1.Expression of Interleukin-10 and Interleukin-15 in Patients with Multiple Myeloma and Its Clinical Significance.
Xiao-Dong ZHANG ; Meng LI ; Hai-Xia LIU ; Dan-Feng ZHANG
Journal of Experimental Hematology 2025;33(3):828-833
OBJECTIVE:
To investigate the expression and clinical significance of interleukin-10 (IL-10) and interleukin-15 (IL-15) in patients with multiple myeloma (MM).
METHODS:
Eighty newly diagnosed MM patients in Department of Hematology in Nanyang First People's Hospital from September 2020 to January 2023 were selected as observation group, and 80 healthy people in our hospital were selected as control group. The expression of IL-10 and IL-15 of the two groups were detected, and survival analysis was conducted for the MM patients.
RESULTS:
The levels of IL-10 and IL-15 in the observation group before treatment were significantly higher than those in the control group (both P <0.05). The levels of IL-10 and IL-15 in different DS stage had significant differences in MM patients (both P <0.05). The levels of IL-10 and IL-15 in stage II and stage III had no significant differences, which were both significantly higher than those in stage I (both P <0.05). The levels of IL-10 and IL-15 in sCR+CR group after treatment were significantly lower than those before treatment (both P <0.05). The levels of IL-10 and IL-15 in VGPR+PR group after treatment were also significantly lower than those before treatment (both P <0.05), but higher than those in the sCR+CR group (both P <0.05). The progression-free survival (PFS) and overall survival (OS) of patients with IL-10>22.01 pg/ml were significantly shorter than those with IL-10≤22.01 pg/ml (both P <0.001). The PFS and OS of patients with IL-15>48.56 pg/ml were also shorter than those with IL-15≤48.56 pg/ml (both P <0.05).
CONCLUSIONS
The levels of IL-10 and IL-15 in MM patients are closely related to efficacy and prognosis, and both decreased after treatment. The more reduction, the better effect. Patients with IL-10 and IL-15 below the threshold have longer median PFS and OS.
Humans
;
Interleukin-10/metabolism*
;
Multiple Myeloma/metabolism*
;
Interleukin-15/metabolism*
;
Prognosis
;
Female
;
Male
;
Middle Aged
;
Clinical Relevance
2.The Expression and Significance of PD-1, Th1, Th2, and Th17 Cytokines in Multiple Myeloma.
Di LIU ; Qian CHEN ; Ling LI ; Hua-Xin JIANG
Journal of Experimental Hematology 2025;33(5):1366-1373
OBJECTIVE:
To explore the expression and clinical significance of programmed death receptor 1 (PD-1), Th1, Th2, and Th17 cytokines in multiple myeloma (MM).
METHODS:
A total of 76 MM patients treated in the Tengzhou Central People's Hospital from May 2021 to May 2023 were collected as MM group, and 48 healthy individuals who underwent physical examination during the same period were included as control group. The expression of PD-1 on the surface of CD4+ and CD8+ T cells and the levels of serum Th1 cytokines [interleukin (IL) -2, interferon γ (IFN-γ), tumor necrosis factor α (TNF-α)], Th2 cytokines (IL-4, IL-6, IL-10) and Th17 cytokines (IL-17) were detected in the two groups. Spearman correlation was used to examine the relationship between PD-1, Th1, Th2 and Th17 cytokines and clinical stage and immune typing of MM patients. Multivariate logistic regression analysis was used to analyze the related factors affecting the efficacy of chemotherapy in MM patients, and the factors were tested for multicollinearity. Receiver operating characteristic (ROC) curve was drawn to analyze the predictive value of PD-1, Th1, Th2 and Th17 cytokines in chemotherapy efficacy of MM patients.
