1.Huanglian Jiedutang Improves Myelin Damage and Agitated Behavior in Vascular Dementia by Regulating Microglial Polarization via CD22/SHP-1/p-Akt Signaling Pathway
Chen CHEN ; Xiaoxia FENG ; Shiting LIANG ; Xinxian SHI ; Guang YANG ; Jing QIU
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(2):25-33
ObjectiveTo investigate the mechanisms by which Huanglian Jiedutang (HLJDT) modulates microglial (MG) phenotypes through the sialic acid-binding Ig-like lectin 2 (SIGLEC2/CD22)/Src-homology-2-domain-containing protein tyrosine phosphatase-1 (SHP-1)/phosphorylated protein kinase B (p-Akt) signaling pathway, thereby promoting myelin repair and alleviating agitation-like behaviors in vascular dementia (VAD). MethodsSixty C57BL/6J mice were randomly assigned to a sham (normal) group, model group, HLJDT low-, medium-, and high-dose groups (2.5, 5, and 10 g·kg-1·d-1), and a risperidone group (2 mg·kg-1·d-1), with 10 mice per group. VAD was induced by bilateral common carotid artery stenosis (BCAS). From day 42, mice received drug interventions for 2 weeks. Agitation-like behaviors were assessed using the resident-intruder test. After behavioral testing, ventrolateral part of the ventromedial hypothalamus (VMHvl) tissues were collected. Western blot was used to measure protein levels of myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), proteolipid protein (PLP), inducible nitric oxide synthase (iNOS), arginase-1 (Arg1), CD86, CD206, and CD22, SHP-1, and p-Akt. Immunofluorescence was used to evaluate myelin-associated glycoprotein (MAG) intensity and the proportion of iNOS+/ionized calcium-binding adapter molecule 1 (Iba1)+ cells. ELISA was used to detect tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β. ResultsCompared with the normal group, the model group exhibited markedly increased biting and aggressive behaviors and shortened attack latency (P<0.01). MOG, MBP, and PLP protein levels and MAG fluorescence intensity were significantly reduced (P<0.05, P<0.01). INOS and CD86 expression and TNF-α, IL-6, and IL-1β levels were significantly elevated (P<0.01). CD22 and SHP-1 expression increased significantly (P<0.01), whereas p-Akt expression decreased (P<0.01). Compared with the model group, the medium- and high-dose HLJDT groups and the risperidone group showed markedly reduced biting and aggression (P<0.05, P<0.01) and prolonged attack latency (P<0.01). MOG, MBP, and PLP levels and MAG fluorescence intensity were significantly increased (P<0.05, P<0.01). INOS, CD86, TNF-α, IL-6, and IL-1β levels decreased significantly (P<0.05, P<0.01). CD22 and SHP-1 expression decreased, while p-Akt expression increased significantly (P<0.05, P<0.01). ConclusionHLJDT may modulate CD22/SHP-1/p-Akt signaling in the VMHvl, promote the shift of MG toward an anti-inflammatory and phagocytic phenotype, enhance myelin repair, and improve agitation-like behaviors in VAD mice.
