OBJECTIVE: To explore the therapeutic effects and underlying mechanisms of Dendrobium officinale (Tiepi Shihu) extract (DOE) on insomnia. METHODS: Forty-two male Sprague-Dawley rats were randomly divided into 6 groups (n=7 per group): normal control, model control, melatonin (MT, 40 mg/kg), and 3-dose DOE (0.25, 0.50, and 1.00 g/kg) groups. Rats were raised in a strong-light (10,000 LUX) and -noise (>80 db) environment (12 h/d) for 16 weeks to induce insomnia, and from week 10 to week 16, MT and DOE were correspondingly administered to rats. The behavior tests including sodium pentobarbital-induced sleep experiment, sucrose preference test, and autonomous activity test were used to evaluate changes in sleep and emotions of rats. The metabolic-related indicators such as blood pressure, blood viscosity, blood glucose, and uric acid in rats were measured. The pathological changes in the cornu ammonis 1 (CA1) region of rat brain were evaluated using hematoxylin and eosin staining and Nissl staining. Additionally, the sleep-related factors gamma-aminobutyric acid (GABA), glutamate (GA), 5-hydroxytryptamine (5-HT), and interleukin-6 (IL-6) were measured using enzyme linked immunosorbent assay. Finally, we screened potential sleep-improving receptors of DOE using polymerase chain reaction (PCR) array and validated the results with quantitative PCR and immunohistochemistry. RESULTS: DOE significantly improved rats' sleep and mood, increased the sodium pentobarbital-induced sleep time and sucrose preference index, and reduced autonomic activity times (P<0.05 or P<0.01). DOE also had a good effect on metabolic abnormalities, significantly reducing triglyceride, blood glucose, blood pressure, and blood viscosity indicators (P<0.05 or P<0.01). DOE significantly increased the GABA content in hippocampus and reduced the GA/GABA ratio and IL-6 level (P<0.05 or P<0.01). In addition, DOE improved the pathological changes such as the disorder of cell arrangement in the hippocampus and the decrease of Nissel bodies. Seven differential genes were screened by PCR array, and the GABA_A receptors (Gabra5, Gabra6, Gabrq) were selected for verification. The results showed that DOE could up-regulate their expressions (P<0.05 or P<0.01). CONCLUSION DOE demonstrated remarkable potential for improving insomnia, which may be through regulating GABA_A receptors expressions and GA/GABA ratio.
Animals ; Dendrobium/chemistry* ; Rats, Sprague-Dawley ; Male ; Sleep Initiation and Maintenance Disorders/blood* ; Plant Extracts/therapeutic use* ; Receptors, GABA-A/metabolism* ; Noise/adverse effects* ; Light/adverse effects* ; gamma-Aminobutyric Acid/metabolism* ; Sleep/drug effects* ; Rats ; Receptors, GABA/metabolism*

AIM To explore the mechanism of Yiqi Wenyang Huwei Decoction on airway inflammation improvement of rats with bronchial asthma based on IL-25/NF-κB signaling pathway.METHODS 60 rats were randomly divided into the control group,the model group,the dexamethasone group(0.2 mg/mL),the low-dose,medium-dose and high-dose Yiqi Wenyang Huwei Decoction groups(1,2,4 g/mL),with 10 rats in each group.Intraperitoneal injection of ovalbumin(OVA)and aluminum hydroxide suspension was applied to establish the rat asthma model,followed by 2-week corresponding dosing of the drugs.The rats of each group had their daily diet,mental status,hair growth and respiration observed;their differential count of inflammatory cells in bronchoalveolar lavage fluid(BALF)detected by automatic hematology analyzer;their pathological changes of lung tissue observed by HE staining;their pulmonary IL-25 protein expression detected by immunohistochemistry(IHC);their levels of IL-4,IL-5 and IL-13 in BALF measured by ELISA;their pulmonary expression of IL-25 and TRAF6 mRNA detected by RT-qPCR;and their pulmonary protein expressions of IL-25,TRAF6,IκBα,p-IκBα,NF-κB p65 and p-NF-κB p65 detected by Western blot.RESULTS Compared with the control group,the model group displayed severe damage of the lung tissue and infiltration of a large number of inflammatory cells;increased number of inflammatory cells and levels of IL-4,IL-5 and IL-13 in BALF(P<0.01);increased mRNA expressions of IL-25 and TRAF6,and pulmonary protein expressions of IL-25,TRAF6,p-IκBα/IκBα and p-NF-κB p65/NF-κB p65(P<0.01).Compared with the model group,all of the Yiqi Wenyang Huwei Decoction groups shared improved pulmonary infiltration of inflammatory cells;decreased number of inflammatory cells and levels of IL-4,IL-5 and IL-13 in BALF(P<0.05,P<0.01);and decreased mRNA expressions of IL-25 and TRAF6,and pulmonary protein expressions of IL-25,TRAF6,p-IκBα/IκBα and p-NF-κB p65/NF-κB p65(P<0.01).CONCLUSION Yiqi Wenyang Huwei Decoction can inhibit the airway inflammation in the rat model of bronchial asthma,which may be related to the inhibited activation of IL-25/NF-κB signaling pathway and the reduced expression of inflammatory factors.