RESULTS:
The levels of CD4+T PD-1, CD8+T PD-1, IL-4, IL-6, IL-10, and IL-17 in the MM group were higher than those in the control group, while the levels of IL-2, IFN-γ, and TNF-α were lower (all P <0.001). The levels of CD4+T PD-1, CD8+T PD-1, IL-4, IL-6, IL-10, and IL-17 in R-ISS stage III patients were higher than those in stage II and I patients, and the levels in stage II patients were higher than those in stage I patients (all P <0.05). The IL-2 level in R-ISS stage III patients was lower than that in stage II and I patients, and IL-2 level in R-ISS stage II patients was lower than that in stage I patients (all P <0.05). The levels of CD4+T PD-1, CD8+T PD-1, IL-4, IL-6, IL-10, and IL-17 in IgG patients were higher than those in IgA, light chain, and non secretory patients, while the level of IL-2 was lower (all P <0.05). Correlation analysis showed that CD4+T PD-1, CD8+T PD-1, IL-4, IL-6, IL-10, and IL-17 were positively correlated with R-ISS staging in MM patients (r =0.623, 0.635, 0.728, 0.330, 0.742, 0.412), and negatively correlated with immune classification (r =-0.664, -0.756, -0.642, -0.479, -0.613, -0.323). IL-2 was negatively correlated with R-ISS staging in MM patients (r =-0.280), and positively correlated with immune classification (r =0.483). The levels of CD4+T PD-1, CD8+T PD-1, IL-4, IL-6, IL-10, and IL-17 in the non-remission group were higher than those in the remission group, while the level of IL-2 was lower (all P <0.001). Multivariate logistic regression analysis showed that the increased CD4+T PD-1, CD8+T PD-1, IL-4, IL-6, IL-10 and IL-17 were risk factors for the efficacy of chemotherapy in MM patients (OR >1, P <0.05), while the increased IL-2 was a protective factor (OR < 1, P <0.05). The results of multicollinearity test showed that the tolerance of the seven factors included was between 0.714-0.885, and the variance inflation factor was between 1.130-1.400. There was no multicollinearity. The ROC curve analysis results showed that the area under the curve for the combined prediction of chemotherapy efficacy in MM patients by the above 7 factors was 0.942, with specificity of 0.741 and sensitivity of 0.909.
CONCLUSION
The expression levels of PD-1 on the surface of CD4+ and CD8+ T cells and serum Th2 and Th17 cytokines in MM patients are high, while Th1 cytokines are low. PD-1, Th1, Th2, and Th17 cytokines are related to clinical stage and immune classification of MM patients. The combined detection of these indicators can help predict the chemotherapy efficacy of MM patients.
Humans
;
Programmed Cell Death 1 Receptor/metabolism*
;
Multiple Myeloma/blood*
;
Cytokines/metabolism*
;
Th17 Cells/metabolism*
;
Th1 Cells/metabolism*
;
Th2 Cells/metabolism*
;
Female
;
Male
;
Interleukin-10
;
Interferon-gamma
;
Middle Aged
;
Interleukin-17
;
Interleukin-2
;
Interleukin-4
;
Tumor Necrosis Factor-alpha
;
Interleukin-6
;
Aged
3.Clinical characteristics and prognosis analysis of 108 cases of recurrent nasopharyngeal carcinoma from a single center.
