Objective To investigate the effect of Ophiopogonin D on lipopolysaccharide(LPS)-induced apoptosis of alveolar epithelial cells by regulating the interleukin-6(IL-6)/Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3)signaling pathway.Methods A549 AT Ⅱ cells cultured in vitro were randomly divided into four groups:control group,LPS group,LPS+Ophiopogonin D group,LPS+Ophiopogonin D+colivelin(JAK2/STAT3 signal activator)group,except for the control group,and cells in all other groups were established injury models while being grouped with Ophiopogonin D and colivelin for treatment.Cell counting kit-8(CCK-8)experiment and flow cytometry were applied to detect cell proliferation and apoptosis in each group;Western blotting was applied to detect the expression of IL-6/JAK2/STAT3 signaling pathway proteins of cells in each group.Results The apoptosis rates of A549 cells in control group,LPS group,LPS+Ophiopogonin D group and LPS+Ophiopogonin D+colivelin group were(2.52±0.73)％,(52.43±4.14)％,(1.67±0.52)％and(47.94±3.43)％;IL-6 protein levels were 0.14±0.03,0.49±0.05,0.17±0.04 and 0.45±0.06,and p-JAK2/JAK2 protein levels were 0.17±0.04,0.64±0.08,0.19±0.06 and 0.61±0.07;p-STAT3/STAT3 protein levels were 0.20±0.06,0.69±0.10,0.22±0.07 and 0.65±0.09;the apoptosis rates of AT Ⅱ cells were(3.01±0.69)％,(55.16±3.94)％,(2.35±0.71)％and(50.28±3.78)％;the levels of IL-6 protein were 0.11±0.03,0.87±0.13,0.19±0.04 and 0.84±0.12;the p-JAK2/JAK2 protein levels were 0.13±0.04,0.56±0.08,0.15±0.03 and 0.53±0.07;p-STAT3/STAT3 protein levels were 0.30±0.08,0.79±0.14,0.33±0.09 and 0.75±0.13.The above indexes:control group,LPS+Ophiopogonin D group compared with LPS group,LPS+Ophiopogonin D+colivelin group compared with LPS+Ophiopogonin D group,the differences were statistically significant(all P＜0.05).Conclusion Ophiopogonin D can reduce LPS induced inflammation and oxidative stress levels by inhibiting the activation of IL-6/JAK2/STAT3 signaling pathway,ultimately reducing LPS-induced apoptosis of alveolar epithelial cells.

Background::Although some well-established oncogenes are involved in cancer initiation and progression such as prostate cancer (PCa), the long tail of cancer genes remains to be defined. Goosecoid ( GSC) has been implicated in cancer development. However, the comprehensive biological role of GSC in pan-cancer, specifically in PCa, remains unexplored. The aim of this study was to investigate the role of GSC in PCa development. Methods::We performed a systematic bioinformatics exploration of GSC using datasets from The Cancer Genome Atlas, Genotype-Tissue Expression, Gene Expression Omnibus, German Cancer Research Center, and our in-house cohorts. First, we evaluated the expression of GSC and its association with patient prognosis, and identified GSC-relevant genetic alterations in cancers. Further, we focused on the clinical characterization and prognostic analysis of GSC in PCa. To understand the transcriptional regulation of GSC by E2F transcription factor 1 ( E2F1), we performed chromatin immunoprecipitation quantitative polymerase chain reaction (qPCR). Functional experiments were conducted to validate the effect of GSC on the tumor cellular phenotype and sensitivity to trametinib. Results::GSC expression was elevated in various tumors and significantly correlated with patient prognosis. The alterations of GSC contribute to the progression of various tumors especially in PCa. Patients with PCa and high GSC expression exhibited worse progression-free survival and biochemical recurrence outcomes. Further, GSC upregulation in patients with PCa was mostly accompanied with higher Gleason score, advanced tumor stage, lymph node metastasis, and elevated prostate-specific antigen (PSA) levels. Mechanistically, the transcription factor, E2F1, stimulates GSC by binding to its promoter region. Detailed experiments further demonstrated that GSC acted as an oncogene and influenced the response of PCa cells to trametinib treatment. Conclusions::GSC was highly overexpressed and strongly correlated with patient prognosis in PCa. We found that GSC, regulated by E2F1, acted as an oncogene and impeded the therapeutic efficacy of trametinib in PCa.

