Quercetin represses cholesterol metabolism and mitigates resistance to cisplatin in oral squamous cell carcinoma by regulating AGR2/AKT/SREBP2 axis.

Purpose: This study aimed to explore the effects of quercetin on cholesterol metabolism and cisplatin sensitivity in oral squamous cell carcinoma (OSCC) cell line (CAL27) and investigate the potential molecular mechanisms. Methods: CAL27 cells were exposed to quercetin or cisplatin after upregulation or downregulation of AGR2. The expression of proteins and genes associated with cholesterol metabolism were assessed. The levels of cholesterol and LDL were also measured, and the cisplatin sensitivity of CAL27 cells was analyzed. Results: RNA high-throughput sequencing revealed that after treatment with quercetin, the expression of AGR2 was significantly reduced in cisplatin-resistant CAL27 cells (CAL-27R), which was associated with lipid metabolism. AGR2 deletion ameliorated but its overexpression exacerbated cisplatin resistance and cholesterol metabolism, evidenced by changes in SQLE, HMGCS, LDLR, and n-SREBP2 expression and cholesterol and LDL levels. Moreover, AGR2 promoted cisplatin resistance by activating the AKT signaling pathway and enhancing SREBP2-mediated cholesterol metabolism. Quercetin increased cisplatin sensitivity by repressing cholesterol metabolism but suppressed the AGR2/AKT/SREBP2 signaling pathway in a concentration-dependent manner. These effects were partly reversed by AGR2 overexpression and AKT activation. Conclusion: Our findings demonstrated that quercetin inhibits cholesterol metabolism and cisplatin resistance in CAL27 cells by modulating the AGR2/AKT/SREBP2 axis.

[Mineralization Characteristics of Soil Organic Carbon and Its Relationship with Organic Carbon Components in Artificial Robinia pseudoacacia Forest in Loess Hilly Region].

In order to explore the characteristics of organic carbon mineralization and the variation law of organic carbon components of an artificial forest in a loess hilly area, an artificial Robinia pseudoacacia forest restored for 13 years and the adjacent slope farmland were selected as the research objects, and indoor culture experiments under three different temperature treatments (15, 25, and 35℃) were carried out. The results indicated that the mineralization rate of soil organic carbon decreased sharply at first and then stabilized. The cumulative release of organic carbon increased rapidly in the initial stage of culture and gradually slowed in the later stage. Soil organic carbon mineralization in sloping farmland was more sensitive to temperature change, and its temperature sensitivity coefficient Q10 was 1.52, whereas that in R. pseudoacacia forest land was only 1.38. According to the fitting of the single reservoir first-order dynamic equation, the soil mineralization potential Cp of R. pseudoacacia forest land and slope farmland was between 2.02-4.32 g·kg-1 and 1.25-3.17 g·kg-1, respectively, that is, the mineralization potential of the R. pseudoacacia forest was higher. During the cultivation period, the content of various active organic carbon components decreased with time, and that in the R. pseudoacacia forest land was greater than that in the slope land. The cumulative carbon release of soil was significantly positively correlated with the contents of MBC and DOC (P<0.05), and Q10 (15-25℃) was negatively correlated with the contents of SOC, EOC, and SWC (P<0.05). These results could provide some reference for the study of soil carbon sequestration in loess hilly regions under climate change.

[Changes in Soil Microbial Carbon-Degrading Enzymes and Their Relationships with Carbon Pool Components During the Restoration Process of Robinia pseudoacacia].

