Quinpirole ameliorates nigral dopaminergic neuron damage in Parkinson's disease mouse model through activating GHS-R1a/D2R heterodimers.

Growth hormone secretagogue receptor 1a (GHS-R1a) is an important G protein-coupled receptor (GPCR) that regulates a variety of functions by binding to ghrelin. It has been shown that the dimerization of GHS-R1a with other receptors also affects ingestion, energy metabolism, learning and memory. Dopamine type 2 receptor (D2R) is a GPCR mainly distributed in the ventral tegmental area (VTA), substantia nigra (SN), striatum and other brain regions. In this study we investigated the existence and function of GHS-R1a/D2R heterodimers in nigral dopaminergic neurons in Parkinson's disease (PD) models in vitro and in vivo. By conducting immunofluorescence staining, FRET and BRET analyses, we confirmed that GHS-R1a and D2R could form heterodimers in PC-12 cells and in the nigral dopaminergic neurons of wild-type mice. This process was inhibited by MPP+ or MPTP treatment. Application of QNP (10 µM) alone significantly increased the viability of MPP+-treated PC-12 cells, and administration of quinpirole (QNP, 1 mg/kg, i.p. once before and twice after MPTP injection) significantly alleviated motor deficits in MPTP-induced PD mice model; the beneficial effects of QNP were abolished by GHS-R1a knockdown. We revealed that the GHS-R1a/D2R heterodimers could increase the protein levels of tyrosine hydroxylase in the SN of MPTP-induced PD mice model through the cAMP response element binding protein (CREB) signaling pathway, ultimately promoting dopamine synthesis and release. These results demonstrate a protective role for GHS-R1a/D2R heterodimers in dopaminergic neurons, providing evidence for the involvement of GHS-R1a in PD pathogenesis independent of ghrelin.

Identification of Flavonoids from Scutellaria barbata D. Don as Inhibitors of HIV-1 and Cathepsin L Proteases and Their Structure-Activity Relationships.

Scutellaria barbata D. Don (SB, Chinese: Ban Zhi Lian), a well-known medicinal plant used in traditional Chinese medicine, is rich in flavonoids. It possesses antitumor, anti-inflammatory, and antiviral activities. In this study, we evaluated the inhibitory activities of SB extracts and its active components against HIV-1 protease (HIV-1 PR) and SARS-CoV2 viral cathepsin L protease (Cat L PR). UPLC/HRMS was used to identify and quantify the major active flavonoids in different SB extracts, and fluorescence resonance energy transfer (FRET) assays were used to determine HIV-1 PR and Cat L PR inhibitions and identify structure-activity relationships. Molecular docking was also performed, to explore the diversification in bonding patterns of the active flavonoids upon binding to the two PRs. Three SB extracts (SBW, SB30, and SB60) and nine flavonoids inhibited HIV-1 PR with an IC50 range from 0.006 to 0.83 mg/mL. Six of the flavonoids showed 10~37.6% inhibition of Cat L PR at a concentration of 0.1 mg/mL. The results showed that the introduction of the 4'-hydroxyl and 6-hydroxyl/methoxy groups was essential in the 5,6,7-trihydroxyl and 5,7,4'-trihydroxyl flavones, respectively, to enhance their dual anti-PR activities. Hence, the 5,6,7,4'-tetrahydroxyl flavone scutellarein (HIV-1 PR, IC50 = 0.068 mg/mL; Cat L PR, IC50 = 0.43 mg/mL) may serve as a lead compound to develop more effective dual protease inhibitors. The 5,7,3',4'-tetrahydroxyl flavone luteolin also showed a potent and selective inhibition of HIV-1 PR (IC50 = 0.039 mg/mL).

The effect of a full-time infection control nursing service in the prevention of multidrug-resistant organism in the orthopedic ward.