Qing WANG ; Fusheng LIN ; Ran ZHANG ; Lin GAO ; Xingqian ZHAO ; Jie YANG ; Xiaojiang LI
Journal of Clinical Otorhinolaryngology Head and Neck Surgery 2025;39(8):743-748
Objective:Retrospective analysis of the correlation between clinicopathologic features and related indexes and prognosis in patients with recurrent nasopharyngeal carcinoma. Methods:One hundred and eight nasopharyngeal cancer(NPC) patients with post-treatment recurrence in Yunnan Cancer Hospital from January 2013 to January 2018 were collected, and the survival time was estimated by Kaplan-Meier method, and clinicopathological characteristics were analyzed by log-rank test; risk factors and prognosis were analyzed by Cox proportional risk model for single-factor and multifactorial analysis. A P-value <0.05 was considered statistically significant. Results:The median survival of all patients was 54 months, with a 3-year survival rate of 80.2% and a 5-year survival rate of 39.8%. The 5-year overall survival rate was 50.2% for patients >46 years old and 27.9% for patients ≤46 years old(P<0.05), a statistically significant difference. Univariate analysis showed that overall survival was associated with age, chemotherapy regimen, EBV early antigen IgA, plasma D-dimer, glycan antigen-125, γ-interferon, α-tumor necrosis factor, IL-10, and IL-4(P<0.05). Multifactorial analysis revealed that age, chemotherapy regimen, EBV early antigen IgA, plasma D-dimer, glycan antigen-125, and interleukin 10 were independent influences on the prognosis of recurrent nasopharyngeal carcinoma(P<0.05). Conclusion:Differences in chemotherapy regimens affect the prognosis of recurrent nasopharyngeal carcinoma. Elevated plasma D-dimer, glycan antigen 125, and interleukin 10 levels affect the overall survival of recurrent nasopharyngeal carcinoma, which may be a valid independent prognostic factor, and are expected to provide new biomarkers for nasopharyngeal carcinoma in the clinic.
Humans
;
Prognosis
;
Nasopharyngeal Neoplasms/mortality*
;
Nasopharyngeal Carcinoma
;
Retrospective Studies
;
Neoplasm Recurrence, Local
;
Male
;
Middle Aged
;
Female
;
Survival Rate
;
Adult
;
Risk Factors
;
Interleukin-10/blood*
;
Aged
;
Proportional Hazards Models
4.High glucose induces pro-inflammatory polarization of macrophages by inhibiting immune-responsive gene 1 expression.
Wei LUO ; Yuhang WANG ; Yansong LIU ; Yuanyuan WANG ; Lei AI
Journal of Southern Medical University 2025;45(1):1-9
OBJECTIVES:
To investigate the effect of high glucose on macrophage polarization and the role of immune-responsive gene 1 (IRG1) in mediating its effect.
METHODS:
RAW264.7 cells were transfected with IRG1-overexpressing plasmid or IRG1 siRNA via electroporation and cultured in either normal or high glucose for 72 h to observe the changes in cell viability and morphology using CCK-8 assay and phase contrast microscopy. The protein levels of IRG1, iNOS, Arg-1, IL-1β and IL-10 in the treated cells were detected with Western blotting, and the fluorescence intensities of iNOS and Arg-1 were detected using immunofluorescence assay. The protein levels of IL-1β and IL-10 in the culture medium were determined with ELISA.
RESULTS:
High glucose exposure significantly reduced IRG1 and Arg-1 expressions, increased iNOS and IL-1β expressions and IL-1β secretion, and decreased IL-10 level in RAW264.7 cells. Transfection with the IRG1-overexpressing plasmid provided the cells with obvious resistance to high glucose-induced changes in iNOS, Arg-1, IL-1β and IL-10, whereas IRG1 knockdown further enhanced the effects of high glucose exposure on Arg-1 expression and the expression and secretion of IL-10.
CONCLUSIONS
High glucose promotes M1 polarization of the macrophages possibly through a mechanism to inhibit the expression of IRG1 protein, thus leading to chronic inflammatory response.
Animals
;
Mice
;
Macrophages/drug effects*
;
Glucose/pharmacology*
;
Interleukin-10/metabolism*
;
Nitric Oxide Synthase Type II/metabolism*
;
RAW 264.7 Cells
;
Interleukin-1beta/metabolism*
;
Arginase/metabolism*
;
RNA, Small Interfering/genetics*
;
Transfection
;
Inflammation
5.Schistosoma japonicum cystatin has protective effects against "two-hit" sepsis in mice by regulating the inflammatory microenvironment.