2.Clinical Efficacy and Mechanism of Bupi Qingfei Prescription in Treating Stable Bronchiectasis
Zi YANG ; Guangsen LI ; Bing WANG ; Bo XU ; Jianxin WANG ; Sheng CAO ; Xinyan CHEN ; Xia SHI ; Qing MIAO
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(2):162-169
ObjectiveTo explore the clinical efficacy and mechanism of Bupi Qingfei prescription (BPQF) in treating stable bronchiectasis in the patients with syndromes of lung-spleen Qi deficiency and phlegm-heat accumulation in the lungs. MethodsA randomized, double-blind, placebo-controlled trial was conducted. Patients were randomized into BPQF and placebo control (PC) groups. On the basis of conventional Western medicine treatment, the BPQF granules and placebo were respectively administered at 10 g each time, twice a day, for a course of 24 weeks. The TCM symptom scores, Quality of Life Questionnaire for Bronchiectasis (QOL-B) scores, lung function indicators, T lymphocyte subsets, level of inflammatory factors in the sputum, level of neutrophil elastase (NE) in the sputum, and occurrence of adverse reactions were observed before and after treatment in the two groups. ResultsA total of 64 patients completed the study, encompassing 32 in the BPQF group and 32 in the PC group. After treatment, the BPQF group showed decreased TCM symptom scores (P<0.01), increased QOL-B scores (P<0.01), and declined levels of tumor necrosis factor (TNF)-α and NE (P<0.05, P<0.01). The PC group showed decreased TCM symptom (except spleen deficiency) scores (P<0.01), increased the QOL-B health cognition and respiratory symptom domain scores (P<0.05, P<0.01), and a declined TNF-α level (P<0.01). Moreover, the BPQF group had lower TCM symptom (except chest tightness) scores (P<0.05, P<0.01), higher QOL-B (except treatment burden) scores (P<0.05, P<0.01), and lower levels of interleukin-6 and TNF-α (P<0.05) than the PC group. Neither group showed serious adverse reactions during the treatment process. ConclusionBPQF can ameliorate the clinical symptoms of stable bronchiectasis patients who have lung-spleen Qi deficiency or phlegm-heat accumulation in the lungs by regulating the immune balance and inhibiting airway inflammatory responses.
3.Jingmaiyan Granules Combined with External Application of Jinhuang Ointment in Treatment of Acute Stage Blood Heat Stasis Type Superficial Thrombophlebitis of Lower Extremities: A Randomized, Double-blind, Placebo-controlled Clinical Trial
Qiaoyilan LIANG ; Hong CHEN ; Weijing FAN ; Hongshuo SHI ; Fangfang WU ; Guobin LIU
Chinese Journal of Experimental Traditional Medical Formulae 2026;32(5):196-202
ObjectiveTo evaluate the clinical efficacy and safety of Jingmaiyan granules (composed of Lonicerae Japonicae Flos, Sedi Herba, Paeoniae Radix Rubra, Moutan Cortex, Rhei Radix et Rhizoma Praeparata, and Glycyrrhizae Radix et Rhizoma) combined with external application of Jinhuang Ointment in treating acute-stage blood heat stasis type superficial thrombophlebitis (ST) of lower extremities, and to explore their effects on hemorheology and serum inflammatory factors. MethodsA randomized, double-blind, placebo-controlled clinical trial was conducted. A total of 124 patients with lower extremity ST were randomized into two groups(62 cases in each group). The control group received external application of Jinhuang ointment and oral placebo treatment, while the observation group received external application of Jinhuang ointment and oral Jingmaiyan granules. Both groups were treated for 2 weeks. The clinical symptom scores, therapeutic efficacy of traditional Chinese medicine (TCM) syndrome, pain visual analog scale (VAS) scores, hemorheological indices [including whole blood high-shear, medium-shear, and low-shear viscosity, as well as plasma viscosity (PV)], and inflammatory factors [C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α)] were compared before and after treatment. ResultsAfter 2 weeks of treatment, the total effective rate in the observation group (98.3%, 60/62) was significantly higher than that in the control group (83.8%, 52/62), with a statistically significant difference (Z=3.512 1, P<0.05). Compared with pre-treatment scores, the scores for skin color, skin temperature, swelling, pain, and cord or nodules were significantly reduced in both groups (P<0.05), with more pronounced improvement in the observation group (P<0.05). Additionally, compared with pre-treatment levels, the whole blood viscosity (low-, medium-, and high-shear) significantly improved in both groups after treatment (P<0.05), with more marked improvement in the observation group (P<0.05). Furthermore, the plasma viscosity, CRP, IL-6, and TNF-α levels were significantly reduced in both groups after treatment (P<0.05), with more pronounced improvement observed in the observation group (P<0.05). ConclusionThe combination of external application of Jinhuang ointment and oral Jingmaiyan granules effectively improves clinical symptoms, hemorheological abnormalities, and inflammatory responses in patients with acute stage blood heat stasis type ST of lower extremities. The treatment is safe and holds clinical promotion value.
