circ_SIRT1 upregulates ATG12 to facilitate Imatinib resistance in CML through interacting with EIF4A3.

Imatinib is the current gold standard for patients with chronic myeloid leukemia (CML). However, the primary and acquired drug resistance seriously limits the efficacy. To identify novel therapeutic target in Imatinib-resistant CML is of crucial clinical significance. CircRNAs have been demonstrated the essential regulatory roles in the progression and drug resistance of cancers. In this study, we identified a novel circRNA (circ_SIRT1), derived from the SIRT1, which is up-regulated in CML. The high expression of circ_SIRT1 is correlated with drug resistance in CML. Knockdown of circ_SIRT1 regulated K562/R cells viability, invasion and apoptosis. Besides, the inhibition of circ_SIRT1 attenuated autophagy level and reduced IC50 to Imatinib of K562/R cells. Mechanistically, circ_SIRT1 directly binds to the transcription factor Eukaryotic Translation Initiation Factor 4A3(EIF4A3) and regulated EIF4A3-mediated transcription of Autophagy Related 12 (ATG12), thereby affecting Imatinib resistance and autophagy level. Overexpression of ATG12 reversed the regulative effects induced by knockdown of circ_SIRT1. Taken together, our findings revealed circ_SIRT1 acted as a potential tumor regulator in CML and unveiled the underlying mechanism on regulating Imatinib resistance. circ_SIRT1 may serve as a novel therapeutic target and provide crucial clinical implications for Imatinib-resistant CML treatment.

Dose-individualization Efficiently Maintains Sufficient Exposure to Methotrexate without Additional Toxicity in High-dose Methotrexate Regimens for Pediatric Acute Lymphoblastic Leukemia.

OBJECTIVE: Methotrexate (MTX) can be safely administered to most patients but may cause severe toxicity in others. This study aimed to summarize the characteristics of high-dose methotrexate (HD-MTX) chemotherapy and to evaluate whether the modified dose-adjustment program was able to improve the maintenance of sufficient MTX exposure levels while minimizing toxicities. METHODS: We evaluated 1172 cycles of high-dose MTX chemotherapy from 294 patients who were treated according to the CCCG-ALL-2015 protocol (clinical trial number: ChiCTR-IPR-14005706) and analyzed the data of actual MTX dosage, MTX concentration, toxicity, and prognosis. We compared data between the dose-adjustment Program 1 (fixed 20% reduction in dose) and the dose-adjustment Program 2 (dose-individualization based on reassessment of the creatine clearance rate and the MTX concentration-monitoring point at 16 h), which were applied if the MTX clearance was delayed in the previous cycle. RESULTS: The patients who used Program 2 had higher actual MTX infusion doses and infusion rates and were able to better maintain the MTX concentration at 44 h at the established target value than those on Program 1 (P<0.001). No significant differences in toxicities were found between these two programs except that abnormal serum potassium levels and prolonged myelosuppression in intermediate-risk/high-risk patients were more frequently observed in patients using Program 2 (P<0.001). No significant correlations were observed between the MTX dose, dose-adjustment programs, or MTX concentrations and relapse-free survival. CONCLUSION: Adjusting the MTX dose using Program 2 is more efficient for maintaining sufficient MTX exposure without significantly increasing the toxicity.

A New Immunosuppressive Therapy for Very Severe Aplastic Anemia in Children with Autoantibodies.

