Neferine inhibits BMECs pyroptosis and maintains blood-brain barrier integrity in ischemic stroke by triggering a cascade reaction of PGC-1α.

Blood-brain barrier disruption is a critical pathological event in the progression of ischemic stroke (IS). Most studies regarding the therapeutic potential of neferine (Nef) on IS have focused on neuroprotective effect. However, whether Nef attenuates BBB disruption during IS is unclear. We here used mice underwent transient middle cerebral artery occlusion (tMCAO) in vivo and bEnd.3 cells exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) injury in vitro to simulate cerebral ischemia. We showed that Nef reduced neurobehavioral dysfunction and protected brain microvascular endothelial cells and BBB integrity. Molecular docking, short interfering (Si) RNA and plasmid transfection results showed us that PGC-1α was the most binding affinity of biological activity protein for Nef. And verification experiments were showed that Nef upregulated PGC-1α expression to reduce mitochondrial oxidative stress and promote TJ proteins expression, further improves the integrity of BBB in mice. Intriguingly, our study showed that neferine is a natural PGC-1α activator and illustrated the mechanism of specific binding site. Furthermore, we have demonstrated Nef reduced mitochondria oxidative damage and ameliorates endothelial inflammation by inhibiting pyroptosis to improve BBB permeability through triggering a cascade reaction of PGC-1α via regulation of PGC-1α/NLRP3/GSDMD signaling pathway to maintain the integrity of BBB in ischemia/reperfusion injury.

Membrane P-glycoprotein expression in lymphocytes from adult patients with refractory glomerulonephritis.

OBJECTIVES: To investigate the expression of P-glycoprotein in T-cell subpopulations of lymphocytes from adult patients with refractory glomerulonephritis (GN). MATERIALS AND METHODS: Flow cytometry was used to analyze the T-cell subpopulations of lymphocytes from adult patients with refractory GN and healthy individuals. The CD243 antibody marked the membrane P-glycoprotein of immune cells. RESULTS: The mean ± standard deviation (SD) values of percentages of CD3+, CD3+CD4+, CD3+CD8+ cells in lymphocytes from patients with refractory GN were 63.94 ± 26.98, 55.16 ± 4.78, and 37.79 ± 6.01%, respectively. These values in healthy individuals were 74.88 ± 3.75, 56.60 ± 9.22, and 34.20 ± 5.21%, respectively. No significant differences were observed between the patients with refractory GN and healthy individuals. The mean ± SD values of percentages of CD3+CD4+CD243+ and CD3+CD8+CD243+ cells in the lymphocytes of patients with refractory GN were 0.14 ± 0.11 and 0.11 ± 0.07%, respectively. These values in healthy individuals were 0.05 ± 0.02 and 0.04 ± 0.02%, respectively. The difference in CD3+CD8+CD243+ percentage between patients with refractory GN and healthy individuals was significant (p = 0.0216). CONCLUSION: These findings suggest that P-glycoprotein expression on CD3+CD8+ T cells is a promising marker and a suitable target of drug resistance in patients with refractory GN.

Cepharanthine synergistically promotes methylprednisolone pharmacodynamics against human peripheral blood mononuclear cells possibly via regulation of P-glycoprotein/glucocorticoid receptor translocation.