ObjectiveTo investigate the effect and mechanism of Guilu Erxiangao on Alzheimer's disease （AD） model rats induced by hydrocortisone and amyloid β-protein（Aβ） based on the theory of kidney-brain correlation. MethodIntraperitoneal injection of hydrocortisone and intracerebroventricular injection of Aβ were performed to induce AD in rats， and different concentrations of Guilu Erxiangao were used for intervention. The indexes of hippocampus， kidney and adrenal gland were measured， and the spatial learning and memory ability of AD rats was observed by Morris water maze experiment. The levels of testosterone （T） and corticosterone （CORT） in serum samples were determined by enzyme-linked immunosorbent assay （ELISA）. Liquid chromatography-mass spectrometry （LC-MS） was used to collect and analyze the serum metabolic data of model rats. The active components and corresponding targets of Guilu Erxiangao were collected using Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP）， a Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine （BATMAN-TCM） and Traditional Chinese Medicine Integrated Database（TCMID）. GeneCards and Online Mendelian Inheritance in Man （OMIM） were retrieved to obtain AD-related targets， and protein-protein interaction （PPI） network was constructed to perform gene ontology （GO）and Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis. The expression level of interleukin-6 （IL-6） in the hippocampus of rats was detected by Western blot. ResultCompared with the model group， the low-， medium- and high-dose groups of Guilu Erxiangao exhibited significantly increased hippocampus index， kidney index and adrenal gland index， reduced CORT levels in serum and down-regulated IL-6 levels in hippocampal tissues. According to the results of water maze experiment， as compared with the model group， the platform crossing times of rats was significantly increased in the low- and high-dose groups of Guilu Erxiangao， with evidently prolonged distance traveled in quadrant Ⅲ （%） and time in quadrant Ⅲ （%）. A total of 24 serum differential metabolites associated with AD were identified by LC-MS， and 50 high-frequency common compounds and 187 high-frequency common targets for AD treatment were screened by network pharmacology method. Results demonstrated phosphatidylinositol 3-kinases（PI3K）/protein kinase B（Akt） signaling pathway plays an important role in the complex AD pathological mechanism. ConclusionGuilu Erxiangao can significantly improve the cognitive dysfunction of AD model rats induced by hydrocortisone and Aβ， reduce serum CORT levels and IL-6 levels in hippocampal tissues， and regulate the metabolic level， which provides a reference for its clinical application.

ObjectiveMetabolomics was used to identify biomarkers of chronic alcoholism， and to evaluate the neuroprotective effect of geniposide， providing reference for the diagnosis and treatment of chronic alcoholism. MethodThe rat model of chronic alcoholism was established by intragastric administration of 50% ethanol with 8 mL·kg^-1 for 14 days， and then increased to 12 mL·kg^-1 for 21 days. Meanwhile， the intervention was performed by continuous gavage of geniposide （15 mg·kg^-1） for 35 days. At the end of the experiment， the biochemical indexes and histopathological morphology of liver and brain tissues of rats were detected. Ultra performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry （UPLC-Q-TOF-MS） was used for urine metabonomics. The chromatographic conditions was as follows：ACQUITY UPLC™ HSS T3 column （2.1 mm×100 mm， 1.8 μm）， mobile phase of 0.1% formic acid acetonitrile solution （A）-0.1% formic acid aqueous solution （B） for gradient elution （0-2.5 min， 1%-11%A； 2.5-4.5 min， 11%-21%A； 4.5-7.0 min， 21%-40%A； 7.0-8.5 min， 40%-99%A； 8.5-10.5 min， 99%A； 10.5-10.6 min， 99%-1%A； 10.6-13.0 min， 1%A）， the flow rate of 0.4 mL·min^-1. The conditions of mass spectrometry were electrospray ionization （ESI）， positive and negative ion modes， scanning range of m/z 50-1 200. Progenesis QI 2.0 and MassLynx 4.1 were used for data analysis， and biomarkers were identified by matching element composition and secondary fragments with Human Metabolome Database （HMDB）. ResultThe pathological results showed that on the 35^th day of model replication， compared with the model group， the cortical neurons in the geniposide group showed a significantly improved state of disorder， nuclear pyknosis， hyperchromatism and cell membrane boundary blurred necrosis. The biochemical results showed that geniposide could significantly increase the activities of glutathione peroxidase （GSH-Px） and superoxide dismutase （SOD）， decrease the activity of acetylcholinesterase （AChE）， decrease the levels of β-endorphin （β-EP） and malondialdehyde （MDA）. A total of 48 biomarkers of chronic alcoholism were identified by metabonomics， involving seven metabolic pathways of tryptophan metabolism， phenylalanine metabolism， pentose and glucuronate interconversions， pyrimidine metabolism， ascorbate and aldarate metabolism， steroid hormone biosynthesis and purine metabolism. The main pathway is 5-hydroxytryptamine pathway of tryptophan metabolism. ConclusionBiomarkers related to nerve injury in chronic alcoholism are mainly derived from the 5-hydroxytryptamine metabolic pathway. Geniposide can regulate this pathway so as to improve oxidative stress in the brain and play a neuroprotective role.