To reveal the change in the characteristics of soil microbial C-degrading enzyme activities and the response to the components of C during the restoration process of Robinia pseudoacacia forests in the Loess Plateau, the components of the soil C pool, C-degrading enzyme activities, and microbial metabolic entropy of R. pseudoacacia in different restoration stages were studied, and the response relationship between C-degrading enzymes and soil C components was explored. The results showed that the microbial respiration (MR) first increased and then decreased with the restored years. We found that the microbial metabolic entropy (qCO2) decreased significantly with the restored years, but the microbial entropy (qMB) increased. Soil C-degrading enzymes increased significantly in the early-stage restoration of R. pseudoacacia; however, oxidizing enzymes (PO and PER) and cellobiohydrolase (CBH) decreased in the late stage of restoration. The soil organic C and recalcitrant organic C increased significantly with the restored years; however, there was no significant difference for the labile organic C. Correlation analysis and the partial least squares-path model (PLS-PM) showed that soil C-degrading enzymes and C components were significantly correlated with microbial respiration and entropy (qCO2 and qMB), respectively. The hydrolytic enzyme (BG+CBH) was significantly positively correlated with SOC, microbial biomass C, qMB, and recalcitrant and labile organic C. The oxidizing enzyme (PO+PER) was significantly positively correlated with the soil clay and qCO2. In addition, the recalcitrant organic C was the key driver of soil microbial metabolism affected by vegetation restoration. Overall, the ecosystem of R. pseudoacacia plantations would gradually stabilize with the increase in restored years and significantly increase the sequestration effect of soil C. These results will be helpful to understand the transformation rule and regulation mechanism of the soil C pool in vulnerable habitats and provide scientific basis for the restoration and management of vegetation in the Loess Plateau.

Saikosaponin D Alleviates DOX-induced Cardiac Injury In Vivo and In Vitro.

ABSTRACT: As a highly efficient anticancer agent, doxorubicin (DOX) is used for treatment of various cancers, but DOX-induced oxidative damages contribute to a degenerative irreversible cardiac toxicity. Saikosaponin D (SSD), which is a triterpenoid saponin with many biological activities including anti-inflammatory effects and antioxidant properties, provides protection against pathologic cardiac remodeling and fibrosis. In the present study, we investigated the work of SSD for DOX-induced cardiotoxicity and the involved mechanisms. We observed that DOX injection induced cardiac injury and malfunction and decreased survival rate. Besides, DOX treatment increased lactate dehydrogenase leakage, cardiomyocyte apoptosis, and myocardium fibrosis and decreased the size of cardiomyocytes. Meanwhile, all the effects were notably attenuated by SSD treatment. In vitro, we found that 1 µM SSD could enhance the proliferation of H9c2 cells and inhibit DOX-induced apoptosis. It was found that the levels of malondialdehyde (MDA) and reactive oxygen species were significantly reduced by improving the activities of the endogenous antioxidative enzymes including catalase and glutathione peroxidase. Furthermore, SSD treatment could downregulate the DOX-induced p38 phosphorylation. Our results suggested that SSD efficiently protected the cardiomyocytes from DOX-induced cardiotoxicity by inhibiting the excessive oxidative stress via p38-MAPK (mitogen-activated protein kinase, MAPK) signaling pathway.

[Synergistic Mechanism of Interferon alpha-1b, Interleukin-2 and Thalidomide for Immune Regulation in Patients with Acute Myeloid Leukemia].