BACKGROUND: Cepharanthin® alone or in combination with glucocorticoid (GC) has been used to treat chronic immune thrombocytopenia (ITP) since the 1990s. Cepharanthine (CEP) is one of the main active components of Cepharanthin®. The purpose of this study was to investigate the effects of CEP on GC pharmacodynamics on immune cells and analyse the possible action mechanism of their interactions. METHODS: Peripheral blood mononuclear cells (PBMCs), T lymphocytic leukemia MOLT-4 cells and daunorubicin resistant MOLT-4 cells (MOLT-4/DNR) were used to evaluate the pharmacodynamics and molecular mechanisms. Drug pharmacodynamics was evaluated by WST-8 assay. P-glycoprotein function was examined by rhodamine 123 assay. CD4+CD25+Foxp3+ regulatory T cells and Th1/Th2/Th17 cytokines were detected by flow cytometry. P-glycoprotein expression and GC receptor translocation were examined by Western blot. RESULTS: CEP synergistically increased methylprednisolone (MP) efficacy with the suppressive effect on the cell viability of PBMCs. 0.3 and 1 µM of CEP significantly inhibited P-glycoprotein efflux function of CD4+ cells, CD8+ cells, and lymphocytes (P<0.05). 0.03~3 µM of CEP also inhibited the P-glycoprotein efflux function in MOLT-4/DNR cells in a concentration-dependent manner (P<0.001). However, 0.03~3 µM of CEP did not influence P-glycoprotein expression. 0.03~0.3 µM of CEP significantly increased the GC receptor distribution from the cytoplasm to the nucleus in a concentration-dependent manner in MOLT-4/DNR cells. The combination did not influence the frequency of CD4+, CD4+CD25+ and CD4+CD25+Foxp3+ T cells or the secretion of Th1/Th2/Th17 cytokines from PBMCs. In contrast, CEP alone at 1 µM decreased the percentage of CD4+ T cell significantly (P<0.01). It also inhibited the secretion of IL-6, IL-10, IL-17, TNF-α, and IFN-γ. CONCLUSIONS: CEP synergistically promoted MP pharmacodynamics to decrease the cell viability of the mitogen-activated PBMCs, possibly via inhibiting P-glycoprotein function and potentiating GC receptor translocation. The present study provides new evidence of the therapeutic effect of Cepharanthin® alone or in combination with GC for the management of chronic ITP.

Tetrandrine inhibits the proliferation of mesangial cells induced by enzymatically deglycosylated human IgA1 via IgA receptor/MAPK/NF-κB signaling pathway.

Objective: Despite the use of renin-angiotensin system blockade and immunosuppressive drugs, including corticosteroids, the current treatment regimens for Immunoglobulins A nephropathy (IgAN) are severely limited. The proliferation of mesangial cell and deposition of deglycosylated human IgA1 immune complex are the most common pathologic features of IgAN. We examined the tetrandrine potential of suppressing the proliferation of mesangial cells and explored its underlying mechanisms with a focus on IgA receptor/MAPK/NF-κB signaling pathway. Methods: Standard human IgA (native IgA) were enzymatically desialylated (deS IgA) or further degalactosylated (deS/deGal IgA) using neuraminidase and ß-galactosidase. Rat glomerular mesangial cells (HBZY-1) and human renal mesangial cells (HRMC) stimulated by IgA were used to observe the suppressive effect of tetrandrine. The MTT assay was used to detect the cell viability. The protein expression of IgA receptor/MAPK/NF-κB signaling pathway was examined by Western blot. Cell cycle analysis was measured by flow cytometer. Results: Native IgA and deS IgA showed limited stimulation effect on both HBZY-1 cells and HRMCs, whereas deS/deGal IgA significantly stimulated the proliferation of both HBZY-1 cells and HRMCs (p < 0.05). Compared with non-stimulation of deS/deGal IgA, 1-3 µM of tetrandrine had stronger inhibitory effect on the proliferation of HBZY-1 cells and HRMCs with the stimulation of deS/deGal IgA (p < 0.05), suggesting that tetrandrine possibly inhibited the proliferation of mesangial cells induced by deglycosylated human IgA1 specifically. Molecular mechanism study revealed that tetrandrine decreased the expression of IgA1 receptor, CD71 and ß4GALT1, and inhibited the activation of MAPK/NF-κB significantly (p < 0.05). Moreover, these inhibitory effect of tetrandrine caused cell cycle arrest and stopped the cell growth in the S phase companied with the upregulating of cyclin A2 and downregulating of cyclin D1. Conclusion: Taken together, tetrandrine inhibited the proliferation of mesangial cells induced by enzymatically deglycosylated human IgA1 via IgA receptor/MAPK/NF-κB signaling pathway. Based on these potential molecular mechanisms, tetrandrine would be an appealing therapeutic option for IgAN.

Function of the algicidal bacterium Pseudomonas sp. Go58 isolated from the biofilm on a water plant, and its active compounds, pyoluteorins.