4.Effect of Modified Shibaotang on Serum Sex Hormone Levels in Patients with Male Late-onset Hypogonadism of Kidney Essence Deficiency Syndrome Complicated with Diabetes Mellitus
Yi SHAN ; Shaokang CHEN ; Zhenfu SHI ; Haifeng XU ; Yi LU
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(1):227-233
ObjectiveTo observe the effect of modified Shibaotang on serum sex hormone levels in patients with male late-onset hypogonadism of kidney essence deficiency syndrome complicated with diabetes mellitus. MethodsA total of 60 patients with male late-onset hypogonadism of kidney essence deficiency syndrome complicated with diabetes mellitus,who met the inclusion criteria and were admitted to Yinchuan Hospital of Traditional Chinese Medicine from October 2022 to October 2023,were selected and randomly divided into an observation group and a control group,with 30 patients in each group. Both groups continued their original treatments,including blood glucose lowering and blood lipid regulation. The observation group was treated with modified Shibaotang,while the control group was treated with testosterone undecanoate capsules. The treatment lasted for 12 weeks. The changes in traditional Chinese medicine (TCM)syndrome scores,partial androgen deficiency in aging males (PADAM)symptom scores,glucose metabolism indexes [fasting plasma glucose (FPG),2-hour postprandial glucose (2 h PG),glycosylated hemoglobin (HbA1c)],and serum sex hormone indexes [sex hormone-binding globulin (SHBG),free testosterone (FT),total testosterone (TT),prolactin (PRL),luteinizing hormone (LH),follicle stimulating hormone (FSH),estrogen (E2)] were compared between the two groups before and after treatment. Safety was also evaluated. Results(1)Clinical efficacy comparison:After treatment,the clinical efficacy in both groups was similar,and there was no statistically significant difference between the two groups. (2)TCM syndrome score and PADAM symptom score comparison:After treatment,both groups showed a significant reduction in TCM syndrome scores and PADAM symptom scores (P<0.01),and the observation group showed a significantly greater reduction compared to the control group (P<0.05). (3)Glucose metabolism indexes comparison:After treatment,the levels of FPG,2 h PG,and HbA1c were significantly reduced in both groups (P<0.01),and there was no statistically significant difference between the two groups regarding FPG,2 h PG,and HbA1c levels after treatment. (4)Serum sex hormone indexes comparison:After treatment,the levels of FT,TT,PRL,LH,and FSH were significantly increased in both groups (P<0.01),while E2 levels were significantly decreased (P<0.01). There was no statistically significant difference between the two groups in the levels of FT,TT,PRL,LH,FSH,and E2 after treatment. There was also no significant difference in SHBG levels within or between the groups before and after treatment. During the clinical observation,neither group exhibited any obvious adverse reactions. ConclusionModified Shibaotang can significantly improve the clinical symptoms of male late-onset hypogonadism of kidney essence deficiency syndrome complicated with diabetes mellitus,reduce blood glucose,and increase sex hormone levels. The mechanism may involve the inhibition of aromatase transformation in adipocytes,promotion of GnRH production,and regulation of the hypothalamic-pituitary-gonadal axis function.
5.Clinical Efficacy of Tangning Tongluo Tablets for Nonproliferative Diabetic Retinopathy
Fuwen ZHANG ; Junguo DUAN ; Wen XIA ; Tiantian SUN ; Yuheng SHI ; Shicui MEI ; Xiangxia LUO ; Xing LI ; Yujie PAN ; Yong DENG ; Chuanlian RAN ; Hao CHEN ; Li PEI ; Shuyu YANG
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(3):132-139
ObjectiveTo observe the clinical efficacy and safety of Tangning Tongluo tablets in the treatment of nonproliferative diabetic retinopathy (DR). MethodsFourteen research centers participated in this study, which spanned a time interval from September 2021 to May 2023. A total of 240 patients with nonproliferative DR were included and randomly assigned into an observation group (120 cases) and a control group (120 cases). The observation group was treated with Tangning Tongluo tablets, and the control group with calcium dobesilate capsules. Both groups were treated for 24 consecutive weeks. The vision, DR progression rate, retinal microhemangioma, hemorrhage area, exudation area, glycosylated hemoglobin (HbA1c) level, and TCM syndrome score were assessed before and after treatment, and the safety was observed. ResultsThe vision changed in both groups after treatment (P<0.05), and the observation group showed higher best corrected visual acuity (BCVA) than the control group (P<0.05). The DR progression was slow with similar rates in the two groups. The fundus hemorrhage area and exudation area did not change significantly after treatment in both groups, while the observation group outperformed the control group in reducing the fundus hemorrhage area and exudation area. There was no significant difference in the number of microhemangiomas between the two groups before treatment. After treatment, the number of microhemangiomas decreased in both the observation group (Z=-1.437, P<0.05) and the control group (Z=-2.238, P<0.05), and it showed no significant difference between the two groups. As the treatment time prolonged, the number of microhemangiomas gradually decreased in both groups. There was no significant difference in the HbA1c level between the two groups before treatment. After treatment, the decline in the HbA1c level showed no significant difference between the two groups. The TCM syndrome score did not have a statistically significant difference between the two groups before treatment. After treatment, neither the TCM syndrome score nor the response rate had significant difference between the two groups. With the extension of the treatment time, both groups showed amelioration of TCM syndrome compared with the baseline. ConclusionTangning Tongluo tablets are safe and effective in the treatment of nonproliferative DR, being capable of improving vision and reducing hemorrhage and exudation in the fundus.