Objective To investigate the role of HK2 and VDAC1 in diacetylmorphine-induced cardiomyocyte apoptosis.Methods A dose-escalation method was used to establish a rat model of diacetylmorphine addiction.Forty SD rats were randomly divided into three groups,the normal group(n=10)was injected with an equal amount of saline subcutaneously,the model group(n=15)was injected with 5 mg/kg of diacetylmorphine for the first time,and then the dose was increased by 2.5 mg/(kg·d)day by day for 20 days,and the group of model +10 D(n=15)continued to increase the dose based on the model group up to the 10th day.Lactate dehydrogenase(LDH)and glutamic oxaloacetic transaminase(GOT)were detected by ELISA;HE staining was used to observe the pathological changes of myocardial tissues in each group;TUNEL staining was used to detect apoptosis in myocardial tissues in each group;and immunohistochemistry,RT-q-analysis,and immunochemistry were used to detect apoptosis in myocardial tissues in each group.Immunohistochemistry,RT-qPCR and Western bl-ot were used to detect the mRNA and protein expression of HK2,VDAC1 and apoptosis-related factors.Results HE staining revealed that myocardial tissues exhibited different degrees of damage with the prolongation of diacetylmorphine intervention.Compared with the normal group,serum LDH,GOT content and myocardial apoptosis rate increased in the model group,mRNA and protein levels of HK2 and anti-apoptotic factor Bcl-2 decreased,mRNA and protein levels of VDAC1 and pro-apoptotic factors Bax and Caspase-3 increased,and the protein level of Clevead Caspase-3 increased;in the model +10 D group the above indexes,there was a statistically significant difference(P<0.05).Conclusion Diacetylmorphine can cause cardiomyocyte apoptosis,and VDAC1 may be involved in the process of cardiomyocyte apoptosis caused by diacetylmorphine.

Objective: To analyze the disease burden of pancreatic cancer in major Asian countries and forecast the burden of that in China, which helps to provide reference for the prevention and control of pancreatic cancer. Methods: Data on disease burden of pancreatic cancer among global and major Asian countries from on the Global Burden of Disease (GBD) 2019 were collected to describe burden distribution through the absolute numbers or standardized rates of incidence, death and disability adjusted life years (DALY) by year, sex and socio-demographic index. Estimated annual percentage changes (EAPC) was used to assess the trend of standardized rate. The proportion of deaths attributable to risk factors for pancreatic cancer in 2019 was used to compare by age, sex and region. ARIMA model was performed with R language to predict change of age-standardized incidence and death rates of pancreatic cancer from 2020 to 2029. Results: From 1990 to 2019, the standardized incidence rates of pancreatic cancer in China increased from 3.17/100 000 to 5.78/100 000, and the standardized death rate increased from 3.34/100 000 to 5.99/100 000. The increases exceeded other high-income Asia countries. In the past three decades, the standardized incidence, death and DALY rates of pancreatic cancer in global have increased year by year. Among the major countries in Asia, China has the highest growth rate of disease burden (EAPC of standardized incidence rates=2.32%, 95% CI: 2.10%-2.48% and EAPC of standardized death rate=2.25%, 95% CI: 2.03%-2.42%). In addition, incidence and death rates of pancreatic cancer in China are expected to continue on the rise between 2000 and 2029 by ARIMA model. Incidence rate is expected to increase 15.92% and death rate is expected to increase 15.86%. Conclusions: The standardized incidence and death rates of pancreatic cancer in China increase year by year with an increasing trend for the burden of disease. The disease burden of pancreatic cancer is expected to rise due to the increase and aging of the population. Preventive measures should be adopted to decrease the burden of the pancreatic cancer.
Asia/epidemiology* ; Global Burden of Disease ; Humans ; Incidence ; Pancreatic Neoplasms/epidemiology* ; Risk Factors