OBJECTIVE: To explore the synergistic immunomodulatory mechanism of interferon alpha-1b, interleukin-2 and thalidomide (ITI) regimen on patients with acute myeloid leukemia (AML). METHODS: Sixty eight untreated de novo or relapsed or refractory or maintenance therapy patients with AML admitted in the Affiliated Cancer Hospital of Zhengzhou University and the other 11 medical units from March 2016 to May 2019 were treated with ITI regimen. Peripheral blood specimen per patient was collected into EDTA-K3 anticoagulation vacuum tube before the administration of ITI and 3 months after the treatment; peripheral blood lymphocyte subsets and perforin and Granzyme B expression were analyzed by using flow cytometry; the levels of VEGF, IFN-γ, TNF-α and IL-6 in the plasma were detected by using a cytometric bead array. Thirty-five healthy subjects from the hospital physical examination centre were selected as normal controls. RESULTS: The ratio of CD4+/CD8+ T cells, the percentage of NK cells, the expression of perforin and Granzyme B of NK cells in the peripheral blood of patients with hematological malignancies were lower than those of healthy controls. The level of VEGF, IL-6 and TNF-α in the peripheral plasma were higher than those of the healthy control group, and the difference was statistically significant. The level of IFN-γ was lower, and the difference was not statistically significant. The ratio of CD4+/CD8+ T cells, the percentage of NK cells, the expression of Granzyme B and Perforin of NK cells in peripheral blood were higher after the therapy of thalidomide combined with rhIFNα-1b for 3 months as compared with those before treatment of ITI, the level of the IFN-γ in peripheral plasma was higher while that of VEGF was lower, the difference was statistically significant; after treatment, the ratio of CD3+ CD4+ and CD3+ CD8+ lymphocytes and the level of TNF-α in peripheral blood were higher those that before treatment, IL-6 was lower, while the difference was not statistically significant. CONCLUSION: The ITI regimen can raise the ratio of CD4+/CD8+ T cells and the percentage of natural killer cells, also, can enhance the generation of perforin and granzyme B and the concentration of IFN-γ as well as inhibit the generation of VEGF, suggesting that these activities may enhance the antitumour capacity of patients with AML.

[Expression of Ki-67 in Adult and Children Acute Lymphoblastic Leukemia and Its Clinical Significance].

OBJECTIVE: To investigate the relationship of Ki-67 level with clinical features, immunophenotype, gene mutation, curative efficacy and prognosis in patients with acute lymphoblastic leukemia(ALL). METHODS: Flow cytometry gated at CD45/SSC was used to detect the expression of Ki-67, and the correlation of Ki-67 expression with clinical manifestation, laboratorial indexes, curative efficacy and prognosis was analysed. RESULTS: Ki-67 expression level increased in ALL patients, the median expression rate was 29.22%, there was significant difference as compared with the healthy control (P<0.01). In adult ALL, the median expression rate of Ki-67 in the high-risk group was 31.49%, and the difference was statistically significant as compared with the low-risk group (P<0.05). In children ALL, the median expression rate of Ki-67 in high-risk group was 42.28%, and the difference was statistically significant (P<0.05). The results of unvariate analysis showed that the age, WBC count at newly diagnosed and extramedullary invasion were adverse factors affecting OS and DFS; the results of multivariate analysis showed that age and extramedullary invasion were independent risk factors for OS and DFS in patients. CONCLUSION: Age≥14 years old, intramedullary invasion are the poor factors for prognosis; the Ki-67 level is not an independent factor for the prognosis of patients.

[Effect of RNA Interference-silenced TAK1 on Kasumi-1 cell Proliferation Inhibition Induced by As2O3 and Its Mechanism].

