The Role of PI3K/AKT Pathway and NADPH Oxidase 4 in Host ROS Manipulation by Toxoplasma gondii

Dendritic cell is one of the first innate immune cell to encounter T. gondii after the parasite crosses the host intestinal epithelium. T. gondii requires intact DC as a carrier to infiltrate into host central nervous system (CNS) without being detected or eliminated by host defense system. The mechanism by which T. gondii avoids innate immune defense of host cell, especially in the dendritic cell is unknown. Therefore, we examined the role of host PI3K/AKT signaling pathway activation by T. gondii in dendritic cell. T. gondii infection or T. gondii excretory/secretory antigen (TgESA) treatment to the murine dendritic cell line DC2.4 induced AKT phosphorylation, and treatment of PI3K inhibitors effectively suppressed the T. gondii proliferation but had no effect on infection rate or invasion rate. Furthermore, it is found that T. gondii or TgESA can reduce H2O2-induced intracellular reactive oxygen species (ROS) as well as host endogenous ROS via PI3K/AKT pathway activation. While searching for the main source of the ROS, we found that NADPH oxidase 4 (NOX4) expression was controlled by T. gondii infection or TgESA treatment, which is in correlation with previous observation of the ROS reduction by identical treatments. These findings suggest that the manipulation of the host PI3K/AKT signaling pathway and NOX4 expression is an essential mechanism for the down-regulation of ROS, and therefore, for the survival and the proliferation of T. gondii.

Dipenyleneiodonium Induces Growth Inhibition of Toxoplasma gondii through ROS Induction in ARPE-19 Cells

Based on the reactive oxygen species (ROS) regulatory properties of diphenyleneiodonium (DPI), we investigated the effects of DPI on host-infected T. gondii proliferation and determined specific concentration that inhibit the intracellular parasite growth but without severe toxic effect on human retinal pigment epithelial (ARPE-19) cells. As a result, it is observed that host superoxide, mitochondria superoxide and H2O2 levels can be increased by DPI, significantly, followed by suppression of T. gondii infection and proliferation. The involvement of ROS in anti-parasitic effect of DPI was confirmed by finding that DPI effect on T. gondii can be reversed by ROS scavengers, N-acetyl-L-cysteine and ascorbic acid. These results suggest that, in ARPE-19 cell, DPI can enhance host ROS generation to prevent T. gondii growth. Our study showed DPI is capable of suppressing T. gondii growth in host cells while minimizing the un-favorite side-effect to host cell. These results imply that DPI as a promising candidate material for novel drug development that can ameliorate toxoplasmosis based on ROS regulation.

Modulated Gene Expression of Toxoplasma gondii Infected Retinal Pigment Epithelial Cell Line (ARPE-19) via PI3K/Akt or mTOR Signal Pathway

Due to the critical location and physiological activities of the retinal pigment epithelial (RPE) cell, it is constantly subjected to contact with various infectious agents and inflammatory mediators. However, little is known about the signaling events in RPE involved in Toxoplasma gondii infection and development. The aim of the study is to screen the host mRNA transcriptional change of 3 inflammation-related gene categories, PI3K/Akt pathway regulatory components, blood vessel development factors and ROS regulators, to prove that PI3K/Akt or mTOR signaling pathway play an essential role in regulating the selected inflammation-related genes. The selected genes include PH domain and leucine- rich-repeat protein phosphatases (PHLPP), casein kinase2 (CK2), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), glutamate-cysteine ligase (GCL), glutathione S-transferase (GST), and NAD(P)H: quinone oxidoreductase (NQO1). Using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), we found that T. gondii up-regulates PHLPP2, CK2β, VEGF, GCL, GST, and NQO1 gene expression levels, but down-regulates PHLPP1 and PEDF mRNA transcription levels. PI3K inhibition and mTOR inhibition by specific inhibitors showed that most of these host gene expression patterns were due to activation of PI3K/Akt or mTOR pathways with some exceptional cases. Taken together, our results reveal a new molecular mechanism of these gene expression change dependent on PI3K/Akt or mTOR pathways and highlight more systematical insight of how an intracellular T. gondii can manipulate host genes to avoid host defense.

Treatment of Steroid-induced Osteonecrosis of Femoral Head by Porous Tantalum Rod and Gugutou Huaisiyu Capsule / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the curative effect of porous tantalum rod and Gugutou Huaisiyu Capsule (GHC) for steroid-induced osteonecrosis of femoral head (SONFH).</p><p><b>METHODS</b>A total 60 hips of 50 SONFH patients were randomly assigned to the treatment group and the control group according to grouping time, 25 in each group (30 hips). Patients in the control group were implanted with porous tantalum rod, while those in the treatment group additionally took GHC (5 pills each time, three time per day for 2 successive months; and then twice per day for 4 successive months). Then all patients were followed-up to observe Harris hip score. The curative effect and the femoral head survival time were assessed.</p><p><b>RESULTS</b>A total of 49 patients (59 hips) were followed-up. The Harris hip score of the two groups at the final follow-up was significantly improved after treatment, with statistical difference when compared with before treatment (P < 0.01). Besides, it was higher in the treatment group than in the control group. The curative effect and the survival time were superior in the treatment group, with statistical difference when compared with the control group (P < 0.05).</p><p><b>CONCLUSIONS</b>Porous tantalum rod combined GHC got better effect in treating SONFH. It could significantly improve the function of affected hips and prolong the survival time of femoral head.</p>