Objective:To observe effect of Zeqi Tang in intervening mice with orthotopic lung cancer model, in order to observe its anti-tumor mechanism. Method:An in situ mouse model of non-small cell lung cancer was established through intrapulmonary injection with 1×10⁵ LLC-luc cells. The model mice were intragastrically administered with Zeqi Tang(0.171 g·mL^-1) or normal saline for 35 days. Appearance (spirit, hair, appetite, sleep), survival period and Zeqi Tang anti-tumor effect were observed, weekly vital imaging was performed to detect the fluorescence signal in the lungs of mice. Flow cytometry was used to detect the NK cell content in the spleen of the model mice. CD107α was used to detect the degranulation of NK cells in the spleen of mice after administration of Zeqi Tang. Kromasil 100 5 C₁₈ column was used and eluted with acetonitrile-0.025%phosphoric acid in a gradient mode, with flow rate at 1.0 mL·min^-1, column temperature at 35℃ and detection wavelength of 265 nm, as to establish the fingerprint of Zeqi Tang. The fingerprints of 10 batches of samples was evaluated by using the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System Software (2012 Edition) recommended by the Chinese Pharmacopoeia Commission, in order to complete the quality control of Zeqi Tang. Result:Zeqi Tang could significantly inhibit the lung fluorescence signal of lung cancer in situ model mice and prolong the survival of mice(P<0.05, P<0.01). After the intervention with Zeqi Tang, the NK cells in the tumors increased significantly(P<0.01), and the degranulation of CD107α also increased significantly(P<0.01). Moreover, the HPLC-DAD fingerprint of Zeqi Tang showed a significant increase in the fingerprint similarity of 10 batches of lacquer soup aqueous extract. Moreover, the HPLC-DAD fingerprint of Zeqi Tang showed that the fingerprint similarity of 10 batches of lacquer soup aqueous extract was ≥ 0.9, indicating that small differences between the batches. Conclusion:Zeqi Tang may enhance the tumor growth and prolong the survival period of mice by up-regulating the number of NK cells in mice and enhancing their degranulation function. The evaluation of similarity of HPLC fingerprint of Zeqi Tang reflects the quality of lacquer soup to a certain extent, and can provide reference for further study.

Objective:To study the mechanism of aloesin in inducing apoptosis in human non-small cell lung cancer (NSCLC) A549 cells, so as to inhibit its proliferation. Method:A549 cells in logarithmic growth phase were collected, and cell counting kit-8 (CCK-8) was used to detect the effect of different concentrations of aloesin (2, 4, 8, 16, 32, 64, 128 μmol·L^-1) on the proliferation of A549. Effect of aloesin (0, 16 μmol·L^-1) on the number of clones formed in A549 cells and the size of clone formation was determined by crystal violet staining. effect of aloesin on apoptosis of A549 cells was detected by annexin V/propidium iodide(PI)apoptosis kit staining. Hoechst staining was used to detect the phenomenon of apoptotic nuclear pyknosis. Western blot was used to detect aloesin's effect on death-related protein expressions of Bcl-xl/Bcl-2 associated death promoter (Bad), cleaved-Caspase-3,cl-Caspase-3(Asp175), Caspase-3, cleaved poly ADP-ribose polymerase (cl-PARP), poly ADP-ribose polymerase (PARP) in A549 cells. In vivo, 5-week-old nude mice were subcutaneously inoculated with 2×10⁶ A549 cells, and randomly divided into the medication group and the blank group. aloesin or normal saline was intraperitoneally injected for 4 weeks, and the tumor volume of nude mice was measured weekly. The body weight of the mice was observed, and the appearance of the nude mice was observed. Result:Aloesin inhibited the proliferation and cloning of A549 cells in a concentration-dependent manner (P<0.05). After treatment with aloesin, the number of apoptosis and the phenomenon of nuclear pyknosis in A549 cells increased significantly (P<0.01). At the same time, aloesin significantly down-regulated the expression of apoptosis-related protein Bcl-xl (P<0.05), and increased the expression of Bad protein (P<0.01). The expression levels of cl-PARP (P<0.01) and cl-Caspase-3 (P<0.05) were also significantly increased. In addition, in vivo, aloesin significantly shrank the volume of subcutaneous tumors in mice, reduced tumor weight, with a better appearance than that of the control group. Conclusion:Aloesin may inhibit the expression of NSCLC by inducing apoptosis of A549 cells, and is safe to use, with no inhibitory effect on the body weight of mice.