6.Textual Research on Classical Formula Mulisan
Dongsen HU ; Xiangyang ZHANG ; Canran XIE ; Jiawei SHI ; Ziyi WANG ; Zhuoyan ZHOU ; Lin ZHANG ; Yexin CHEN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):191-200
The classic formula Mulisan is the 45th of the 93 formulas in the Catalogue of Ancient Classic Formulas (second batch) of Han medicine published by the National Administration of Traditional Chinese Medicine. It consists of Ostreae Concha, Astragali Radix, Ephedrae Radix et Rhizoma, and wheat, with the effect of replenishing qi and stopping sweating. It is a common formula in the clinical treatment with traditional Chinese medicine. This study analyzes the historical evolution, composition, dosage, original plants and their processing methods, decocting method, efficacy, indications, and modern clinical application of Mulisan by tracing, comparative analysis, and bibliometric methods. The results showed that Mulisan firstly appeared in the Pulse Classic written by WANG Shuhe in the Western Jin Dynasty. The formulation idea can be traced back to the Important Prescriptions Worth a Thousand Gold for Emergency in the Tang Dynasty. The herb composition, dosage, efficacy, and indications of Mulisan were first recorded in the Treatise on Diseases, Patterns, and formulas Related to Unification of the Three Etiologies in the Southern Song dynasty. In terms of original plants and their processing methods, Ostreae Concha is the shell of Ostrea rivularis, which should be calcined before use. Astragali Radix and Ephedrae Radix et Rhizoma are the dried roots of Astragalus membranaceus var. mongholicus and Ephedra sinica, respectively, the raw material of which should be used. Wheat is the dried mature fruit of T. aestivum, which can be used without processing, while the stir-fried fruit, being thin and deflated, demonstrates better effect. The composition of Mulisan is Ostreae Concha 8.26 g, Astragali Radix 8.26 g, Ephedrae Radix et Rhizoma 8.26 g, and wheat 7.92 g. The medicinal materials should be ground into coarse powder and decocted with 450 mL water to reach a volume of 240 mL, and the decoction should be taken warm. In modern clinical practice, Mulisan has a wide range of indications, including spontaneous sweating and night sweating caused by Yang deficiency or Qi deficiency. The clinical disease spectrum treated by Mulisan involves endocrine system diseases, neurological diseases, respiratory system diseases, and cancer. This formula plays a significant role in the treatment of internal medicine diseases in traditional Chinese medicine. This study aims to provide a scientific basis for the subsequent research, development, and clinical application of Mulisan.