Background::Gut-resident macrophages (gMacs) supplemented by monocytes-to-gMacs differentiation play a critical role in maintaining intestinal homeostasis. Activating transcription factor 4 (ATF4) is involved in immune cell differentiation. We therefore set out to investigate the role of ATF4-regulated monocytes-to-gMacs differentiation in sepsis-induced intestinal injury.Methods::Sepsis was induced in C57BL/6 wild type (WT) mice and Atf4-knockdown ( Atf4+/-) mice by cecal ligation and puncture or administration of lipopolysaccharide (LPS). Colon, peripheral blood mononuclear cells, sera, lung, liver, and mesenteric lymph nodes were collected for flow cytometry, hematoxylin and eosin staining, immunohistochemistry, quantitative reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay, respectively. Results::CD64, CD11b, Ly6C, major histocompatibility complex-II (MHC-II), CX3CR1, Ly6G, and SSC were identified as optimal primary markers for detecting the process of monocytes-to-gMacs differentiation in the colon of WT mice. Monocytes-to-gMacs differentiation was impaired in the colon during sepsis and was associated with decreased expression of ATF4 in P1 (Ly6C hi monocytes), the precursor cells of gMacs. Atf4 knockdown exacerbated the impairment of monocytes-to-gMacs differentiation in response to LPS, resulting in a significant reduction of gMacs in the colon. Furthermore, compared with WT mice, Atf4+/- mice exhibited higher pathology scores, increased expression of inflammatory factor genes ( TNF-α, IL-1β), suppressed expression of CD31 and vascular endothelial-cadherin in the colon, and increased translocation of intestinal bacteria to lymph nodes and lungs following exposure to LPS. However, the aggravation of sepsis-induced intestinal injury resulting from Atf4 knockdown was not caused by the enhanced inflammatory effect of Ly6C hi monocytes and gMacs. Conclusion::ATF4, as a novel regulator of monocytes-to-gMacs differentiation, plays a critical role in protecting mice against sepsis-induced intestinal injury, suggesting that ATF4 might be a potential therapeutic target for sepsis treatment.

Bile Duct Diseases ; Biliary Atresia/surgery* ; Cysts ; Humans ; Infant ; Liver Failure ; Portoenterostomy, Hepatic ; Retrospective Studies