BACKGROUND: Our aim was to evaluate the effect of setting up a full-time infection control nursing service on reducing the prevalence of multidrug-resistant organism (MDRO) in the orthopedic ward. METHODS: From January 2015 to March 2019, routine prevention and control measures were taken for patients infected/colonized with MDRO in this ward, which was set as the pre-intervention period. The intervention period was from April 2019 to June 2021. The study was designed to evaluate whether the establishment of a full-time infection control nursing service could reduce the positive density of MDRO in the hospital by using an interrupted time-series model of a quasi experimental study. RESULTS: There were 11,759 patients during pre-intervention period and 8142 patients during intervention period. The total number of MDRO isolated before intervention was 177, of which 145 were obtained in hospital and 32 were brought in from outside hospital. The total number of MDRO isolated after intervention was 47, of which 29 were obtained in hospital and 18 were brought in from outside hospital. Before intervention, the positive density of MDRO in the orthopedic ward showed an increasing trend (ß1 = 0.02, P = 0.003). After intervention, the positive density of MDRO showed a downward trend (ß3 = - 0.05, P = 0.018). CONCLUSIONS: The establishment of the full-time infection control nursing service in the orthopedic ward can effectively reduce the nosocomial prevalence of MDRO.

Cancer-related fatigue in hospitalised patients treated for lymphoma and its burden on family caregivers.

OBJECTIVE: To investigate the prevalence of cancer-related fatigue (CRF) in patients with lymphoma and to explore the burden of CRF on the family caregivers (FCs). METHODS: A cross-sectional study was conducted in a university-affiliated tertiary care hospital in China. Patients with lymphoma who received treatment in the in-patient ward of the Haematology Department were consecutively recruited. Face-to-face interviews were conducted to gather information related to the patients' sociodemographic characteristics and perceived CRF and its burden on the FCs. Cochran-Armitage trend analysis and Multivariable logistic regression analyses were employed to determine the association between CRF and the FCs' burden. RESULTS: Of the 116 cancer patient-FC dyads, about 70% of patients experienced some level of fatigue, while 51% of unpaid family members suffered some degree of depression. The Cochran-Armitage trend analysis showed that the FCs' burden significantly increased with the severity of CRF. Logistic regression indicated that the FCs of the patients reporting fatigue experienced a higher burden in both the unadjusted and adjusted models. CONCLUSION: The prevalence of CRF appeared to be high among patients with lymphoma. It might be important to design innovative health-promoting practices for ameliorating or preventing the impact of fatigue.

Author Correction: Plumbagin attenuates cancer cell growth and osteoclast formation in the bone microenvironment of mice.

During re-read of our previously article Plumbagin attenuates cancer cell growth and osteoclast formation in the bone microenvironment of micepublished in Acta Pharmacologica Sinica, we were regretted to point out a mistake shown in Fig. 2a. The representative figure chosen to indicate the inhibitory effect of 4 mg/kg of plumbagin treatment at 1 week against MDA-MB-231SArfp cells localization within bone environment was incorrect due to the mishandling in manuscript preparation. Although this correction does not affect the results and conclusion of the paper, all the authors agree on the correction of our negligence as providing the corrected Fig. 2a presented below. We feel sorry and apologize for all the inconvenience it caused.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Pathologic T-cell response in ischaemic failing hearts elucidated by T-cell receptor sequencing and phenotypic characterization.

AIMS: A persistent cardiac T-cell response initiated by myocardial infarction is linked to subsequent adverse ventricular remodelling and progression of heart failure. No data exist on T-cell receptor (TCR) repertoire changes in combination with phenotypic characterization of T cells in ischaemic failing human hearts. METHODS AND RESULTS: Analysis of TCR repertoire with high-throughput sequencing revealed that compared with T cells in control hearts, those in ischaemic failing hearts showed a clonally expanded TCR repertoire but similar usage patterns of TRBV-J rearrangements and V gene segments; compared with T cells in peripheral blood, those in ischaemic failing hearts exhibited a restricted and clonally expanded TCR repertoire and different usage patterns of TRBV-J rearrangements and V gene segments, suggesting the occurrence of tissue-specific T-cell expansion in ischaemic failing hearts. Consistently, TCR clonotype sharing was prominent in ischaemic failing hearts, especially in hearts of patients who shared human leucocyte antigen (HLA) alleles. Furthermore, ischaemia heart failure (IHF) heart-associated clonotypes were more frequent in peripheral blood of IHF patients than in that of controls. Heart-infiltrating T cells displayed memory- and effector-like characteristics. Th1 cells were the predominant phenotype among CD4+ T cells; CD8+ T cells were equally as abundant as CD4+ T cells and produced high levels of interferon-γ, granzyme B, and perforin. CONCLUSION: We provide novel evidence for a tissue-specific T-cell response predominated by Th1 cells and cytotoxic CD8+ T cells in ischaemic failing human hearts that may contribute to the progression of heart failure.