7.Textual Research on Classical Formula Mulisan
Dongsen HU ; Xiangyang ZHANG ; Canran XIE ; Jiawei SHI ; Ziyi WANG ; Zhuoyan ZHOU ; Lin ZHANG ; Yexin CHEN
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(7):191-200
The classic formula Mulisan is the 45th of the 93 formulas in the Catalogue of Ancient Classic Formulas (second batch) of Han medicine published by the National Administration of Traditional Chinese Medicine. It consists of Ostreae Concha, Astragali Radix, Ephedrae Radix et Rhizoma, and wheat, with the effect of replenishing qi and stopping sweating. It is a common formula in the clinical treatment with traditional Chinese medicine. This study analyzes the historical evolution, composition, dosage, original plants and their processing methods, decocting method, efficacy, indications, and modern clinical application of Mulisan by tracing, comparative analysis, and bibliometric methods. The results showed that Mulisan firstly appeared in the Pulse Classic written by WANG Shuhe in the Western Jin Dynasty. The formulation idea can be traced back to the Important Prescriptions Worth a Thousand Gold for Emergency in the Tang Dynasty. The herb composition, dosage, efficacy, and indications of Mulisan were first recorded in the Treatise on Diseases, Patterns, and formulas Related to Unification of the Three Etiologies in the Southern Song dynasty. In terms of original plants and their processing methods, Ostreae Concha is the shell of Ostrea rivularis, which should be calcined before use. Astragali Radix and Ephedrae Radix et Rhizoma are the dried roots of Astragalus membranaceus var. mongholicus and Ephedra sinica, respectively, the raw material of which should be used. Wheat is the dried mature fruit of T. aestivum, which can be used without processing, while the stir-fried fruit, being thin and deflated, demonstrates better effect. The composition of Mulisan is Ostreae Concha 8.26 g, Astragali Radix 8.26 g, Ephedrae Radix et Rhizoma 8.26 g, and wheat 7.92 g. The medicinal materials should be ground into coarse powder and decocted with 450 mL water to reach a volume of 240 mL, and the decoction should be taken warm. In modern clinical practice, Mulisan has a wide range of indications, including spontaneous sweating and night sweating caused by Yang deficiency or Qi deficiency. The clinical disease spectrum treated by Mulisan involves endocrine system diseases, neurological diseases, respiratory system diseases, and cancer. This formula plays a significant role in the treatment of internal medicine diseases in traditional Chinese medicine. This study aims to provide a scientific basis for the subsequent research, development, and clinical application of Mulisan.
8.Value of different noninvasive diagnostic models in the diagnosis of esophageal and gastric varices with significant portal hypertension in compensated hepatitis B cirrhosis
Cheng LIU ; Jiayi ZENG ; Mengbing FANG ; Zhiheng CHEN ; Bei GUI ; Fengming ZHAO ; Jingkai YUAN ; Chaozhen ZHANG ; Meijie SHI ; Yubao XIE ; Xiaoling CHI ; Huanming XIAO
Journal of Clinical Hepatology 2025;41(2):263-268
ObjectiveTo investigate the value of different noninvasive diagnostic models in the diagnosis of esophageal and gastric varices since there is a high risk of esophageal and gastric varices in patients with compensated hepatitis B cirrhosis and significant portal hypertension, and to provide a basis for the early diagnosis of esophageal and gastric varices. MethodsA total of 108 patients with significant portal hypertension due to compensated hepatitis B cirrhosis who attended Guangdong Provincial Hospital of Traditional Chinese Medicine from November 2017 to November 2023 were enrolled, and according to the presence or absence of esophageal and gastric varices under gastroscopy, they were divided into esophageal and gastric varices group (GOV group) and non-esophageal and gastric varices group (NGOV group). Related data were collected, including age, sex, imaging findings, and laboratory markers. The chi-square test was used for comparison of categorical data between groups; the least significant difference t-test was used for comparison of normally distributed continuous data between groups, and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between groups. The