Objective:To investigate the effect of astragaloside on the macrophage polarization and the possible anti-tumor immunity mechanism of astragaloside. Method:The cytotoxic effect of different concentrations of astragaloside at different time points on macrophage was measured by methylthiazolyldiphenyl-tetrazolium bromide (MTT), in order to choose the suitable concentration of astragaloside, macrophages were co-cultured with tumor cells at the ratio 1:1, and the effect of astragaloside on macrophage-mediated lysis of tumor cells was performed by biophotonic cytotoxicity assay after the mixed cells were effected with 0.1 mg·L^-1 astragaloside for 24 h. Macrophages were dealt with 0.1 mg·L^-1 astragaloside for 24h, the expressions of CD16/32 and CD206 in macrophages were performed by flow cytometry, the mRNA expressions of macrophage inducible nitric oxide synthase (iNOS), Arginine-1 (Arg-1), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), interleukin-12 (IL-12), interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) were measured by Real-time PCR, the protein expressions of macrophage signal transducers and activators of transcription 1 (STAT1) and phosphorylation signal transducers and activators of transcription 1 (p-STAT1) were determined by Western blot. Result:Astragaloside had no effect on the viability of macrophages with 0.1 mg·L^-1. Compared with control group, astragaloside obviously enhanced the macrophage-mediated lysis of tumor cells according to the biophotonic cytotoxicity assay, induced the M1 macrophage marker CD16/32 expression according to flow cytometry, increased the mRNA expressions of iNOS, IL-1β, TNF-α and IL-12 according to the Real-time PCR, and promoted the phosphorylation of STAT1 in macrophages on the basis of Western blot. Conclusion:Astragaloside could induce M1 macrophage polarization by increasing the phosphorylation of STAT1, and initiate macrophage-related anti-tumor immunity response.

Objective To assess the influence of glycolytic pathway on the proportion and numbers of regulatory T cells dur-ing Schistosoma japonicum infection. Methods A S. japonicum-infected mouse model was established,and C57/BL6 male mice infected with S.japonicum were subjected to intraperitoneal injections of with the glycolytic inhibitor 2-Deoxy-D-glucose (2DG)or PBS for 6 times,and then the cells from spleen or mesenteric lymph nodes(LNs)were isolated and analyzed by flow cytometry(FCM)to detect the percentage of Glut1+CD4+T cells and Treg cells. Results The proportions of Glut1+CD4+T cells in the spleen(43.58%±2.50% vs.21.15%±0.96%;t=8.834,P<0.01)and mesenteric LNs(38.97%±1.97% vs.28.40%± 2.11%;t=3.662,P<0.05)were higher in the normal mice than those in the infected mice,and the percentages of Treg cells in the spleen(6.83% ± 0.21% vs. 13.30% ± 0.35%;t = 15.65,P < 0.01)and LNs(8.26% ± 0.15% vs. 14.37% ± 0.44%;t =13.14,P<0.01)were lower in the normal mice than those in the infected mice.In addition,the proportions of Treg cells in the spleen(15.50%±0.76% vs.13.07%±0.15%;t=3.130,P<0.05)and LNs(17.00% ± 0.41% vs.13.83% ± 0.18%;t=6.947, P<0.01)were higher in the infected mice injected intraperitoneally with 2DG than those in the infected mice injected intraperi-toneally with PBS. Conclusion Glycolytic pathway inhibits Treg differentiation in the spleen and mesenteric LNs of S.japoni-cum-infected mice.

Objective To investigate the role of TIGIT signal in Th1/Th2 balance in the process of Schistosoma japonicum in-fection.Methods Male C57BL/6 mice were infected with cercariae of S.japonicum,and normal uninfected mice served as the controls.The percentages of TIGIT+cells,Ki67+CD3+CD4+TIGIT+cells,Ki67+CD3+CD4+TIGIT-cells,IFN-γ+CD3+CD4+TIGIT+cells,IFN-γ+CD3+CD4+TIGIT- cells,IL-4+CD3+CD4+TIGIT+cells and IL-4+CD3+CD4+TIGIT- cells were evaluated in mouse spleen by flow cytometry.Results The proportion of TIGIT+CD4+T cells was higher in the spleen of S.japonicum-infected mice than in the normal uninfected mice(29.30%±0.70% vs.3.09%±0.50%;t=8.834,P<0.01).However,no significant differ-ence in the percentages of TIGIT+CD8+T cells between the infection group and normal controls(3.61% ± 0.26% vs. 3.58% ± 0.16%;t=0.108,P>0.05),and no significant difference was detected in the percentages of TIGIT+cells in non-T cells be-tween the infection group and controls(1.86%±0.19% vs.1.37%±0.17%;t=1.931,P>0.05).In addition,the proportion of Ki67 in the TIGIT+cells was higher than that in the TIGIT-cells(17.03%±0.64% vs.6.59%±0.37%;t=14.09,P<0.01).The Th2/Th1 ratio was higher in the TIGIT+CD4+T cells than in the TIGIT-CD4+T cells(39.28%±3.75% vs.11.79%±1.64%;t=6.721,P<0.01).Conclusion The TIGIT signaling may be involved in the development of Th2 responses in the mice infected with S.japonicum.