OBJECTIVE: At present, a number of very severe aplastic anemia (VSAA) patients cannot receive hematopoietic stem cell transplantation (HSCT) or standard immunosuppressive therapy (IST) due to the high cost of therapy, shortage of sibling donors, and lack of resources to support the HSCT. In addition, some VSAA patients with autoantibodies have no life-threatening infections or bleeding at the time of initial diagnosis. Considering the disease condition, economics and other factors, the present study designed a new and relatively mild treatment strategy: cyclosporine A plus pulsed high-dose prednisone (CsA+HDP). METHODS: The present study retrospectively analyzed 11 VSAA patients, who were treated with CsA+HDP in our hospital from August 2017 to August 2019. RESULTS: The median follow-up time for these patients was 24.9 months. The overall response rate was 54.5% (6/11) at six months after the initiation of IST and 81.8% (9/11) at deadline. Five patients achieved complete remission and four patients met the criteria for partial response at the last follow-up. The median time to response for responders was 110 days. Three patients underwent HSCT due to the poor effect of CsA+HDP or to find a suitable transplant donor. Recurrence and clonal evolution were not found in any of these patients. The estimated 3-year overall survival rate and 3-year failure-free survival rate were 100.0% and 72.7%, respectively. In addition, the results revealed that the cyclosporine-prednisone-associated toxicity was mild and well-tolerated by most patients. CONCLUSION: The novel CsA+HDP regimen has good therapeutic effect and safety for VSAA patients with autoantibodies, who have no serious life-threatening infections or bleeding at the time of initial diagnosis.

PM2.5 promotes Drp1-mediated mitophagy to induce hepatic stellate cell activation and hepatic fibrosis via regulating miR-411.

BACKGROUND: Particulate matter≤ 2.5 µm (PM2.5) is a type of environmental agent associated with air pollution, which induces hepatic fibrosis. However, the function and mechanism of PM2.5 on hepatic stellate cell (HSC) proliferation and fibrosis remain largely unknown. METHODS: Human HSC line (LX-2) and murine HSCs were exposed to various doses of PM2.5. microRNA (miR)-411 expression was detected via quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell proliferation, fibrosis, mitochondrial dynamics dysfunction and mitophagy were determined via cell counting kit-8 (CCK-8), qRT-PCR, Western blotting and immunofluorescence. RESULTS: PM2.5 facilitated HSC proliferation and fibrosis via increasing the levels of ACTA2, Collagen 1, TIMP1 and TGF-ß1. PM2.5 reduced miR-411 expression, and contributed to mitochondrial dynamics dysfunction via increasing Drp1 and decreasing OPA1, TOM20 and PGC-1α levels. PM2.5 promoted mitophagy by upregulating the levels of Beclin-1, LC3II/I, PINK1 and Parkin. miR-411 overexpression or autophagy blockage using 3-methyladenine (3-MA) relieved PM2.5-mediated cell proliferation and fibrosis-associated factor expression in HSCs. Drp1 was targeted by miR-411. miR-411 mitigated PM2.5-induced mitophagy via targeting Drp1. Drp1 overexpression abolished the inhibitory role of miR-411 in cell proliferation and fibrosis-associated factor levels in HSCs. CONCLUSION: PM2.5 induced HSC activation and fibrosis via promoting Drp1-mediated mitophagy by decreasing miR-411, thereby causing liver fibrosis.

Short-chain fatty acids exert opposite effects on the expression and function of p-glycoprotein and breast cancer resistance protein in rat intestine.