In the development of applications to mitigate nuisance cyanobacterial blooms, environmentally friendly approaches have gained much attention. Recently, we found a bacterial strain Go58, which was isolated from the biofilm of a water plant, that showed potent algicidal activity against the bloom-forming cyanobacterium Microcystis aeruginosa. Whole genome sequencing of strain Go58 suggested that it is potentially a novel species closely related to Pseudomonas protegens. Pyoluteorins were obtained from the culture broth of this strain, and they demonstrated high toxicity against cultured cyanobacterial species, including M. aeruginosa and Anabaena cylindrica, but less toxicity against eukaryotic microalgae and other aquatic organisms. The production of pyoluteorin was enhanced by the presence of the target cyanobacterium. When a wild-caught microalgal consortium was treated with either strain Go58 or pyoluteorin, both efficiently suppressed the growth of harmful wild cyanobacteria, but promoted the growth of some specific eukaryotic microalgae. Since P. protegens is globally ubiquitous and highly anticipated to be a biopesticide for infectious diseases in the field of agriculture, the similar bacterial group identified in this study may also have potential as a safe on-site collectable biological countermeasure for controlling cyanobacterial blooms.

Mechanisms and biological effects of organic amendments on mercury speciation in soil-rice systems: A review.

Mercury (Hg) pollution is a well-recognized global environmental and health issue and exhibits distinctive persistence, neurotoxicity, bioaccumulation, and biomagnification effects. As the largest global Hg reservoir, the Hg cumulatively stored in soils has reached as high as 250-1000 Gg. Even more concerning is that global soil-rice systems distributed in many countries have become central to the global Hg cycle because they are both a major food source for more than 3 billion people worldwide and the central bridge linking atmospheric and soil Hg circulation. In this review, we discuss the form distribution, transformation, and bioavailability of Hg in soil-rice systems by focusing on the Hg methylation and demethylation pathways and distribution, uptake, and accumulation in rice plants and the effects of Hg on the community structure and ecological functions of microorganisms in soil-rice systems. In addition, we clarify the mechanisms through which commonly used humus and biochar organic amendments influence Hg and its environmental effects in soil-rice systems. The review also elaborates on the advantages of sulfur-modified biochars and their critical role in controlling Hg migration and bioavailability in soils. Finally, we provide key information about Hg pollution in soil-rice systems, which is of great significance for developing appropriate strategies and mitigation planning to limit Hg bioconcentration in rice crops and achieving key global sustainable development goals, such as the guarantee of food security and the promotion of sustainable agriculture.

Roles of H2S and NO in regulating the antioxidant system of Vibrio alginolyticus under norfloxacin stress.

Antioxidant system is of great importance for organisms to regulate the level of excessive reactive oxygen species (ROS) under the environmental stresses including antibiotics stress. Effects of norfloxacin (NOR) on cystathionine-ß-synthase (CBS), nitric oxide synthase (NOS) and antioxidant enzymes were investigated, and interaction between NO and H2S and their regulation on the antioxidant system of Vibrio alginolyticus under NOR were determined as well in the present study. After treated with 2 µg/mL NOR (1/2 MIC), CBS content, H2S and NO contents decreased while H2O2 accumulation and the antioxidant-related genes mRNA level increased. Additionally, the endogenous H2S content in V. alginolyticus was increased by the exogenous NO, while H2O2 accumulation and the relative expression level of SOD (Superoxide dismutase gene) decreased under exogenous NO or H2S. And the content of endogenous NO and NOS in V. alginolyticus increased under the exogenous H2S as well. Taken together, these results showed that anti-oxidative ability in V. alginolyticus was respectively enhanced by the gas molecules of H2S and NO under NOR-induced stress, and there may be a crosstalk regulative mechanism between H2S and NO. These results lay a foundation for the research of regulation network of H2S and NO, and provide a hint to synthesize anti-vibrio drugs in the future.

Effects of vitamin K2 combined with methotrexate against mitogen-activated peripheral blood mononuclear cells of healthy subjects and rheumatoid arthritis patients.