Dihydromyricetin Inhibits Inflammation of Fibroblast-Like Synoviocytes through Regulation of Nuclear Factor-κB Signaling in Rats with Collagen-Induced Arthritis.

Dihydromyricetin (DMY), the main flavonoid of Ampelopsis grossedentata, has potent anti-inflammatory activity. However, the effect of DMY on chronic autoimmune arthritis remains undefined. In this study, we investigated the therapeutic effects of DMY on collagen-induced arthritis (CIA). Wistar rats were immunized with bovine type II collagen to establish CIA and were then administered DMY intraperitoneally (5, 25, and 50 mg/kg) every other day for 5 weeks. Paw swelling, clinical scoring, and histologic analysis were assessed to determine the therapeutic effects of DMY on the development of arthritis in CIA rats. The results showed that treatment with DMY significantly reduced erythema and swelling in the paws of CIA rats. Pathologic analysis of the knee joints and peripheral blood cytokine assay results confirmed the antiarthritic effects of DMY on synovitis and inflammation. Fibroblast-like synoviocytes (FLSs) were isolated from the synovium of CIA rats and treated with 10 ng/ml interleukin (IL)-1ß DMY significantly inhibited the proliferation, migration, and inflammation of IL-1ß-induced FLSs, whereas it significantly increased IL-1ß-induced FLS apoptosis in a dose-dependent manner (6.25-25 µM). Moreover, DMY suppressed phosphorylation of IκB kinase (IKK) and inhibitor of NF-κB α and subsequently reduced the IL-1ß-induced nucleus translocation of NF-κB in FLSs. Through a molecular docking assay, we demonstrated that DMY could directly bind to the Thr9 and Asp88 residues in IKKα and the Asp95, Asn142, and Gln167 residues in IKKß These findings demonstrate that DMY could alleviate inflammation in CIA rats and attenuate IL-1ß-induced activities in FLSs through suppression of NF-κB signaling.

Author Correction: Synergistic suppression of human breast cancer cells by combination of plumbagin and zoledronic acid in vitro.

The authors regretted to find the mis-representative images in Fig. 3a, c and Fig. 4a, c when re-read our previously published article Synergistic suppression of human breast cancer cells by combination of plumbagin and zoledronic acid In vitro (DOI: 10.1038/aps.2015.42) in the journal of Acta Pharmacologica Sinica. This mistake occurred due to the careless compilation when the authors tried to show the synergistic effect against tumor apoptosis during figure presentation process. The right Fig. 3a, c and Fig. 4a, c were provided below. Despite that this correction does not affect the results and conclusions of the aforementioned paper, all the authors still consent on the correction of this negligence. We apologize to the Editor and the readership of the journal for any inconvenience caused. Your thoughtful understanding is highly appreciated.

Defective Circulating Regulatory B Cells in Patients with Dilated Cardiomyopathy.

BACKGROUND/AIMS: Newly identified IL-10-producing regulatory B cells (Bregs) have been shown to play an important role in the suppression of immune responses. Chronic immune activation participates in the pathogenesis of dilated cardiomyopathy (DCM) but whether Bregs are involved in its development remains unclear. We aimed to investigate the circulating frequency and function of Bregs in DCM. METHODS: In total, 35 DCM patients (20 men and 15 women) and 44 healthy controls (23 men and 21 women) were included in the experiment, and the frequency of Bregs was detected using flow cytometry. RESULTS: According to our results, the frequency of circulating IL-10-producing Bregs was significantly lower in DCM patients compared with healthy controls. Furthermore, the CD24hiCD27+ B cell subset in which IL-10-producing Bregs were mainly enriched from DCM patients showed impaired IL-10 expression and a decreased ability to suppress the TNF-α production of CD4+CD25- Tconv cells and to maintain Tregs differentiation. Correlation analysis showed that the frequency of IL-10-producing Bregs and the suppressive function of CD24hiCD27+ B cells were positively correlated with left ventricular ejection fraction and negatively correlated with NT-proBNP in DCM patients. CONCLUSIONS: In conclusion, the reduced frequency and impaired functions suggest a potential role of Bregs in the development of DCM.