P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are involved in intestinal barrier. Short-chain fatty acids (SCFAs) play important roles in maintaining intestinal barrier. In this study we explored how SCFAs affected the expression and function of intestinal P-gp and BCRP in rats. Rats received 150 mM acetate, propionate or butyrate in drinking water for 4 weeks. In SCFA-treated rats, the expression and function of intestinal P-gp were decreased, but those of intestinal BCRP were increased; intestinal p-p65 was also decreased, which was positively related to P-gp protein expression. Among the three SCFAs tested, butyrate exhibited the strongest induction or inhibitory effect, followed by propionate and acetate. Similar results were observed in mouse primary enterocytes and Caco-2 cells treated with acetate (5 mM), propionate (2 mM), or butyrate (1 mM). In Caco-2 cells, addition of butyrate, vorinostat, and valproate (two classic HDAC inhibitors), Bay117082 (selective inhibitor of NF-κB activation) or NF-κB p65 silencing significantly decreased the expression of P-gp and the level of phosphorylated p65 (p-p65). Furthermore, butyrate attenuated the expression of P-gp and p-p65 induced by TNF-α (NF-κB activator) and theophylline (HDAC activator). However, vorinostat, valproate, Bay117082, TNF-α or p65 silencing hardly affected BCRP protein expression. But GW9662 (selective PPARγ antagonist) or PPARγ silencing abolished BCRP induction by butyrate and troglitazone (PPARγ agonist). SCFAs-treated rats showed higher intestinal protein expression of PPARγ, which was positively related to BCRP protein expression. Butyrate increased plasma exposure of fexofenadine but decreased that of rosuvastatin following oral dose to rats. In conclusion, SCFAs exert opposite effects on the expression and function of intestinal P-gp and BCRP; butyrate downregulated P-gp expression and function possibly via inhibiting HDAC/NF-κB pathways; butyrate induced BCRP expression and function partly via PPARγ activation.

[Effect of Huaier Aqueous Extract Combined with Routine Chemo-therapeutic Drugs on Human Acute Lymphoblastic Leukemia Cells Nalm-6 and Sup-B15].

OBJECTIVE: To investigate the proliferation inhibition and pro-apoptotic effect of Huaier aqueous extract combined with routine chemotherapeutic drugs including Vincristine (VCR), Daunorubicin (DNR), L-aspartase (L-Asp) on human acute lymphoblastic leukemia cell lines Nalm-6 and Sup-B15. METHODS: Nalm-6 and Sup-B15 cell lines were treated with different concentrations of Huaier aqueous extract and chemotherapeutics including VCR, DNR, L-Asp alone or in combination for 48 h, and the growth inhibitory effect and IC50 values (the half maximal inhibitory concentration) were detected by CCK-8. Jin's formula was used to estimated the synergistic effect of these combinations. Apoptosis rates of Nalm-6 and Sup-B15 cells and expression of apoptosis-related proteins BAX, BCL-2, cleaved Caspase-3 were determined by flow cytometry and Western blot respectivcly. RESULTS: Huaier aqueous extract, VCR, DNR and L-Asp had inhibition effect on Nalm-6 and Sup-B15 cell lines. The inhibition rate of Huaier aqueous extract combined with VCR, DNR and L-Asp were all higher than those of each dug alone (P＜0.05) and the combination index (q) was between 0.85 and 1.15 or greater than 1.15. The two kinds of drugs showed had additive or synergistic effects. The results of flow cytometry showed that the cell apoptosis rates in combined treatment group were higher than those of each drug alone (P＜0.05). The results of Western blot revealed that Huaier aqueous extract and VCR all decreased protein expression of BCL-2 (P＜0.05) and increase protein expression of BAX (P＜0.05) and cleaved Caspase-3 (P＜0.05) in Nalm-6 and Sup-B15 cells. Compared with Huaier aqueous extract or VCR alone, the effect of two drug combination were more significant. DNR down-regulated protein expression of BCL-2 (P＜0.05) and up-regulated cleaved Caspase-3 (P＜0.05). However, it had no effect on the expression of BAX in Nalm-6 and Sup-B15 cells. When it was combined with Huaier aqueous extract, the expression of cleaved Caspase-3 and BCL-2 showed more significant changes. The expression of BAX in combined treated group did not show significant difference, compared with group treated with Huaier aqueous extract in Nalm-6 and Sup-B15 cells. L-Asp did not show significant effect on the three apoptosis-related proteins and there was no significant difference between the combination group and the Huaier aqueous extract group. CONCLUSION: the combination of Huaier aqueous extract and VCR, DNR, L-Asp shows additive or synergistic effects on human acute lymphoblastic leukemia cell lines Nalm-6 and Sup-B15.