BACKGROUND: Methotrexate (MTX) is used as anchor drug for patients with early and established rheumatoid arthritis (RA). Vitamin K2 administration was also reported to be associated with decreased disease activity in RA. OBJECTIVES: Immunosuppressive pharmacodynamics of vitamin K2 combined with MTX was investigated. METHODS: Mitogen-activated peripheral blood mononuclear cells (PBMCs) were used to evaluate immunosuppressive pharmacodynamics of drugs in vitro. RESULTS: Vitamin K2 alone dose-dependently suppressed T cell mitogen-activated proliferation of PBMCs of both healthy subjects and RA patients. 446.5 and 2232.5 ng/mL vitamin K2 significantly decreased the IC50 values of MTX on the proliferation of PBMCs of RA patients, with little influences on the pharmacodynamics of MTX in the healthy PBMCs. 4465 ng/mL vitamin K2 potentiated the pharmacodynamics of MTX in both RA patients and healthy PBMCs. The additional effects of vitamin K2 to potentiate the suppressive effects of MTX seemed not to be related to the regulation of CD4+ CD25+ T cells or CD4+ CD25+ Foxp3+ Treg cells. MTX alone at 100 ng/mL significantly decreased the percentage of CD4+ T cells in PBMCs of healthy subjects (p < 0.001) with a slight influence in that of RA patients (not significant) and the combination did not show synergistic inhibitory effect. Vitamin K2 alone tended to suppress the secretion of IL-17, IFN-γ, and TNF-α from the activated PBMCs of RA patients with smaller influences on the cytokine productions from healthy PBMCs. These additional effects of vitamin K2 were also observed in combination with MTX. CONCLUSION: The above information may partially elucidate the potentiation effects of vitamin K2 on the immunosuppressive efficacy of MTX.

Effects of sinomenine on the proliferation, cytokine production, and regulatory T-cell frequency in peripheral blood mononuclear cells of rheumatoid arthritis patients.

Sinomenine (SN) is a plant-derived alkaloid isolated from Caulis Sinomenii. It has been approved by the State Food and Drug Administration of China for treating rheumatoid arthritis (RA) nearly 20 years ago. To investigate the anti-RA mechanism of SN, a lot of scholars reported the immunosuppressive effect of SN on T lymphocytes. We continued to evaluate the suppressive function of SN by using human peripheral blood mononuclear cells (PBMCs) isolated from RA patients. As the positive control, 10 ng/ml of methylprednisolone (MP) showed the antiproliferation effect on mitogen-activated PBMCs of RA patients significantly (*p < .05). Meanwhile, MP decreased the frequency of CD4+ CD25+ T cells and suppressed the secretion of inflammatory Th1/Th2/Th17 cytokines such as IL-4, IL-6, IL-10, IL-17, IFN-γ, and TNF-α. However, SN at concentrations of 0.3-30 µM, showed little suppressive effects on the proliferation of PBMCs of RA patients. We did not observe any suppressive effects of SN on percentages of CD4+ T cells and CD4+ CD25+ T cells in the mitogen-activated PBMCs of RA patients. The influence of SN on the percentage of CD4+ CD25+ Foxp3+ T cells was also limited. Finally, even 30 µM of SN did not influence the secretion of Th1/Th2/Th17 cytokine significantly. The present study provided evidence that anti-RA mechanism of SN seems not to be related with the suppressive effects on peripheral T cells.

Suppressive effect of vitamin K2 against mitogen-activated peripheral blood mononuclear cells of rheumatoid arthritis patients.

OBJECTIVE: To investigate the immunosuppressive effect of vitamin K2 against mitogen-activated peripheral blood mononuclear cells (PBMCs) of rheumatoid arthritis (RA) patients. MATERIALS AND METHODS: Concanavalin A-stimulated PBMC culture procedure was used to evaluate the pharmacodynamics of vitamin K2 in vitro. Methotrexate was set up as the positive control. The proliferation of PBMCs was detected by MTT assay. Relationship between IC50 values of drugs on PBMC proliferation and patient-related factors including laboratory data was analyzed by nonparametric Spearman correlation test. RESULTS: Vitamin K2 inhibited the proliferation of mitogen-activated PBMCs of RA patients with an IC50 value of 3,288.47 ± 4,910.02 ng/mL (mean ± SD). There was a significant correlation between IC50 values of vitamin K2 and patient-related factors of RA patients (p < 0.05), such as C-reactive protein (CRP), rheumatoid factor, anti-cyclic citrullinated peptide antibody (ACPA), matrix metalloproteinase-3, Pre-DAS-28 (CRP), and ∆DAS-28 (CRP). It would be possible to predict the pharmacodynamics of vitamin K2 in RA patients according to the above factors. Methotrexate inhibited the proliferation of mitogen-activated PBMCs of RA patients with a IC50 value of 22.83 ± 12.47 ng/mL (mean ± SD). IC50 values of methotrexate only showed significant correlation with ACPA (p = 0.0158, r = 0.6905), which suggests that ACPA might be a suitable predictor of the pharmacodynamics of methotrexate. CONCLUSION: The above information suggests that vitamin K2 could provide a benefit for the treatment of RA patients via its immunosuppressive function.