receiver operating characteristic (ROC) curve was plotted to evaluate the diagnostic value of five scoring models, i.e., fibrosis-4 (FIB-4), LOK index, LPRI, aspartate aminotransferase-to-platelet ratio index (APRI), and aspartate aminotransferase/alanine aminotransferase ratio (AAR). The binary logistic regression method was used to establish a combined model, and the area under the ROC curve (AUC) was compared between the combined model and each scoring model used alone. The Delong test was used to compare the AUC value between any two noninvasive diagnostic models. ResultsThere were 55 patients in the GOV group and 53 patients in the NGOV group. Compared with the NGOV group, the GOV group had a significantly higher age (52.64±1.44 years vs 47.96±1.68 years, t=0.453, P<0.05) and significantly lower levels of alanine aminotransferase [42.00 (24.00 — 17.00) U/L vs 82.00 (46.00 — 271.00) U/L, Z=-3.065, P<0.05], aspartate aminotransferase [44.00 (32.00 — 96.00) U/L vs 62.00 (42.50 — 154.50) U/L,Z=-2.351, P<0.05], and platelet count [100.00 (69.00 — 120.00)×109/L vs 119.00 (108.50 — 140.50)×109/L, Z=-3.667, P<0.05]. The ROC curve analysis showed that FIB-4, LOK index, LPRI, and AAR used alone had an accuracy of 0.667, 0.681, 0.730, and 0.639, respectively, in the diagnosis of esophageal and gastric varices (all P<0.05), and the positive diagnostic rates of GOV were 69.97%, 65.28%, 67.33%, and 58.86%, respectively, with no significant differences in AUC values (all P>0.05), while APRI used alone had no diagnostic value (P>0.05). A combined model (LAF) was established based on the binary logistic regression analysis and had an AUC of 0.805 and a positive diagnostic rate of GOV of 75.80%, with a significantly higher AUC than FIB-4, LOK index, LPRI, and AAR used alone (Z=-2.773,-2.479,-2.206, and-2.672, all P<0.05). ConclusionFIB-4, LOK index, LPRI, and AAR have a similar diagnostic value for esophageal and gastric varices in patients with compensated hepatitis B cirrhosis and significant portal hypertension, and APRI alone has no diagnostic value. The combined model LAF had the best diagnostic efficacy, which provides a certain reference for clinical promotion and application.
9.Role of Macrophage Activation and Polarization in Diabetes Mellitus and Its Related Complications and Traditional Chinese Medicine Intervention
Zhichao CHEN ; Qiaoni LIN ; Liya SUN ; Jinxi WANG ; Zishan FU ; Yufeng YANG ; Yan SHI
Chinese Journal of Experimental Traditional Medical Formulae 2025;31(6):311-320
The occurrence of diabetes mellitus (DM) is closely related to insulin resistance and islet β cell dysfunction. Modern studies have found that macrophages are widely present in the liver,fat,skeletal muscle,islets, and other tissues and organs. Macrophage M1/M2 polarization plays an important role in the occurrence and development of diabetes mellitus and its related complications by intervening in inflammatory response,improving insulin resistance,and promoting tissue repair. Most of the traditional Chinese medicines that regulate the activation and polarization of macrophages are Qi-replenishing and Yin-nourishing,heat-clearing, and detoxicating medicinal,which are consistent with the etiology and pathogenesis of diabetes and its related complications. Therefore,by summarizing the mechanisms between macrophage activation,polarization, and insulin resistance in various tissues,this paper reviewed traditional Chinese medicine and its effective components and compounds in improving diabetes mellitus and its related complications through multi-channel regulation of macrophage polarization and regulation of M1/M2 ratio,providing references for the future treatment of DM and its related complications with traditional Chinese medicine.
10.In vitro studies of the anti-inflammatory activity of micheliolide on myeloproliferative neoplasm cell lines
Meng CHEN ; Jinqin LIU ; Ying ZHANG ; Zhexin SHI ; Zhijian XIAO
Journal of Beijing University of Traditional Chinese Medicine 2025;48(1):68-79
Objective:
The effects and molecular mechanisms of micheliolide on cytokine expression in myeloproliferative neoplasm cell lines were explored based on the signal transducer and activator of transcription 3 (STAT3)/nuclear factor-kappa B (NF-κB) signaling pathways.