Physiologically based pharmacokinetic-pharmacodynamic modeling for prediction of vonoprazan pharmacokinetics and its inhibition on gastric acid secretion following intravenous/oral administration to rats, dogs and humans.

Vonoprazan is characterized as having a long-lasting antisecretory effect on gastric acid. In this study we developed a physiologically based pharmacokinetic (PBPK)-pharmacodynamic (PD) model linking to stomach to simultaneously predict vonoprazan pharmacokinetics and its antisecretory effects following administration to rats, dogs, and humans based on in vitro parameters. The vonoprazan disposition in the stomach was illustrated using a limited-membrane model. In vitro metabolic and transport parameters were derived from hepatic microsomes and Caco-2 cells, respectively. We found the most predicted plasma concentrations and pharmacokinetic parameters of vonoprazan in rats, dogs and humans were within twofold errors of the observed data. Free vonoprazan concentrations (fu × C2) in the stomach were simulated and linked to the antisecretory effects of the drug (I) (increases in pH or acid output) using the fomula dI/dt = k × fu × C2 × (Imax - I) - kd × I. The vonoprazan dissociation rate constant kd (0.00246 min-1) and inhibition index KI (35 nM) for H+/K+-ATPase were obtained from literatures. The vonoprazan-H+/K+-ATPase binding rate constant k was 0.07028 min-1· µM-1 using ratio of kd to KI. The predicted antisecretory effects were consistent with the observations following intravenous administration to rats (0.7 and 1.0 mg/kg), oral administration to dogs (0.3 and 1.0 mg/kg) and oral single dose or multidose to humans (20, 30, and 40 mg). Simulations showed that vonoprazan concentrations in stomach were 1000-fold higher than those in the plasma at 24 h following administration to human. Vonoprazan pharmacokinetics and its antisecretory effects may be predicted from in vitro data using the PBPK-PD model of the stomach. These findings may highlight 24-h antisecretory effects of vonoprazan in humans following single-dose or the sustained inhibition throughout each 24-h dosing interval during multidose administration.

[Pure red cell aplasia in children: a clinical analysis of 16 cases].

OBJECTIVE: To study the clinical features, treatment, and prognosis of pure red cell aplasia (PRCA) in children. METHODS: A retrospective analysis was performed for the clinical data of 16 children with PRCA. The outcome and prognosis of patients treated with prednisone combined with Huaiqihuang granules versus prednisone alone were evaluated. RESULTS: All the 16 children complained of symptoms of anemia including pale or sallow complexion. Of 12 children undergoing pathogen test, 7 (58%) were found to have pathogen infection, among which human cytomegalovirus was the most common. Lymphocyte subsets were measured for 7 children, among whom 5 (71%) had lymphocyte immune disorder. Six children were found to have abnormalities in immunoglobulin and complement. The 8 children treated with prednisone combined with Huaiqihuang granules had a median follow-up time of 21.5 months, among whom 1 was almost cured, 1 was relieved, and 6 were obviously improved; the median onset time of treatment was 1 month, and 2 children had disease recurrence in the course of drug reduction or withdrawal. The 8 children in the prednisone alone treatment group had a median follow-up time of 34 months, among whom 4 were almost cured, and 4 were obviously improved; the median onset time of treatment was 2.5 months, and 4 children had recurrence during drug reduction or withdrawal. CONCLUSIONS: Children with PRCA usually complain of anemia-related symptoms. Laboratory tests show pathogen infection in some children with PRCA, and most of children have immune disorders. Glucocorticoids have a good therapeutic effect, but some children relapse in the course of drug reduction or withdrawal. Combined treatment with prednisone and Huaiqihuang granules may have a faster onset of action and less possibility of recurrence.

Acute liver failure enhances oral plasma exposure of zidovudine in rats by downregulation of hepatic UGT2B7 and intestinal P-gp.