Absolute configuration of tetrandrine and isotetrandrine influences their anti-proliferation effects in human T cells via different regulation of NF-κB.

Natural compound tetrandrine was reported to inhibit the proliferation of T cells by inhibiting activation of NF-κB. Chemically, isotetrandrine differs from tetrandrine only in the stereochemistry at the chiral centers. The present study aimed to compare their anti-proliferation effects on human T cells with a focus on NF-κB. The IC50 values of tetrandrine against MOLT-4 cells, MOLT-4/DNR cells, and concanavalin A-activated peripheral blood mononuclear cells of healthy subjects and dialysis patients were 4.43 ± 0.22, 3.62 ± 0.22, 1.91 ± 0.22 and 3.03 ± 0.28 µM, respectively. Whereas, the IC50 values of isotetrandrine against the above immune cells were 2.19 ± 0.27, 2.28 ± 0.33, 1.29 ± 0.14 and 1.55 ± 0.26 µM, respectively. The inhibitory effect of isotetrandrine against the proliferation of T cells was stronger than that of tetrandrine significantly (p < 0.05). Molecular mechanism investigation showed that 10 µM of isotetrandrine largely decreased the expression of p-NF-κB and NF-κB in both MOLT-4 and MOLT-4/DNR T cells (p < 0.05), whereas 10 µM of tetrandrine slightly inhibited the phosphorylation of p-NF-κB with little influence on the expression of NF-κB. Taken together, absolute configurations of tetrandrine and isotetrandrine are suggested to influence on their anti-proliferation effects in human T cells via different regulation of NF-κB.

Molecular mechanisms and therapeutic implications of tetrandrine and cepharanthine in T cell acute lymphoblastic leukemia and autoimmune diseases.

Inappropriately activated T cells mediate autoimmune diseases and T cell acute lymphoblastic leukemia (T-ALL). Glucocorticoid and chemotherapeutic agents have largely extended lives of these patients. However, serious side effects and drug resistance often limit the prognosis of considerable number of the patients. The efficient treatment of autoimmune diseases or T-ALL with drug resistance remains an important unmet demand clinically. Bisbenzylisoquinoline alkaloids tetrandrine and cepharanthine have been applied for the treatment of certain types of autoimmune diseases and cancers, while studies on their action mechanisms and their further applications combined with glucocorticoids or chemotherapeutic agents remains to be expanded. This review introduced molecular mechanisms of tetrandrine and cepharanthine in T cells, including their therapeutic implications. Both tetrandrine and cepharnthine influence the growth of activated T cells via several kinds of signaling pathways, such as NF-κB, caspase cascades, cell cycle, MAPK, and PI3K/Akt/mTOR. According to recent preclinical and clinical studies, P-glycoprotein inhibitory effect of tetrandrine and cepharnthine could play a significant role on T cell-involved refractory diseases. Therefore, tetrandrine or cepharanthine combined with glucocorticoid or other anti-leukemia drugs would bring a new hope for patients with glucocorticoid-resistant autoimmune disease or refractory T-ALL accompanied with functional P-glycoprotein. In conclusion, bisbenzylisoquinoline alkaloids tetrandrine and cepharanthine can regulate several signaling pathways in abnormally activated T cells with low toxicity. Bisbenzylisoquinoline alkaloids deserve to be paid more attention as a lead compound to develop new drugs for the treatment of T cell-involved diseases in the future.

[Change laws of water absorption in Chinese herbal pieces and prediction model of relative density for Chinese medicine decoction].