Objective: To construct a new CD123- specific chimeric antigen receptor in order to provide a foundation for immunotherapy of CD123 positive leukemia. Methods: A hybridoma strain (6E11) capable of stably secreting CD123 antibody was obtained by a monoclonal screening technique, and the hybridoma cells were expanded and injected intraperitoneally to the pretreated Balb/c mice. Ascites was collected and purified to obtain the monoclonal antibody (mAb) . The affinity and specificity of 6E11 mAb were measured. The variable regions of the heavy and light chains of the 6E11 mAb were cloned by RT-PCR from the 6E11 mouse hybridoma. We generated a new CD123 specific chimeric antigen receptor with a scFv fragment derived from 6E11 antibody, designated as 6E11 CAR. T cells were transduced with lentiviral supernatant from 293T cells transfected with 6E11 CAR plasmid to generate 6E11 CAR-T cells. The specific cytotoxicity of 6E11 CAR-T against CD123(+) acute myeloid leukemia (AML) cell lines and primary AML cells in vitro were evaluated by co-culture experiments, degranulation experiments and cytokine releasing assay. Results: ① A hybridoma cell line 6E11 stably secreting anti-human CD123 antibody was developed and its variable region sequences were obtained. ② The 6E11 mAb has high affinity for CD123 protein (Kd value: 2.1 nmol/L) . The 6E11 mAb specifically recognizes CD123(+) cell line THP-1 cells and does not respond to CD123(-) cell line Jurkat cells. ③ 6E11 CAR-T cells were successfully generated with a CAR expression rate higher than 60%. ④ 6E11 CAR-T cells could specifically kill CD123(+) MV4-11 cell line but had no killing effect on the CD123(-) K562 cell line. Compared with vector-T cells, 6E11 CAR-T cells have higher killing rate to MV4-11 cells[ (98.60±1.20) %vs (20.28±6.74) %, P<0.001]. ⑤ MV4-11 cells activated 6E11 CAR-T cells significantly but not Vector-T cells[ (26.33±3.30) %vs (1.17±0.06) %, P<0.001]. ⑥ 6E11 CAR-T cells released more cytokines than vector-T cells when co-cultured with MV4-11[IL-2: (92.90±1.51) pg/ml vs (6.05±3.41) pg/ml, P<0.001; TNF-α: (1 407.20±91.95) pg/ml vs (7.86±0.85) pg/ml, P<0.001; IFN-γ: (5 614.60±170.17) pg/ml vs (8.42±2.70) pg/ml, P<0.001]. The IFN-γ, IL-2 and TNF-α in the 6E11 CAR-T group were similar to those in the Vector-T group when co-cultured with K562. ⑦ 6E11 CAR-T cells could be activated by bone marrow mononuclear cells (BMMNC) derived from CD123(+) AML patients and effectively kill these BMMNC cells from CD123(+) AML patients. Conclusion: 6E11 hybridoma cell line can stably secrete highly specific monoclonal antibodies against human CD123, which can be used to detect the expression of human CD123. It can also be used to target human CD123 protein in tumor immunotherapy. CD123 CAR-T cells with 6E11 Ig variable region sequence have specific anti-leukemic activity in vitro, which may provide a new option for further clinical research of AML.
Animals ; Cell Line, Tumor ; Humans ; Interleukin-3 Receptor alpha Subunit ; Leukemia, Myeloid, Acute ; Mice ; Receptors, Chimeric Antigen ; Single-Chain Antibodies

Purpose To investigate the value of application of D2-40/CD34-CK cocktail antibodies by double immunohistochemical staining for assessment of lymphovascular invasion (LVI) and to determine its prognostic significance in colorectal cancer with insufficient lymph node harvest. Methods Specimens from 133 cases of colorectal cancer with less than 12 lymph nodes were selected. HE staining and double immunohistochemical staining of the cocktail antibodies were performed to compare the difference of the two methods in screening for LVI. The The relationship between LVI confirmed by cocktail antibody immunohistochemical staining and clinicopathological characteristics and overall survival (OS) of patients was analyzed. Results (1) The detection rates of cocktail antibody double immunohistochemical staining and HE staining for LVI were 42.9％ (57/133) and 21.8％ (29/133) with statistically significant difference (P < 0.001). (2) The presence of LVI confirmed by double staining was significantly associated with Dukes staging, depth of invasion, clinical stages, lymph node metastasis and tumor budding (P < 0.05). (3) The presence of LVI, the location and extent of LVI, and the number of tumor cells in thrombus ≥5.5 for cases with LVI ≤2 clusters, were significantly associated with OS (P < 0.05). Conclusion D2-40/CD34-CK cocktail antibodies double staining is superior to routine HE staining in assessing LVI. LVI is intimately associated with tumor stage, lymph nodes metastasis and tumor budding, and it is an independent prognostic factor for CRC patients. It should be a supplementary examination for these patients with insufficient lymph node harvest.