OBJECTIVE: To explore the effect of transforming growth factor-ß activated kinase-1(TAK1) gene silenced by RNA interference on proliferation inhibition of Kasumi-1 cells induced by As2O3 and its mechanism. METHODS: The experiments were divided into 4 groups, including control group(Kasumi-1 cells treated with non-specific siRNA), TAK1 specific siRNA treated group (Kasumi 1 treated with TAK specific siRNA), As2O3 treated group (Kasumi 1 cells treated with As2O3) and combined treated group (Kasumi 1 cells treated with TAK1 specific siRNA plus As2O3). The proliferation inhibition rate of Kasami 1 cells was detected by CCK-8 method, the apoptotic rate of cells was detected by flow eytometry, the expressions of TAK1, phosphorylated c-Jun N-terminal kinase(p-JNK) and apoptosis-related proteins were detected by Western blot. RESULTS: As2O3 could inhibit Kasumi-1 cell proliferation in a dose-dependent manner between 0.5 to 20 µmol/L with IC50 of (3.79±0.36) µmol/L at 24 h, and also inhibit Kasumi-1 cell proliferation in a dose-dependent manner between 0.5 to 10 µmol/L with IC50 of (2.38±0.17) µmol/L at 48 h, but then the inhibitory effect reached plateau. After treating Kasumi-1 cells with TAK1 siRNA and 3.5 µmol/L As2O3 for 24 h, the proliferation inhibition rate was (10.86±1.64)% and (49.80±2.19)%, meanwhile the apoptosis rate was (8.47±0.75)% and (24.78±2.14)%, all significantly higher than those in control group (P<0.05, P<0.01). The proliferation inhibition rate and apoptosis rate of the combined treated group were significantly higher than that in control and single treated groups (P<0.05, P<0.01), TAK1 silencing and 3.5 µmol/L of As2O3 could decrease the expression of TAK1, p-JNK, c-Fos, c-Jun and BCL-2 in different degrees, and increase the expression levels of BAX and the activated (cleaved) caspase-3, 9 with statistically significant differences as compared with control group (P< 0.05). When Kasumi-1 cells were treated with TAK1 specific siRNA plus As2O3 for 24 h, protein expression levels were all significantly greater than that in the single-treated groups (P< 0.05). CONCLUSION: TAK1 silencing and As2O3 can separately and synergistically inhibit Kasumi-1 cell proliferation which probably relates with the inducing apoptosis via the JNK and mitochondrial pathway. Meanwhile, TAK1 silencing enhances the inhibitory effect of As2O3 on Kasumi-1 cell proliferation.

Comparison of Intraperitoneal and Intraepididymal Quercetin for the Prevention of Testicular Torsion/Detorsion-induced Injury.

OBJECTIVE: To compare the effects of intraepididymal quercetin (IE-QE) with those of intraperitoneal quercetin (IP-QE) on testicular torsion/detorsion (TD)-induced ischemia/reperfusion (IR) injury of the testes in an experimental rat model. METHODS: Twenty-four rats were divided into 4 groups: sham (S), TD, TD treated with IP-QE, and TD treated with IE-QE. The IP-QE group received 20 mg/kg QE intraperitoneally, whereas the IE-QE group received quercetin (QE) epididymally. After surgically induced TD, sera and testicular tissues were obtained for the analysis of biochemical parameters including glutathione peroxidase (GPx), malondialdehyde, total antioxidant status, total oxidant status, oxidative stress index, histologic changes, and evaluation of germ cell apoptosis. RESULTS: The oxidative stress index and oxidants (malondialdehyde and total oxidant status) were increased with a concomitant decrease in the antioxidants (GPx and total antioxidant status) in the TD group. Severe histopathological damage, indicated by low Johnsen scores and high testicular injury grades, and germ cell apoptosis were found in the TD group compared with the other groups. Rats treated with QE showed significantly less IR injury, with moderately altered biochemical parameters, histopathological damage, and germinal cell apoptosis compared with the TD group. Most importantly, we found no significant differences in the biochemical parameters, histopathological changes, and germinal cell apoptosis between the IP-QE and IE-QE groups. CONCLUSION: IE-QE was comparable to IP-QE in the treatment of testicular TD. Local QE therapy should be considered as a new approach to treating testicular IR injury due to TD.

[The distribution of caspase-3 positive apoptotic cells in 18 months old SD rat's brainstem compared with that in 3 months rat].

OBJECTIVE: To study whether there is apoptosis in brainstem neurons while aging by oberving the distribution of Caspase-3 positive apoptotic cells in in brainstem of young and old SD rats. METHODS: Healthy male SD rats were divided into 2 groups (3 and 18 month-old respectively), 3 rats each group. Brainstem specimens were treated followed the brainstem's common paraffin embedding, sectioning and HE staining procedures (sections were 6 µm in thickness). The sections were also determined by Caspase-3 immunostaining and TUNEL. The Caspase-3 positive cells on the rat stereotaxic atlas were drew, then composed the sections into a 3D model. RESULTS: Compared to 3 month-old rats, there were more Caspase-3 positive neurons in the brainstem and the positive neurons were distributed more extensively in 18 month-old rats spectially in nucleus of solitary tract and pontine reticular nuclei. CONCLUSION: More neurons suffer apoptotic changes in the brainstem while aging.