Wenjuan DUO ; Yixiang WANG ; Jiaxing WANG ; Xinlong XU ; Linxian LI ; Dongchen YANG ; Qili SHEN ; Lichun YANG ; Xiaojing LIU ; Qiwang JING ; Liang CHU ; Xiaodi YANG
Journal of Southern Medical University 2025;45(1):110-117
OBJECTIVES:
To evaluate the protective effect of Schistosoma japonicum cystatin (rSj-Cystatin) in a mouse mode of "two-hit" sepsis.
METHODS:
Sixty male C57BL/6 mice randomized equally into sham-operated group, protein group, "two-hit" modeling group, and protein intervention group. In the former two groups, the mice received an intraperitoneal injection of 100 μL PBS followed by exposure of the cecum and then by intraperitoneal injection of 100 μL PBS or 25 μg rSj-Cystatin 30 min later; In the latter two groups, 100 μL PBS containing LPS (5 mg/kg) was injected intraperitoneally 24 h before cecal ligation and puncture (CLP), and 100 μL PBS or 25 μg rSj-Cystatin were injected 30 min after CLP. At 12 h after rSj-Cystatin treatment, 6 mice from each group were sacrificed for detection of TNF-α, IL-6, IL-10, TGF-β, iNOS and Arg-1 in the serum, spleen, liver, lung and kidney tissues using ELISA, for examinations of liver, lung and kidney pathologies with HE staining, and for analysis of CD3+CD4+CD25+Foxp3+ T cell percentage in the spleen using flow cytometry. The remaining mice were observed for general condition and 72-h survival.
RESULTS:
The 72-h survival rates in the 4 groups were 100%, 100%, 0% and 20%, respectively, showing significant differences between the latter two groups. The mouse models of "two-hit" sepsis exhibited obvious tissue pathologies and significant elevations of TNF-α and IL-6 in both the serum and tissue homogenate, which were significantly ameliorated by rSj-Cystatin treatment. Treatment with rSj-Cystatin also increased IL-10 and TGF-β levels and spleen CD3+CD4+CD25+Foxp3+ T cell percentage. The septic mouse models also showed increased iNOS levels in all the detected tissues and a decreased Arg-1 level in the kidney, and these changes were obviously improved by rSj-Cystatin treatment.
CONCLUSIONS
rSj-Cystatin has a protective effect against "two-hit" sepsis in mice by regulating the inflammatory microenvironment.
Animals
;
Mice
;
Sepsis/drug therapy*
;
Male
;
Schistosoma japonicum/chemistry*
;
Mice, Inbred C57BL
;
Cystatins/therapeutic use*
;
Interleukin-10/metabolism*
;
Interleukin-6/blood*
;
Tumor Necrosis Factor-alpha/blood*
;
Disease Models, Animal
;
Transforming Growth Factor beta/metabolism*
6.Effects of liver fibrosis induced by iron overload on M2 polarization of macrophages in mice.
Jiawen YU ; Yi ZHOU ; Chunmei QIAN ; Lan MU ; Renye QUE
Journal of Southern Medical University 2025;45(4):684-691
OBJECTIVES:
To observe the evolution of intrahepatic macrophage polarization in mice with liver fibrosis induced by iron overload.
METHODS:
Thirty-two C57BL/6 mice (6-8 weeks) were randomized into control group (n=8) and liver fibrosis model group (n=24) induced by aidly intraperitoneal injection of iron dextran. At the 3rd, 5th, and 7th weeks of modeling, 8 mice in the model group were sacrificed for observing liver fibrosis using Masson, Sirius Red and immunohistochemical staining and detecting serum levels of ALT, AST and the levels of serum iron, ferritin, liver total Fe and ferrous Fe. iNOS+/F4/80+ cells and CD206+/F4/80+ cells were detected by double immunofluorescence assay to observe the proportion and distribution of M1 and M2 macrophages. The hepatic expressions of Arg-1, iNOS, IL-6, IL-10, and TNF‑α proteins were detected using Western blotting or ELISA, and the expression of CD206 mRNA was detected using RT-PCR.