HIV infection is often associated with liver failure, which alters the pharmacokinetics of many drugs. In this study we investigated whether acute liver failure (ALF) altered the pharmacokinetics of the first-line anti-HIV agent zidovudine (AZT), a P-gp/BCRP substrate, in rats. ALF was induced in rats by injecting thioacetamide (TAA, 300 mg·kg-1·d-1, ip) for 2 days. On the second day after the last injection of TAA, the pharmacokinetics of AZT was investigated following both oral (20 mg/kg) and intravenous (10 mg/kg) administration. ALF significantly increased the plasma concentrations of AZT after both oral and intravenous doses of AZT, but without affecting the urinary excretion of AZT. AZT metabolism was studied in rat hepatic microsomes in vitro, which revealed that hepatic UGT2B7 was the main enzyme responsible for the formation of AZT O-glucuronide (GAZT); ALF markedly impaired AZT metabolism in hepatic microsomes, which was associated with the significantly decreased hepatic UGT2B7 expression. Intestinal absorption of AZT was further studied in rats via in situ single-pass intestinal perfusion. Intestinal P-gp function and intestinal integrity were assessed with rhodamine 123 and FD-70, respectively. We found that ALF significantly downregulated intestinal P-gp expression, and had a smaller effect on intestinal BCRP. Further studies showed that ALF significantly increased the intestinal absorption of both rhodamine 123 and AZT without altering intestinal integrity, thus confirming an impairment of intestinal P-gp function. In conclusion, ALF significantly increases the oral plasma exposure of AZT in rats, a result partly attributed to the impaired function and expression of hepatic UGT2B7 and intestinal P-gp.

Differential effects of pravastatin on the pharmacokinetics of paroxetine in normal and diabetic rats.

1. Diabetes is often accompanied with depression and hypercholesterolemia. It is possible that paroxetine and pravastatin are co-administered to diabetic patients. The aim of this study was to research the differential effect of pravastatin on plasma exposure of paroxetine in normal and diabetic rats. 2. Pharmacokinetics of paroxetine was investigated following oral administration of paroxetine with and without pravastatin in normal and diabetic rats. Effects of pravastatin on metabolism, intestinal absorption and hepatic uptake of paroxetine were investigated. Activity and expression of hepatic Oatp1 and Oatp2 were also assessed. 3. Pravastatin decreased plasma exposure of paroxetine in normal rats, but increased exposure of paroxetine in diabetic rats. Pravastatin neither affected metabolism nor intestinal absorption of paroxetine. Data from hepatocytes demonstrated that hepatic uptake of paroxetine were involved in Oatp1 and Oatp2. Diabetes suppressed Oatp1 activity and expression, but enhanced Oatp2 activity and expression. Pravastatin stimulated Oatp1 but inhibited Oatp2 activity. 4. We concluded that differential effects of pravastatin on plasma exposure of paroxetine in normal and diabetic rats was partly due to the fact that diabetes suppressed Oatp1 activity and expression but enhanced Oatp2 activity and expression as well as that pravastatin stimulated Oatp1 activity but inhibited Oatp2 activity.

Unconjugated bilirubin elevation impairs the function and expression of breast cancer resistance protein (BCRP) at the blood-brain barrier in bile duct-ligated rats.