This study aims to explore the change laws of water absorption in Chinese herbal pieces and establish the prediction model of relative density for Chinese medicine compound decoction. Firstly, fitted equations of water absorption and decocting time was established by observing the change laws of water absorption in 36 kinds of Chinese herbal pieces in 12 groups(according to the drug-parts) with decocting time. The r value of the mineral group and other type group was 0.691 2 and 0.663 3, respectively. The r value of the remaining 10 groups was 0.802 2-0.925 4. All P values were less than 0.05(n=21). The formula of the amount of water added was optimized by combining the fitted equations with determined water absorption, and the liquid yield could be controlled in a range of 100%±10%. Secondly, it was determined that the liquid density tester could be used for the rapid determination of relative density of Chinese medicine decoction after methodological study and comparison with the pycnometer method. The linear regression equation between the corrected relative density(y) and extraction ratio(%, x) was built by measuring and analyzing the related parameters such as liquid yield, relative density and extraction ratio in 46 kinds of Chinese herbal pieces. The established equation was y=0.041 3x+1.003 7, r=0.930 9(P <0.01, n=46), with linear range of 1.94%-65.75%. Based on this, the prototype model for predicting relative density of Chinese medicine decoction was established, and the relative densities of 8 Chinese medicine decoctions were within the prediction interval of this model in verification. This study lays a foundation for database construction of Chinese medicine decoction, implementation of personalized decocting mode and rapid quality control of Chinese medicine decoction.

Methylprednisolone potentiates tetrandrine pharmacodynamics against human T lymphoblastoid leukemia MOLT-4 cells via regulation of NF-κB activation and cell cycle transition.

Low response to glucocorticoid (GC) predicts therapeutic failure in acute T lymphoblastic leukemia (T-ALL). The efficient and safe strategies are still required for the treatment of relapsed T-ALL. Our previous study revealed that tetrandrine induces apoptosis in human T lymphoblastoid leukemia cells possibly via activation of NF-κB. GCs are recognized as typical NF-κB inhibitors and are used for the treatment of T-ALL patients. In the present study, we examined whether methylprednisolone (MP) potentiates the cytotoxic effect of tetrandrine (TET) via NF-κB regulation by using human T lymphoblastoid leukemia MOLT-4 cells. WST-8 assay data showed that nM grade of MP increased cytotoxicity of TET against MOLT-4 cells in vitro. This effect seemed to be related to the potentiation of TET action by MP to induce apoptosis. Meanwhile, the combination also impeded the transition of cell cycle from G0/G1 phase to S phase. However, the regulation effect of this combination on cell cycle had no relationship with cyclin signaling pathway, since the drug-combination did not influence on the expression of cyclin A2/B1/D1 in MOLT-4 cells. On the other hand, the combination significantly inhibited the phosphorylation of NF-κB (p < 0.01). These results suggest that nM grade of MP potentiates the cytotoxic effect of TET possibly via regulation of NF-κB activation and "G0/G1 to S" phase transition in human T lymphoblastoid leukemia MOLT-4 cells. Combination of TET and MP may provide a new therapeutic strategy for relapsed T-ALL.

Bisbenzylisoquinoline alkaloids and P-glycoprotein function: A structure activity relationship study.

Conflicts with the notion that specific substrate interactions were required in the control of reaction path in active transport systems, P-glycoprotein showed extraordinarily low specificity. Therefore, overexpression P-glycoprotein excluded a large number of anticancer agents from cancer cells, and multidrug resistance happened. Several kinds of bisbenzylisoqunoline alkaloids were reported to modulate P-glycoprotein function and reverse drug resistance. In order to provide more information for their structure activity relationship on P-glycoprotein function, the effects of tetrandrine, isotetrandrine, fangchinoline, berbamine, dauricine, cepharanthine and armepavine on the P-glycoprotein function were compared by using daunorubicin-resistant leukemia MOLT-4 cells in the present study. Among them, tetrandrine exhibited the strongest P-glycoprotein inhibitory effect, followed with fangchinoline and cepharanthine, and subsequently with berbamine or isotetrandrine. However, dauricine and armepavine showed little influence on the P-glycoprotein function. These data revealed that the 18-membered ring of the bisbenzylisoquinoline alkaloids maintained the P-glycoprotein inhibitory activity, suggesting that double isoquinoline units connected by two oxygen bridges were indispensable. Moreover, stereo-configuration of bisbenzylisoquinoline 3D structures determined their inhibitory activities, which provided a new viewpoint to recognize the specificity of binding pocket in P-glycoprotein. Our data also indicated that 3D chemical structure was more sensitive than 2D to predict the P-glycoprotein inhibitory-potencies of bisbenzylisoqunoline alkaloids.