[Clinical significance of dynamic monitoring of thymic recent output function in different stages of treatment in patients with diffuse large B-cell lymphoma].

OBJECTIVE: To detect the changes of naive T cell level of thymic recent output at different stages of treatment in patients with diffuse large B-cell lymphoma (DLBCL), thereby to evaluate the relationship of thymic recent output function with prognosis and the impact of chemotherapy on the potential of immunological recovery. METHODS: The levels of T-cell receptor rearrangement excision circles (TREC) in DNA of peripheral blood mononuclear cells (PBMNC) from 30 DLBCL patients were monitored before, during, until 3 months and 6 months after chemotherapy by real-time PCR (TaqMan), and TREC-level was detected according to the number of CD3 positive(CD3(+)) cells. Twelve normal individuals who matched in age were served as controls. RESULTS: There was a dramatic reduction of TREC values in all DLBCL patients among which TREC values in germinal center B-cell-like-DLBCL (GCB-DLBCL) were higher than those in non-GCB-DLBCL, as compared with TREC values of normal individual in peripheral blood. The mean values of TREC were 0.91 ± 0.15/1000 PBMNCs and (1.22 ± 0.69)/1000 CD3(+) cells in GCB-DLBCL, (0.43 ± 0.29)/1000 PBMNCs and (0.64 ± 0.44)/1000 CD3(+) cells in non-GCB-DLBCL before chemotherapy. TREC values were significantly associated with lower international prognostic index (IPI) grade (r = -0.441, P = 0.015). TREC-level in DLBCL patients was further decreased after chemotherapy, and reached to the lowest level after the 6th cycle of chemotherapy, and during the corresponding period, the mean values of TREC were (0.63 ± 0.34)/1000 PBMNCs and (0.89 ± 0.65)/1000 CD3(+)cells in GCB-DLBCL, (0.19 ± 0.11)/1000 PBMNCs and (0.27 ± 0.25)/1000 CD3(+) cells in non-GCB-DLBCL. TREC-level began to rise obviously 3 months after the last cycle of chemotherapy in most of the DLBCL patients, and came close to normal level in five cases of patients 6 months after the last cycle of chemotherapy. CONCLUSIONS: Thymic recent output function was impaired severely in DLBCL patients. There was an important relationship between thymic recent output function before chemotherapy and prognosis, and chemotherapy had influenced the potential of immunological recovery.

[Expression and clinical significance of VEGF and apoptosis in frozen-thawed mouse ovaries after transplantation].

OBJECTIVE: To investigate expression and clinical significance of VEGF and apoptosis in frozen-thawed mouse ovaries after transplantation. METHODS: Ovaries from B6C2F1 (C57BL/6j x BALB/c) 4 week old mice were cryopreservation and the thawed ovaries were xenografted into kidney capsules of 8-12 week old adult male mice. The grafted were recovered 1 d, 2 d and 7 d after transplantation respectively, the grafts and frozen-thawed were removed for follicle counting and immunohistochemically, ultrastructure, and detection of the mRNA expression of vascular endothelial growth factor(VEGF). RESULTS: The follicle numbers were decreased gradually after transplantation,the cell apoptosis increased especially in 48 h after transplantation. Transmission electron microscopy (TEM) showed the tissue damaged was severest 48 h after transplantation; the expression of VEGF increased after transplantation, peaked on day 7, the mRNA expression of VEGF120 and VEGF188 was more on 48 h after transplantation, decreased on day 7 (P < 0.05). CONCLUSION: The number of follicles was decreased after transplantation, the apoptosis index was increased especially in 48 h after transplantation; the expression of VEGF increased after transplantation, an increase in the VEGF188 and VEGF164 isoform might suggest the positive effect in the early stages of angiogenesis.