RESULTS:
The mice in the model group showed gradual increase of fibrous tissue hyperplasia in the portal area over time, structural destruction of the hepatic lobules and formation of pseudolobules. With the passage of time during modeling, the rat models showed significantly increased hepatic expressions of α-SMA and COL-1, elevated serum levels of ALT, AST, Fe, ferritin, and increased liver total Fe and ferrous Fe levels. The expressions of M1 polarization markers IL-6, TNF‑α, and iNOS all increased with time and reached their peak levels at the 3rd week; The expressions of M2 polarization markers (IL-10 and Arg-1 proteins and CD206 mRNA) significantly increased in the 3rd week and but decreased in the 5th and 7th weeks.
CONCLUSIONS
Iron overload promotes M1 polarization of macrophages in mice. Liver fibrosis in the early stage promotes M2 polarization of macrophages but negatively regulate M2 polarization at later stages.
Animals
;
Mice
;
Mice, Inbred C57BL
;
Iron Overload/pathology*
;
Macrophages/metabolism*
;
Male
;
Liver Cirrhosis/etiology*
;
Nitric Oxide Synthase Type II/metabolism*
;
Interleukin-10/metabolism*
;
Liver/pathology*
;
Interleukin-6/metabolism*
;
Mannose Receptor
;
Tumor Necrosis Factor-alpha/metabolism*
;
Mannose-Binding Lectins/metabolism*
;
Arginase
7.Pirfenidone inhibits bladder cancer xenograft growth in mice by regulating regulatory T cells.
Hongbo ZHANG ; Mengyu YAN ; Jiandong ZHANG ; Peiwang SUN ; Rui WANG ; Yuanyuan GUO
Journal of Southern Medical University 2025;45(7):1513-1518
OBJECTIVES:
To investigate the inhibitory effect of pirfenidone (PFD) on growth of bladder cancer xenograft and its regulatory effect on Treg cells in tumor-bearing mice.
METHODS:
Thirty-two C57BL/6 mice bearing ectopic bladder tumors were randomized into control and PFD groups (n=16). In PFD group, PFD was administered orally at the daily dose of 500 mg/kg, and tumor growth and survival of the mice were monitored. After treatment for 21 days, the tumors and vital organs were harvested for analysis. Immunohistochemistry was used to assess CD3, CD4, CD8, and FOXP3 expressions in the tumors. Flow cytometry and RT-qPCR were used to analyze the percentage of CD4⁺CD25⁺FOXP3⁺ Treg cells and IL-2, IL-10, and IL-35 expressions in the tumors and spleens; organ damage of the mice was examined with HE staining.
RESULTS:
Compared with the control group, the PFD-treated mice exhibited significantly lower tumor growth rate with smaller tumor volumes at day 21, along with improved survival at day 28. Immunohistochemistry revealed no significant differences in the infiltration of CD3⁺ and CD8⁺ cells between the two groups, but the percentages of CD4⁺ and FOXP3⁺ cells were significantly lower in the tumors of PFD-treated mice. Flow cytometric analysis confirmed a decrease in CD4⁺CD25⁺FOXP3⁺ Treg cells in the tumors from PFD-treated mice, which also had reduced expression levels of IL-2, IL-10 and IL-35 mRNAs in the tumors. No significant differences were found in Treg cell populations or cytokine expressions in the spleen tissues between the two groups. HE staining showed obvious organ damage in neither of the groups.
CONCLUSIONS
PFD inhibits bladder cancer growth and enhances survival of tumor-bearing mice possibly by suppressing Treg cells in the tumor microenvironment.