Polymorphisms of the KCNQ1 gene are associated with the therapeutic responses of sulfonylureas in Chinese patients with type 2 diabetes.

KCNQ1 channel is a member of the voltage-gated potassium channel KQT-like subfamily. The KCNQ1 gene has recently been identified as a susceptibility locus for type 2 diabetes mellitus (T2DM). In the present study, we examined the effects of KCNQ1 variants on the therapeutic response to modified-release gliclazide (gliclazide MR) treatment in Chinese patients newly diagnosed with T2DM. A total of 100 newly diagnosed T2DM patients without a history of any anti-diabetic medications were treated with gliclazide MR for 16 weeks, but 91 patients completed the entire study. The anthropometric parameters were determined at baseline and at the final visit, while clinical laboratory tests were performed at baseline and on weeks 2, 4, 6, 12, 16. Two SNPs, rs2237892 and rs2237895, in the region of the KCNQ1 gene were genotyped in all the participants. All calculations and statistical analyses were conducted using SPSS. The rs2237892 TT homozygotes exhibited significantly higher 2-h glucose levels at baseline (P<0.05) and a lower cumulative attainment rate of the target 2-h glucose level (Plog-rank=0.020) than the C allele carriers. Patients with greater numbers of rs2237892 T alleles exhibited larger augmentations (Δ) in the 2-h glucose levels (P=0.027); and patients with the rs2237892 TT genotype exhibited a higher Δ homeostasis model assessment of ß-cell function (HOMA-ß) than CC and CT genotype carriers (P=0.021 and P=0.043, respectively). Moreover, the rs2237895 C allele was associated with a greater decrement in Δ glycated hemoglobin (HbA1c) (P=0.024); and patients with the CC genotype exhibited greater variance than those with the AA and AC genotypes (P=0.005 and 0.021, respectively). Compared with the C allele, the odds ratio for treatment success among carriers of the rs2237892 T allele was 2.533 (P=0.007); and the rs2237895 C allele was associated with a 2.360-fold decrease in HbA1c compared with the A allele (P=0.009). KCNQ1 polymorphisms are associated with gliclazide MR efficacy in Chinese patients with type 2 diabetes.

Circulating MiR-19b-3p, MiR-134-5p and MiR-186-5p are Promising Novel Biomarkers for Early Diagnosis of Acute Myocardial Infarction.

BACKGROUND/AIMS: Recent studies have shown that circulating microRNAs (miRNAs) are emerging as promising biomarkers for cardiovascular diseases. This study aimed to determine whether miR-19b-3p, miR-134-5p and miR-186-5p can be used as novel indicators for acute myocardial infarction (AMI). METHODS: To investigate the kinetic expression of the three selected miRNAs, we enrolled 18 patients with AMI and 20 matched controls. Plasma samples were collected from each participant, and total RNA was extracted. Quantitative real-time PCR and ELISA assays were used to investigate the expression of circulating miRNAs and cardiac troponin I (cTnI), respectively. Plasma samples from another age- and gender-matched cohort were collected to investigate the impact of medications for AMI on the expression of the selected miRNAs. RESULTS: Levels of plasma miR-19b-3p, miR-134-5p and miR-186-5p were significantly increased in early stage of AMI. Plasma miR-19b-3p and miR-134-5p levels reached peak expression immediately after admission (T0), whereas miR-186-5p achieved peak expression at 4 h after T0. All of these times were earlier than the peak for cTnI (8 h after T0). In addition, all three miRNAs were positively correlated with cTnI. Receiver Operating Characteristic (ROC) analysis indicated that each single miRNA showed considerable diagnostic efficiency for predicting AMI. Furthermore, combining all three miRNAs in a panel increased the efficiency of distinguishing between patients with AMI and controls. Moreover, we found that heparin and medications for AMI did not impact the expression of these circulating miRNAs. CONCLUSION: Circulating miR-19b-3p, miR-134-5p and miR-186-5p could be considered promising novel diagnostic biomarkers for the early phase of AMI.

TIMP3 regulates osteosarcoma cell migration, invasion, and chemotherapeutic resistances.