A LC-ESI-MS method for the simultaneous determination of madecassoside and its metabolite madecassic acid in rat plasma: comparison pharmacokinetics in normal and collagen-induced arthritic rats / 中国天然药物

To develop a simple and highly sensitive high performance liquid chromatography with electrospray ionization mass spectrometric (LC-ESI-MS) method for the simultaneous determination of madecassoside and its major metabolite madecassic acid in rat plasma, and compare the pharmacokinetics of the two compounds in normal and collagen-induced arthritis (CIA) rats. Glycyrrhetinic acid was used as the internal standard (IS). Chromatographic separation was accomplished on an Inertsil ODS-3 column, using a gradient elution with the mobile phase composed of acetonitrile and water acidified with 0.1% (V/V) formic acid. Detection was achieved by ESI-MS under the negative selected ion monitoring (SIM) mode. In normal and CIA rats, madecassoside (30 mg·kg(-1)) was orally administered for 21 consecutive days from the day of arthritis onset. For madecassoside, the linear range was 10-1 000 ng·mL(-1) with the square regression coefficient (r) of 0.998 9, while for madecassic acid, the linear range was 10-500 ng·mL(-1) with the square regression coefficient (r) of 0.996 1. The lower limit of quantification was 10 ng·mL(-1) for both analytes. The intra- and inter-day precision ranged from 1.78% to 13.42% for madecassoside and 2.30% to 14.90% for madecassic acid, and the accuracy was between -0.95% and 6.30% for madecassoside and between -1.48% and 5.34% for madecassic acid. The average recoveries of madecassoside, madecassic acid and IS from spiked plasma samples were > 81%. The developed method was successfully applied to the pharmacokinetic study of madecassoside and madecassic acid in rats after an oral administration of madecassoside. During initial 7 days of dosing, the cmax and AUC of madecassoside were greatly decreased and Vd/F was markedly increased in CIA rats, and no significant difference was observed on the first day of dosing. In contrast, the T1/2, cmax and AUC of madecassic acid were significantly increased, and Ke of madecassic acid was greatly decreased in CIA rats compared with normal rats. Along with repeated administration of madecassoside, the differences of pharmacokinetic parameters of both madecassoside and madecassic acid between CIA and normal rats gradually subsided. The pharmacokinetic characteristics of both madecassoside and madecassic acid in rats were significantly altered by arthritis status, and the differences of pharmacokinetic parameters between arthritis and normal rats coincide with the severity of arthritis.

Pre-exposure prophylaxis for HIV high-risk groups: An analysis of clinical compliance / 第二军医大学学报

Objective: To investigate the factors influencing the compliance of pre-exposure prophylaxis (PrEP) among high-risk groups of human immunodeficiency virus (HIV). Methods: A total of 352 subjects were selected into the cohort by non-probability sampling, including 153 men who have sex with men (MSM), 36 female sex workers (FSMs), and 163 fixed sex partners(PARs) of HIV carriers. Each subject was randomly given a medication regimen of either once daily or twice a week. The pre-exposure prophylaxis compliance was evaluated by observing the maintenance of the cohort and medication omission every 4 weeks from week 0 to week 28. Cochran-Mantel-Haenszel (CMH) test was employed to conduct univariate analysis and logistic regression was adopted for multiple variable analysis. Results: It was found that 76. 70% (270/352) of the subjects finished the 28-week-observation in this PrEP clinical trial among high-risk groups of HIV. The medication omission rate fluctuated around 35% during the follow-up, with the total compliance score being 82. 12 ± 27. 48 and the median score being 95. 24. Logistic regression suggested that the monthly income, presence of worries or difficulties during the trials, need for medical service, and the way of follow-up were the factors influencing the maintenance of cohort. The marital status, medication method, presence of worries or difficulties during the trial, and presence of adverse effect were the influencing factors of medication omission. The population typ e, number of sex partners, taking of other drugs at the same time, need of medical service during the trial, and way of follow-up were the factors impacting the overall compliance score. Conclusion: The compliance of the present PrEP clinical trials among high-risk groups of HIV is high, and it has multiple influencing factors. Compliance management should be reinforced among MSM and FSW populations with greater motility. The cohort management model of follow-up by fixed specialists should be continued with more frequent follow-up; meanwhile, classified management should be implemented for individuals with different characters, so as to ensure a good compliance during the clinical trials.

Condom use among spouses/fixed partners of HIV infected people: An analysis of influencing factors / 第二军医大学学报

Objective To investigate the factors influencing condom use among spouses/fixed partners of HIV-infected people. Methods The spouses/fixed partners of HIV-infected people were selected for questionnaire investigation. Rank sum test and logistic regression analysis were employed to study the factors influencing the use of condom among them. Results Totally 395 qualified subjects were recruited, and 72.66% of them used condoms every time when they had sexual behavior and 15.70% never used condom. The results of the logistic regression analysis showed that the factors influencing condom use among the spouses/fixed partners of HIV-infected people included ethnicity, having children or not, knowledge about HIV/AIDS, time when HIV was detected in spouse and self-awareness of the possibility to catch HIV from their spouses/fixed partners. Conclusion Condom use among the spouses/fixed partners of HIV-infected people is not satisfactory in China, which is due to multiple factors. More efforts need to be made to enforce AIDS intervention among target population. High-risk groups should be screened on a regular basis and timely measures should be taken.