OBJECTIVETo study if programmed death-ligand 1 (PL-L1) expression in breast cancer cell activates PD-L1/PD-1 pathway in dendritic cells to inhibit dendritic cell maturation.

METHODSHuman monocytes were induced to differentiate into immature dendritic cells using GM-CSF and IL-4, and further to mature dendritic cells using TNF-α. PD-L1-expressing breast cancer cell line MDA-MB-231 was co-cultured in contact with the dendritic cells to observe the effects of the breast cancer cells on the maturation of the dendritic cells. A PD-L1 blocking antibody was applied to the co-culture, and the changes in the inhibitory effect of the MDA-MB-231 cells on dendritic cell maturation was observed. TNF-α-induced dendritic cells were treated with a recombinant human PD-L1 protein to study the effect of PD-L1/PD-1 pathway activation on the maturation of dendritic cells. The expression of PD-L1 in MDA-MB-231 cells and the dendritic cell maturation marker HLA-DR and CD83 were analyzed using flow cytometry.

RESULTSMDA-MB-231 cell line showed PD-L1 positivity on the cell membrane cells at a rate as high as (99.7∓0.15)%. In mature dendritic cells, the positivity rates for HLA-DR and CD83 were (88.8∓6.96)% and (18.36∓3.07)%, respectively, but in the co-culture system, the positivity rates of the dendritic cells were significantly decreased to (42.76∓10.52)% (P<0.01) and (9.93∓2.74)% (P<0.05), respectively, indicating that MDA-MB-231 cells inhibited the maturation of dendritic cells. Following treatment with a PD-L1 antibody isotype control, the percentages of HLA-DR- and CD83-positive cells in the co-culture were (45.17∓10.19)% and (10.15∓2.54)%, which were significantly increased to (63.46∓1.72)% and (16.46∓2.58)% after treatment with PD-L1 antibody, respectively (both P<0.05). Compared with the mature dendritic cell controls, the cells treated with the recombinant human PD-L1 protein exhibited significantly lowered percentages of HLA-DR-positive [from (84.23∓4.18)% to (2.56∓2.39)%, P<0.05] and CD83-positive cells [(87.26∓1.54)% to (60.67∓1.63)%, P<0.05].

CONCLUSIONThe effect of PD-L1 antibody therapy on triple negative breast cancer can be partially mediated by blocking PD-L1 expression on breast cancer cell membrane, which attenuates the inhibition of dendritic cell maturation in the cancer microenvironment.

Objective:To observe the clinical efficacy and safety of cranial suture acupuncture plus paroxetine in treating depression,and to discuss the action mechanism of this acupuncture method.Methods:One hundred depression patients were allocated to an observation group and a control group according to the random number table,with 50 cases in each group.The control group was intervened by oral administration of paroxetine tablets,20 mg each time,once a day for successive 6 weeks;the observation group was additionally given cranial suture acupuncture,once a day for 6 weeks.They were scored by Hamilton depression scale-17 (HAMD-17) before the treatment and respectively after 1-week,2-week,4-week and 6-week treatment.The clinical efficacy and safety were also observed.Results:After 6-week treatment,the total effective rate was 94.0％ in the observation group versus 78.0％ in the control group,and the between-group difference was statistically significant (P＜0.01).The HAMD-17 scores respectively after 1-week,2-week,4-week and 6-week treatment were significantly lower than the score before the treatment in the observation group (all P＜0.05);the HAMD-17 scores respectively after 2-week,4-week and 6-week treatment were significantly different from the score before the treatment in the control group (all P＜0.05).There were significant differences in the HAMD-17 score between the two groups respectively after 4-week and 6-week treatment (both P＜0.05).Conclusion:Cranial suture acupuncture plus paroxetine can ease the symptoms of depression,with faster onset and more significant therapeutic efficacy compared with paroxetine alone.