Animals
;
Urinary Bladder Neoplasms/drug therapy*
;
Mice
;
T-Lymphocytes, Regulatory/metabolism*
;
Mice, Inbred C57BL
;
Interleukins/metabolism*
;
Interleukin-10/metabolism*
;
Cell Line, Tumor
;
Interleukin-2/metabolism*
;
Xenograft Model Antitumor Assays
;
Female
8.Molecular mechanism of Xixian Pills for improving rheumatoid arthritis in rats: a proteomic analysis.
Yahui LI ; Xin YANG ; Xueming YAO ; Cong HUANG
Journal of Southern Medical University 2025;45(11):2330-2339
OBJECTIVES:
To analyze the molecular mechanism of Xixian Pills for treatment of rheumatoid arthritis (RA).
METHODS:
Forty-eight rats were randomized into 6 groups (n=8), including a normal control group, a collagen-induced arthritis (CIA) model group, 3 Xixian Pills treatment (200, 400 and 800 mg/kg) groups, and a Tripterygium glycosides tablet (TGT) treatment group. In the latter 4 groups, the rats were treated with daily gavage of Xixian Pills or TGT 2 weeks after CIA modeling for 3 consecutive weeks. The differentially expressed proteins in high-dose Xixian Pills group and the model group compared with the normal control group were screened based on the tandem mass spectrometry tag (TMT) technology, and the core targets and signaling pathways were analyzed. The immune cell infiltration and gene expression data were analyzed using ggplot2 and tidyverse packages, and the correlation coefficients between the core targets and the immune cells were calculated.
RESULTS:
The CIA rats showed significantly increased serum levels of TNF-α and IL-6 and lowered serum IL-10 level. Treatments with high- and medium-dose Xixian Pills and TGT all significantly reduced serum TNF‑α and IL-6 and increased IL-10 levels in CIA rats. Proteomic analysis identified 160 differential proteins between the model group and high-dose Xixian Pills group, and the core targets included CCL5, STAT1, GZMB and IL7R. The areas under the ROC curve of CCL5 and STAT1 were both greater than 0.9. Immunohistochemical and immunofluorescence staining revealed increased levels of CCL5 and STAT1 in the ankle joints of CIA rats, which were significantly decreased after treatment with Xixian Pills.
CONCLUSIONS
Treatment with Xixian Pills offers protection of the joints in CIA rats possibly by inhibiting joint inflammation via regulating protein expressions of CCL5 and STAT1.
Animals
;
Drugs, Chinese Herbal/pharmacology*
;
Rats
;
Arthritis, Rheumatoid/metabolism*
;
Proteomics
;
Tripterygium/chemistry*
;
Arthritis, Experimental/metabolism*
;
Tumor Necrosis Factor-alpha/blood*
;
Interleukin-10/blood*
;
Interleukin-6/blood*
;
Male
;
Rats, Sprague-Dawley
;
Signal Transduction
9.Effect of transcutaneous electrical acupoint stimulation on postoperative pain in patients undergoing modified radical mastectomy for breast cancer.
Li YAN ; Bin SUN ; Meiyan ZHOU ; Yan ZHANG ; Fei GAO ; Qianwen ZHAO ; Liwei WANG
Chinese Acupuncture & Moxibustion 2025;45(2):162-166
OBJECTIVE:
To observe the effect of transcutaneous electrical acupoint stimulation (TEAS) on postoperative pain in patients undergoing modified radical mastectomy for breast cancer.
METHODS:
A total of 140 female patients scheduled for unilateral modified radical mastectomy for breast cancer undergoing general anesthesia were randomized into a TEAS group (70 cases) and a sham TEAS group (70 cases, 2 cases dropped out). Patients in both groups received TEAS or sham TEAS at bilateral Neiguan (PC6), Zusanli (ST36), and Danzhong (CV17), respectively, from 30 min before anesthesia induction until the end of surgery, and on 1st, 2nd, and 3rd days after surgery for 30 min a time, once a day. On 1st, 2nd, and 3rd days after surgery, the pain visual analogue scale (VAS) score was observed; on 3, 6, 12 months after surgery, the incidence rate of chronic pain was observed; before surgery, and on 1st, 3rd, and 7th days after surgery, the serum levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10 were detected; the number of analgesia pump press, rescue analgesia, and the occurrence of adverse reaction after surgery were recorded in the two groups.
