectively, while the metformin + fluoxetine group and the high- and low-dose Zuogui Jiangtang Jieyu prescription groups were continuously given intragastric administration for 28 d at the same time of stress modeling. Depression-like behavior was evaluated by open field and forced swimming experiments. The levels of serum insulin and adenosine triphosphate (ATP) in hippocampus were detected by biochemical analysis. The levels of 5-hydroxytryptamine (5-HT) and dopamine (DA) in hippocampus were detected by enzyme-linked immunosorbent assay (ELISA). The autophagosomes of hippocampal neurons were observed by transmission electron microscopy. The morphology and structure of dendrites and spines of hippocampal neurons were evaluated by Golgi staining. Western blot detected the expression levels of GluR2 and Parkin proteins in hippocampus. The expression levels of GluR2, Parkin, regulating synaptic membrane exocytosis protein 3 (RIMS3), and postsynaptic density protein 95 (PSD95) in the dentate gyrus of the hippocampus were detected by immunofluorescence. ResultsCompared with the normal group, the model group exhibited reduced total activity distance in the open field and increased immobility time in forced swimming (P<0.01), lowered levels of serum insulin and ATP, 5-HT, and DA in hippocampus (P<0.01), increased autophagosomes of hippocampal neurons, significantly damaged morphology and structure of dendrites and spines of hippocampal neurons, decreased expression levels of GluR2, RIMS3, and PSD95 in hippocampus, and an increased Parkin expression level (P<0.05, P<0.01). Compared with the model group, the GluR2 blocker group and the GluR2 agonist group showed aggravation and alleviation of the above abnormal changes, respectively (P<0.05, P<0.01). The above depression-like behavior was significantly improved in the high- and low-dose Zuogui Jiangtang Jieyu prescription groups to different degrees. Specifically, the two groups saw elevated levels of serum insulin and ATP, 5-HT, and DA in hippocampus (P<0.05, P<0.01), restrained increase in autophagosomes and damage to morphology and structure of dendrites and spines of hippocampal neurons, up-regulated protein expression levels of GluR2, RIMS3, and PSD95, and down-regulated Parkin expression level (P<0.05, P<0.01). ConclusionZuogui Jiangtong Jieyu prescription can ameliorate the mitophagy-mediated damage to hippocampal synaptic microenvironment in DD rats, the mechanism of which might be related to the regulation of GluR2/Parkin signaling pathway.
8.Effect of Epimedium brevicornu Ethanol Extract on Aging of Castrated Rats by Intervening in Mesenchymal Adipose-derived Stem Cells
Zuyu MENG ; Haiquan LIU ; Shaozi LIN ; Mei WANG ; Yiyao ZHANG ; Fang LIU ; Menghan LI ; Hongling CHEN ; Jiajia QIN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(1):174-181
ObjectiveTo explore the mechanism by which the ethanol extract of Epimedium brevicornu (EEBM) intervenes in mesenchymal adipose-derived stem cells (ADSCs) to delay aging in castrated rats. MethodsForty-five 3-month-old SPF female SD rats were ovariectomized and randomly divided into model group, ADSCs treatment group, and ADSCs groups treated with low, medium, and high concentrations of EEBM (1, 50, 100 μg·L-1), referred to as the AE low, medium, and high concentration groups, with 9 rats in each group. After tail vein injection of 200 μL of the corresponding stem cell suspension, aging-related indicators including cyclin-dependent kinase inhibitor (p21), tumor suppressor gene (p53), interleukin-6 (IL-6), interleukin-8 (IL-8), superoxide dismutase (SOD), malondialdehyde (MDA), B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), cysteine-aspartic acid protease-3 (Caspase-3), and lipofuscin were measured using enzyme-linked immunosorbent assay (ELISA) and Western blot. ResultsCompared with the model group, the IL-6 content in the AE low, medium, and high concentration groups was significantly decreased (P<0.05). Lipofuscin, MDA, and IL-8 levels in the ADSCs treatment group and AE low, medium, and high concentration groups were significantly reduced (P<0.01), while SOD content was significantly increased (P<0.05, P<0.01). Compared with the ADSCs treatment group, lipofuscin and IL-8 levels in the AE low, medium, and high concentration groups were significantly reduced (P<0.05, P<0.01). The MDA content was significantly decreased in the AE medium concentration group (P<0.01). Compared with the model group, protein levels of p21, p53, Bax, and Caspase-3 in the ADSCs treatment group and AE low, medium, and high concentration groups were significantly reduced (P<0.05, P<0.01), while the Bcl-2 protein level was significantly increased (P<0.01). Compared with the ADSCs treatment group, protein levels of p21, p53, Bax, and Caspase-3 in the AE low, medium, and high concentration groups were significantly reduced (P<0.05, P<0.01), and the Bcl-2 protein level in the AE low concentration group was significantly increased (P<0.01). ConclusionThe results of this experiment show that EEBM-treated ADSCs or ADSCs may delay aging in castrated rats by inhibiting cell apoptosis, reducing cell cycle inhibitors and pro-inflammatory factors, enhancing antioxidant capacity, and reducing oxidative reactions. Moreover, EEBM-treated ADSCs demonstrate stronger anti-aging effects than ADSCs alone. This study provides experimental evidence supporting the clinical use of EEBM to intervene in ADSCs and delay aging.