Tetrandrine enhances glucocorticoid receptor translocation possibly via inhibition of P-glycoprotein in daunorubicin-resistant human T lymphoblastoid leukemia cells.

Glucocorticoids are used as anticancer and immunosuppressive agents, whereas glucocorticoid resistance has been observed in a significant fraction of patients due to overexpression of P-glycoprotein encoded by multi-drug resistance-1 gene. Tetrandrine is a bisbenzylisoquinoline alkaloid isolated from traditional herb Fangji. According to our previous report, tetrandrine potentiated glucocorticoid pharmacodynamics partially via inhibiting P-glycoprotein function. In the present study, we investigated whether glucocorticoid receptor translocation was influenced indirectly by tetrandrine via P-glycoprotein inhibition, using human T lymphoblastoid leukemia MOLT-4 cell line with little P-glycoprotein expression and its multidrug resistant sub-line MOLT-4/DNR exhibiting a large amount of P-glycoprotein. Molecular mechanism investigation suggested that overexpressed P-glycoprotein weakened the glucocorticoid receptor translocation in MOLT-4/DNR cells comparing with the parent MOLT-4 cells. Our data also suggested that tetrandrine enhanced nuclear glucocorticoid receptor translocation in MOLT-4/DNR cells indirectly by dual influences on P-glycoprotein, inhibiting the efflux function and downregulating the protein expression. Therefore, tetrandrine potentiated the cytotoxic effect of methylprednisolone against MOLT-4/DNR cells with less effects on MOLT-4 cells. These effects of tetrandrine were suggested to be beneficial for the treatment of glucocorticoid resistant diseases induced by the overexpression of P-glycoprotein.

Cytotoxic effects of vitamins K1, K2, and K3 against human T lymphoblastoid leukemia cells through apoptosis induction and cell cycle arrest.

The present study was undertaken to evaluate cytotoxic effects of vitamin K1 (phylloquinone), vitamin K2 (menaquinones), and vitamin K3 (menadione) against human T lymphoblastoid leukemia cells, Jurkat T cells, MOLT-4 cells, and P-glycoprotein-expressing multidrug-resistant MOLT-4/DNR cells. Vitamins K2 and K3, but not vitamin K1, reduced viabilities of Jurkat, MOLT-4, and MOLT-4/DNR cells. The influence potency of vitamin K3 was larger than that of vitamin K2 in all of the three cell lines. MOLT-4/DNR cells seemed to be more sensitive toward the effects of vitamins K2 and K3. The cytotoxicity of vitamins K2 and K3 on these leukemia cells seems to be related to apoptosis induction and cell cycle arrest. Vitamin K2 and K3 treatment induced cleavage of PARP obviously. Moreover, vitamins K2 and K3 specifically down-regulated the expressions of cyclin A2 in all of the three cell lines. However, the effects of vitamins K2 and K3 on the cell cycle profiling in Jurkat, MOLT-4, and MOLT-4/DNR cells varied with the cell type. Vitamins K2 and K3 also decreased the viability of mitogen-activated human peripheral blood mononuclear cells. Our observations suggest that vitamins K2 and K3 have bilateral cytotoxic effects on activated human peripheral lymphocytes and the human leukemic T cells.

[Pharmacokinetics of eight constituents in rat plasma after oral administration of Modified Xiaochaihu Granules for gastric ulcer based on UPLC-MS/MS].

An ultra-performance liquid chromatography-tandem mass spectrometric method (UPLC-MS/MS) was developed in this study to simultaneously determine the contents of eight effective constituents in rat plasma, including baicalin, wogonoside, baicalein, liquiritin, glycyrrhizic acid, berberine hydrochloride, saikosaponin a and saikosaponin d in plasma of gastric ulcer rats, and investigate the pharmacokinetics of Modified Xiaochaihu Granules. Chromatographic separation was conducted on Zorbax SB-C18 column (2.1 mm×100 mm, 1.8 µm) with acetonitrile -0.1% formic acid aqueous solution as the mobile phase for gradient elution, at a flow rate of 0.4 mL·min⁻¹ and column temperature of 40 °C. Detection was performed in the multiple reaction monitoring (MRM) mode with ESI ion source. All calibration curves showed good linearity (r>0.996) over a wide concentration range for all constituents. RSDs of intra-day and inter-day precision were all within 15% and the extraction recoveries of all the constituents were in the range of 81.92% to 104.8%. The time to peak (tmax) of these eight constituents was (2.69±2.02), (5.17±2.04), (0.25±0), (0.83±0.26), (0.92±0.20), (0.92±0.20), (0.58±0.20), and (0.083±0) h, respectively; the half-life (t1/2) of them was (7.85±0.34), (10.16±2.21), (6.79±0.21), (8.32±0.48), (11.05±1.78), (11.56±3.46), (15.30±1.84), and (5.54±1.91) h, respectively; the peak concentration (Cmax) of them was (55.02±1.67), (213.66±4.62), (62.61±0.69), (68.43±1.42), (62.22±0.39), (30.17±1.89), (61.79±4.81), and (38.02±1.75) µg·L⁻¹, respectively. This established method is simple and accurate with good repeatability and strong specificity, which could provide modern experimental basis for modified Xiaochaihu granules in clinical treatment of gastric ulcer.