RESULTS:
In the TEAS group, the VAS scores on 1st and 2nd days after surgery, and the incidence rates of chronic pain on 3 and 6 months after surgery were lower than those in the sham TEAS group (P<0.05). On 1st, 3rd, and 7th days after surgery, the serum levels of TNF-α, IL-6, and IL-10 were increased compared with those before surgery in both groups (P<0.05, P<0.01); the above indexes in the TEAS group were lower than those in the sham TEAS group (P<0.05). The number of analgesia pump press and the incidence rate of rescue analgesia after surgery in the TEAS group were lower than those in the sham TEAS group (P<0.05). There was no statistically significant difference in the incidence of adverse reactions after surgery between the two groups (P>0.05).
CONCLUSION
TEAS can effectively improve both the postoperative acute pain and chronic pain in patients undergoing modified radical mastectomy for breast cancer, the mechanism may relate to inhibiting the inflammatory reaction.
Humans
;
Female
;
Acupuncture Points
;
Pain, Postoperative/blood*
;
Middle Aged
;
Breast Neoplasms/surgery*
;
Adult
;
Transcutaneous Electric Nerve Stimulation
;
Mastectomy, Modified Radical/adverse effects*
;
Interleukin-6/blood*
;
Tumor Necrosis Factor-alpha/blood*
;
Interleukin-10/blood*
;
Aged
10.Effects of electroacupuncture at changbing fang on autophagy of colonic cells and gut microbiota in ulcerative colitis of rats.
Huichao XU ; Tian WU ; Jianheng HAO ; Ronglin WU ; Bingbei YAN ; Haijun WANG ; Laixi JI
Chinese Acupuncture & Moxibustion 2025;45(5):657-669
OBJECTIVE:
To observe the effects of electroacupuncture (EA) at changbing fang (prescription for intestinal disease) on autophagy of colonic cells and gut microbiota in rats with ulcerative colitis (UC), and to explore the mechanism of EA in the treatment of UC.
METHODS:
Thirty-two SD male rats were randomly divided into a control group, a model group, an EA group and a sham-EA group, with 8 rats in each group. Except the control group, the UC rat model was established by free drinking of 5% dextran sulfate sodium solution for 7 days in the other groups. In the EA group, changbing fang was adopted, in which, EA was applied at "Tianshu" (ST25) and "Shangjuxu" (ST37), at disperse-dense wave and frequency of 10 Hz/50 Hz, for 20 min in each intervention. In the sham-EA group, shallow transcutaneous puncture was performed at the sites, 5 mm away from the points as the EA group, with the same parameters as the EA group. The intervention was delivered once daily for 3 consecutive days. The body weight was measured daily and the disease activity index (DAI) score was calculated before and after intervention. After intervention completion, the colon length was measured. Using HE staining, the colon morphology was observed and the score of colonic pathology was assessed. With ELISA adopted, the contents of tumor necrosis factor (TNF-α), interleukin (IL)-1β, IL-2 and IL-10 in the serum of the rats were detected. The ultrastructure of the colon tissue was observed under electron microscopy. Using Western blotting, the protein expression was detected for microtubule-associated protein 1 light chain 3 (LC3)Ⅱ, LC3Ⅰ, autophagy-related genes (ATG) 5, ATG12, sequestosome 1 (p62), phosphatidylinositol 3-kinase (PI3K), phosphorylated protein kinase B (p-AKT), protein kinase B (AKT), and phosphorylated mammalian target of rapamycin (p-mTOR), mammalian target of rapamycin (mTOR) in the colon tissue. The mRNA expression of PI3K, AKT and m-TOR in the colon tissue was detected by real-time fluorescence quantitative PCR. The 16S rRNA gene sequencing was used to analyze the structure of gut flora in the feces of rats.