AIM: Liver failure is associated with dyshomeostasis of efflux transporters at the blood-brain barrier (BBB), which contributes to hepatic encephalopathy. In this study we examined whether breast cancer resistance protein (BCRP), a major efflux transporter at the BBB, was altered during liver failure in rats. METHODS: Rats underwent bile duct ligation (BDL) surgery, and then were sacrificed after intravenous injection of prazosin on d3, d7 and d14. The brains and blood samples were collected. BCRP function at the BBB was assessed by the brain-to-plasma prazosin concentration ratio; Evans Blue extravasation in the brain tissues was used as an indicator of BBB integrity. The protein levels of BCRP in the brain tissues were detected. Human cerebral microvessel endothelial cells (HCMEC/D3) and Madin-Darby canine kidney cells expressing human BCRP (MDCK-BCRP) were tested in vitro. In addition, hyperbilirubinemia (HB) was induced in rats by intravenous injection of unconjugated bilirubin (UCB). RESULTS: BDL rats exhibited progressive decline of liver function and HB from d3 to d14. In the brain tissues of BDL rats, both the function and protein levels of BCRP were progressively decreased, whereas the BBB integrity was intact. Furthermore, BDL rat serum significantly decreased BCRP function and protein levels in HCMEC/D3 cells. Among the abnormally altered components in BDL rat serum tested, UCB (10, 25 µmol/L) dose-dependently inhibit BCRP function and protein levels in HCMEC/D3 cells, whereas 3 bile acids (CDCA, UDCA and DCA) had no effect. Similar results were obtained in MDCK-BCRP cells and in the brains of HB rats. Correlation analysis revealed that UCB levels were negatively correlated with BCRP expression in the brain tissues of BDL rats and HB rats as well as in two types of cells tested in vitro. CONCLUSION: UCB elevation in BDL rats impairs the function and expression of BCRP at the BBB, thus contributing to hepatic encephalopathy.

Laminaria japonica increases plasma exposure of glycyrrhetinic acid following oral administration of Liquorice extract in rats.

The present study was designed to investigate the effects of Laminaria japonica (Laminaria) on pharmacokinetics of glycyrrhetinic acid (GA) following oral administration of Liquorice extract in rats. Following oral administrations of single-dose and multi-dose Liquorice extract and Liquorice-Laminaria extract, respectively, plasma samples were obtained at various times and the concentrations of GA, liquiritigenin, and isoliquiritigenin were measured by LC-MS. The effects of Laminaria extract on pharmacokinetics of GA were also investigated, following single-dose and multidose of glycyrrhizic acid (GL). The effects of Laminaria extract on intestinal absorption of GA and GL were studied using the in situ single-pass intestinal perfusion model. The metabolism of GL to GA in the contents of small and large intestines was also studied. The results showed Liquorice-Laminaria extract markedly increased the plasma concentration of GA, accompanied by a shorter Tmax. Similar alteration was observed following multidose administration. However, pharmacokinetics of neither liquiritigenin nor isoliquiritigenin was affected by Laminaria. Similarly, Laminaria markedly increased concentration and decreased Tmax of GA following oral GL were observed. The data from the intestinal perfusion model showed that Laminaria markedly increased GL absorption in duodenum and jejunum, but did not affect the intestinal absorption of GA. It was found that Laminaria enhanced the metabolism of GL to GA in large intestine. In conclusion, Laminaria increased plasma exposures of GA following oral administration of liquorice or GL, which partly resulted from increased intestinal absorption of GL and metabolism of GL to GA in large intestine.

[Dynamics of soil water under Eucommia ulmodes plantation in hilly red soil region of southern China].

By using time domain reflectometry (TDR), a fixed-position monitoring research on the dynamics of soil water under Eucommia ulmoides plantation was conducted in a hilly red soil region of southern China. The results showed that there was a significant difference in the soil water storage among different months, and the dynamics of soil water could be divided into the phases of reduction, increase, and more reduction. Soil water varied significantly in its vertical distribution, and the variation pattern also differed with seasons. The vertical distribution of soil water could be divided into two phases, i.e., accumulation and depletion based on the seasonal variation of soil water, or rainy season and dry season based on the monthly variation of rainfall. Soil water was correlated significantly (P < 0.05) with relative humidity (RH), air temperature (t), vapor pressure deficit (VPD) and rainfall (R), and regulated by these meteorological factors synthetically, among which, rainfall was the most important factor, followed by air temperature. The soil water loss rate after rain had a significant hyperbolic relationship with durative droughty days, while soil water storage had a significant linear negative relationship with this duration (P < 0.05). With the extension of drought duration after rain, soil water loss tended to vary gently with increasing soil depth.