Mechanisms of Antiulcer Effect of an Active Ingredient Group of Modified Xiao Chaihu Decoction.

The present study aimed to investigate the antiulcer activities and mechanisms of action of an active ingredient group (AIG) of Modified Xiao Chaihu Decoction (MXCD). The gastroprotective action of the AIG was studied in ethanol-induced, pylorus ligature-induced, and acetic acid-induced in vivo gastric ulcer models. The enzyme-linked immunoadsorbent assay (tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), and epidermal growth factor (EGF)), nitrate reductase assay (nitric oxide (NO)), western blot analysis (Bax, Bcl-2, cleaved-caspase-3, and cleaved-PARP (poly (ADP-Ribose) polymerase)), histological analysis (HE), and immunohistochemical analysis (HSP-70, p-AKT, and PCNA) were used to evaluate the anti-inflammatory, antiapoptotic, and healing properties of AIG. Numerous mechanisms are involved in the antiulcer activity of AIG, including the increase of PGE2, NO, and EGF content and a reduction in TNF-α levels. The upregulation of HSP-70, p-AKT, and PCNA seems to be directly linked to the healing effect of AIG. Bax, Bcl-2, cleaved-caspase-3, and cleaved-PARP also play a key role in this process. The AIG exerted gastroprotective effects by reducing antisecretory, anti-inflammatory, and antiapoptotic mechanisms. In addition, it promotes cell proliferation. Therefore, activation of the PI3K/AKT signaling pathway may play an important role in cell proliferation.

Anti-Helicobacter pylori and Anti-Inflammatory Effects and Constituent Analysis of Modified Xiaochaihutang for the Treatment of Chronic Gastritis and Gastric Ulcer.

Chronic gastritis and gastric ulcers are prevalent throughout the world and are considered to be a global health problem. Modified Xiaochaihutang (MXCHT) prescription is broadly used in traditional medicine hospital for the treatment of gastritis. In order to assess the anti-Helicobacter pylori (H. pylori) effect of MXCHT, agar diffusion method in vitro and fluid dilution method for the minimal inhibitory concentration (MIC) were established. The anti-inflammatory effects were then evaluated using mouse ear edema model and rat paw edema model. The ethanol-induced gastric ulcer method was employed to verify the gastroprotective effect of active extracts in MXCHT. HPLC-TOF-MS/MS was used for analyzing the possible active constituents after oral administration of effective extracts in ethanol-induced gastric ulcer models. MXCHT and 4 different extracts of the bacterial inhibition diameter and MIC were dramatically decreased compared with control group, showing anti-Helicobacter pylori effects. High dose groups of MXCHT, water extract, EtOAc extract, and n-BuOH extract displayed significant anti-inflammatory effects in xylene-induced mouse ear edema model and carrageenan-induced rat paw edema model test. MXCHT and all active extracts exhibited gastroprotective activity and prevented gastric lesions induced by ethanol in rats. 4 prototype components and 4 metabolites were identified after oral administration of EtOAc extract. In addition, 6 prototype components and 6 metabolites were identified in n-BuOH extract. MXCHT, EtOAc extract, and n-BuOH extract demonstrate gastroprotective effects through anti-Helicobacter pylori and anti-inflammatory activities. Thus, this prescription may be a suitable natural source for the prevention and treatment of chronic gastritis and gastric ulcers.