RESULTS:
From day 1 to day 7, compared with the control group, the body weight decreased in the model group, EA group, and SEA group (P<0.05, P<0.01). From day 9 to day 10, the EA group showed an increase in body weight compared with the model group and SEA group (P<0.05, P<0.01). Before intervention, the DAI score in the model group, EA group, and SEA group was higher than the score of the control group, respectively (P<0.01). After intervention, the DAI score in the EA group was reduced compared with the model group and SEA group (P<0.01). Compared with the control group, in the model group, the colon length of rats was shorter (P<0.01); it showed the distorted crypts, thinner mucosal layer, reduced goblet cells, inflammatory cell infiltration and the disarranged histological structure; and the pathological score of the colon tissue increased (P<0.01); the serum contents of TNF-α and IL-1β increased (P<0.01), and those of IL-2 and IL-10 decreased (P<0.01). The structure of colon epithelial cells was disarranged, with cilia pelt off, and a large number of vacuoles in the cytoplasm; the mitochondria were swollen, with unclear structure and cristae partially disappeared; and few autophagosomes were observed. The value of LC3Ⅱ/LC3Ⅰand the protein expression of ATG5 and ATG12 in the colon tissues were reduced (P<0.01), the protein expression of p62 and PI3K, and the values of p-AKT/AKT, and p-mTOR/mTOR increased (P<0.01), and mRNA expression of PI3K, AKT and mTOR was elevated (P<0.01). The indexes of Chao1, Ace and Shannon decreased (P<0.01). At the phylum level, the relative abundance of Firmicutes decreased (P<0.05), that of Bacteroidetes and Proteobacteria increased (P<0.05, P<0.01). At the genus level, the relevant abundance of Lactobacillus decreased (P<0.05), while that of Lachnospiraceae_NK4A136_group and Phascolarctobacterium increased (P<0.01, P<0.05 ). Compared with the model group and SEA group, in the EA group, the colon length increased (P<0.01), the infiltration of inflammatory cells was reduced, the arrangement of intestinal epithelial cells was arranged regularly, with a small amount of shedding, and the pathological score of the colon tissue decreased (P<0.01). The serum contents of TNF-α and IL-1β decreased (P<0.01), and those of IL-2 and IL-10 increased (P<0.01). The colonic epithelial cells were arranged relatively, the morphology of organelles was basically normal, and autophagosomes were visible. The value of LC3Ⅱ/LC3Ⅰand the protein expression of ATG5 and ATG12 in colon tissue increased (P<0.01, P<0.05), the protein expression of p62 and PI3K, and the values of p-AKT/AKT, and p-mTOR/mTOR decreased (P<0.01); and mRNA expression of PI3K, AKT, m-TOR was reduced (P<0.01). The indexes of Chao1, Ace and Shannon increased (P<0.01). At the phylum level, the relative abundance of Firmicutes increased (P<0.01), while that of Bacteroidetes decreased (P<0.01). At the genus level, the relative abundance of Lactobacillus increased (P<0.05), whereas that of Lachnospiraceae_NK4A136_group decreased (P<0.01). When compared with the model group, the relative abundance of Proteobacteria decreased (P<0.05), and that of Phascolarctobacterium was reduced (P<0.05) in the EA group.
CONCLUSION
EA at changbingfang alleviates UC symptoms probably through inhibiting the PI3K/AKT/mTOR signaling pathway to regulate colonic autophagy and improve the intestinal flora.
Animals
;
Electroacupuncture
;
Colitis, Ulcerative/metabolism*
;
Male
;
Rats
;
Gastrointestinal Microbiome
;
Rats, Sprague-Dawley
;
Colon/metabolism*
;
Humans
;
Autophagy
;
Acupuncture Points
;
Tumor Necrosis Factor-alpha/genetics*
;
Interleukin-10/genetics*

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