Methods:
The UKE-1 and SET-2 cell lines were investigated, and micheliolide concentrations were screened using the CCK-8 assay. The UKE-1 and SET-2 cells were divided into the control and micheliolide-treated groups at concentrations of 2.5, 5.0, and 10.0 μmol/L. Each group received 1 mL of micheliolide solution at final concentrations of 2.5, 5.0, and 10.0 μmol/L, respectively, whereas the control group only received an equal volume of culture medium. The inhibition rates of interleukin-1β(IL-1β), tumor necrosis factor-α (TNF-α), and chemokine ligand 2 (CCL2) mRNA expression in cells from each group were detected using real-time fluorescent PCR (RT-PCR). Western blotting was used to measure STAT3 and phosphorylated STAT3 (p-STAT3) protein expression levels in cells from each group. Reversal experiments with reduced glutathione and dithiothreitol were performed using UKE-1 cells, which were divided into the control group, micheliolide, micheliolide + glutathione, micheliolide + dithiothreitol, and glutathione + dithiothreitol groups. Western blotting was used to detect the STAT3 and p-STAT3 protein expression levels in the cells of each group. UKE-1 cells were stimulated with TNF-α (5 μg/L) to replicate a pathological model of excessive cytokine secretion. Subsequently, UKE-1 cells were divided into the control, model, and three micheliolide-treated groups at concentrations of 2.5, 5.0, and 10.0 μmol/L. RT-PCR was used to measure the indicators above. An enzyme-linked immunosorbent assay (ELISA) was used to detect the CCL2 content in the cell culture media of each group. Western blotting was performed to assess the protein expression levels of STAT3, p-STAT3, and proteins related to the NF-κB signaling pathway.
Results:
Compared with the control group, the proliferation inhibition rates of UKE-1 cells at 24, 48, and 72 h increased in the micheliolide-treated groups at concentrations of 2.5, 5.0, 10.0, and 20.0 μmol/L. Similarly, the proliferation inhibition rates of SET-2 at 48 and 72 h increased in the micheliolide-treated groups at concentrations of 5.0, 10.0, and 20.0 μmol/L (P<0.05). Concentrations of 2.5, 5.0, and 10.0 μmol/L were selected for further studies to exclude the potential influence of high micheliolide concentrations on subsequent result owing to reduced cell numbers. Compared with the control group, the inhibition rates of TNF-α mRNA expression in UKE-1 and SET-2 cells increased in the micheliolide-treated groups at concentrations of 2.5, 5.0, and 10.0 μmol/L. Similarly, the inhibition rates of IL-1β mRNA expression in UKE-1 and SET-2 cells also increased in the micheliolide-treated groups at concentrations of 5.0 and 10.0 μmol/L. Additionally, the inhibition rate of CCL2 mRNA expression in UKE-1 and SET-2 cells increased in the micheliolide-treated group at a concentration of 10 μmol/L (P<0.05). Compared with the model group, the inhibition rates of TNF-α, IL-1β, and CCL2 mRNA expression in UKE-1 cells increased in the micheliolide-treated groups at concentrations of 2.5, 5.0, and 10.0 μmol/L after stimulation with TNF-α (P<0.05). ELISA showed that compared with the control group, the CCL2 content in UKE-1 cells increased in the model group. Compared with the model group, the CCL2 content in UKE-1 cells decreased in the micheliolide-treated groups at concentrations of 2.5, 5.0, and 10.0 μmol/L (P<0.05). Western blotting showed that compared with the control group, the p-STAT3 protein expression levels in UKE-1 and SET-2 cells were downregulated in the micheliolide-treated groups at concentrations of 5.0 and 10.0 μmol/L, and the protein expression level of STAT3 in SET-2 was also downregulated (P<0.05). Compared with the control group, the p-STAT3 expression level in UKE-1 cells decreased in the micheliolide group in the reductive glutathione and dithiothreitol reversal experiments. Compared with the micheliolide group, the p-STAT3 protein expression levels in UKE-1 cells increased in the micheliolide + dithiothreitol and micheliolide + glutathione groups (P<0.05). Compared with the control group, the model group showed increased p-STAT3, p-IκKα/β, p-IκBα, and p-NF-κB p65 protein expression and decreased IκBα protein expression after stimulation with TNF-α. Compared with the model group, the micheliolide-treated groups showed decreased p-IκKα/β, p-IκBα, p-STAT3, and p-NF-κB p65 protein expression at concentrations of 2.5, 5.0, and 10.0 μmol/L, whereas the micheliolide-treated groups showed increased IκBα protein expression at concentrations of 5.0 and 10.0 μmol/L (P<0.05).
Conclusion
Micheliolide potently suppresses IL-1β, TNF-α, and CCL2 mRNA expression in UKE-1 and SET-2 cells, as well as CCL2 secretion by UKE-1 cells, which may be associated with STAT3 phosphorylation suppression and NF-κB signaling pathway activation.


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