9.Ancient Literature Analysis and Textual Research of Classic Formula Zhishi Shaoyaosan
Chenyu LI ; Cong OUYANG ; Rou ZENG ; Ziyan LIU ; Ye ZHANG ; Jie LIN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(1):234-243
Zhishi Shaoyaosan is the 34th prescription in the Catalogue of Ancient Classic Formulas (Second Batch) published by the National Administration of Traditional Chinese Medicine in 2023. It is widely used in clinical practice and has a definite curative effect. However, there is currently a lack of its ancient literature analysis and textual research, and there is no corresponding Chinese patent medicine preparation. By consulting and combing the relevant ancient books of traditional Chinese medicine, this paper analyzes and conducts textual research of the origin, composition, measurement, administration, and efficacy of Zhishi Shaoyaosan. The results show that Zhishi Shaoyaosan is derived from Essentials from the Golden Cabinet written by Zhang Zhongjing in the Eastern Han Dynasty. It is mainly recorded in the name of Zhishi Shaoyaosan in the literature of the past dynasties. The prescription is composed of Aurantii Fructus Immaturus and Paeoniae Radix Alba. The processing method is stir-frying Aurantii Fructus Immaturus to scorch and using raw Paeoniae Radix Alba. The dose of the prescription recorded in the ancient books is mainly an equal amount of Aurantii Fructus Immaturus and Paeoniae Radix Alba in one square-cun spoon, taken three times a day, which is converted into a modern dose of 1.5 g each time (0.75 g Aurantii Fructus Immaturus and 0.75 g Paeoniae Radix Alba each time). The components of the prescription are ground into powder and taken with barley porridge, three times a day. The efficacy is to break stagnated Qi, harmonize blood, and relieve restlessness and pain. It is mainly used to treat postpartum abdominal pain, acute pelvic inflammatory disease, acute cholecystitis and intestinal diseases, stroke sequelae, and other diseases. This study combs and analyzes the ancient literature recording Zhishi Shaoyaosan and clarifies the key information of the prescription, which provides a basis for promoting the research and development of its patent medicine.
10.Expert Consensus on Clinical Diseases Responding Specifically to Traditional Chinese Medicine: Atopic Dermatitis
Junfeng LIU ; Xiumei MO ; Mei MO ; Hongyi LI ; Ying LIN ; Xiaoxiao ZHANG ; Dacan CHEN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(1):244-252
Atopic dermatitis (AD) is a common pruritic and chronic inflammatory dermatosis in clinical practice and is one of the diseases responding specifically to traditional Chinese medicine (TCM). With the launch of biological agents and small molecule drugs and the development and implementation of guidelines of diagnosis and treatment, clinical pathways of treatment of moderate to severe AD, and consensus on the whole-process management of AD, the clinical efficacy of moderate to severe AD has been significantly improved. However, there are still many unmet clinical needs that require more effective methods to meet. In response to the Opinions of the CPC Central Committee and the State Council on Facilitating the Inheritance, Innovation, and Development of Traditional Chinese Medicine and the spirit of the National Conference on TCM, the China Association of Chinese Medicine organized more than 20 experts in TCM dermatology, Western medicine dermatology, interdisciplinary fields, and industries to discuss the difficulties and advantages of TCM in the treatment of AD. TCM treatment for AD can not only improve rash and relieve itching but also solve many concomitant syndromes. The abundant external treatment methods of TCM have advantages for different special populations and rash characteristics. The concept of treating disease before its onset in TCM is in line with the chronic disease management mode of prevention and treatment of atopic march and prevention of recurrence. In addition, TCM therapy can reduce the use of topical glucocorticoids and has good safety. Regarding the comorbidity of AD, equal emphasis on TCM and Western medicine and multidisciplinary joint treatment should be advocated to achieve maximum benefit for patients. The exchange of TCM and Western medicine has clarified the positioning and advantages of TCM intervention in AD, providing guidance for clinical and scientific research.

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