Tissue inhibitors of metalloproteinases (TIMPs) inhibit matrix metalloproteinases (MMPs) to limit degradation of the extracellular matrix. Low levels of TIMP3 have been demonstrated in cancer tissues at advanced clinical stages, with positive distant metastasis and chemotherapeutic resistance. We examined the role of TIMP3 in osteosarcoma (OS) cell invasiveness and chemoresistance. TIMP3 was overexpressed or knocked down in the human OS cell lines Saos2 and MG63. Cell migration and invasion capacities were then evaluated using Transwell assays, and resistance to cisplatin was assessed by CCK-8 assay and flow cytometry. Real-time PCR and western blotting were used to investigate activation of signaling pathways downstream of TIMP3. Overexpression of TIMP3 inhibited the migration and invasion of Saos2 and MG63 cells, while knockdown of TIMP3 had the opposite effect. Cell survival after exposure to cisplatin was inhibited by TIMP3 overexpression in both Saos2 and MG63 cells. Consistently, downregulation of TIMP3 gene expression significantly decreased the sensitivity of OS cells to cisplatin treatment. MMP1, MMP2, Bcl-2, and Akt1 were all downregulated following TIMP3 overexpression, while Bax and cleaved caspase-3 were upregulated. TIMP3 knockdown had opposite effects on the regulation of these genes. Taken together, our findings suggest TIMP3 as a new target for inhibition of OS progression and chemotherapeutic resistance.

Cathepsin G activity lowers plasma LDL and reduces atherosclerosis.

Cathepsin G (CatG), a serine protease present in mast cells and neutrophils, can produce angiotensin-II (Ang-II) and degrade elastin. Here we demonstrate increased CatG expression in smooth muscle cells (SMCs), endothelial cells (ECs), macrophages, and T cells from human atherosclerotic lesions. In low-density lipoprotein (LDL) receptor-deficient (Ldlr(-/-)) mice, the absence of CatG reduces arterial wall elastin degradation and attenuates early atherosclerosis when mice consume a Western diet for 3months. When mice consume this diet for 6months, however, CatG deficiency exacerbates atherosclerosis in aortic arch without affecting lesion inflammatory cell content or extracellular matrix accumulation, but raises plasma total cholesterol and LDL levels without affecting high-density lipoprotein (HDL) or triglyceride levels. Patients with atherosclerosis also have significantly reduced plasma CatG levels that correlate inversely with total cholesterol (r=-0.535, P<0.0001) and LDL cholesterol (r=-0.559, P<0.0001), but not with HDL cholesterol (P=0.901) or triglycerides (P=0.186). Such inverse correlations with total cholesterol (r=-0.504, P<0.0001) and LDL cholesterol (r=-0.502, P<0.0001) remain significant after adjusting for lipid lowering treatments among this patient population. Human CatG degrades purified human LDL, but not HDL. This study suggests that CatG promotes early atherogenesis through its elastinolytic activity, but suppresses late progression of atherosclerosis by degrading LDL without affecting HDL or triglycerides.

Activated regulatory T-cells attenuate myocardial ischaemia/reperfusion injury through a CD39-dependent mechanism.

Regulatory T-cells (Tregs) are generally regarded as key immunomodulators that maintain immune tolerance and counteract tissue damage in a variety of immune-mediated disorders. However, its role in myocardial ischaemia/reperfusion injury (MIRI) remains unknown. The purpose of the present study was to determine whether Tregs exert a beneficial effect on mouse MIRI. We examined the role of Tregs in murine MIRI by depletion using 'depletion of regulatory T-cell' (DEREG) mice and adoptive transfer using Forkhead box P3 (Foxp3)-GFP knockin mice and the mechanisms of cardio protection were further studied in vivo and in vitro. Tregs rapidly accumulated in murine hearts following MIRI. Selective depletion of Tregs in the DEREG mouse model resulted in aggravated MIRI. In contrast, the adoptive transfer of in vitro-activated Tregs suppressed MIRI, whereas freshly isolated Tregs had no effect. Mechanistically, activated Treg-mediated protection against MIRI was not abrogated by interleukin (IL)-10 or transforming growth factor (TGF)-ß1 inhibition but was impaired by the genetic deletion of cluster of differentiation 39 (CD39). Moreover, adoptive transfer of in vitro-activated Tregs attenuated cardiomyocyte apoptosis, activated a pro-survival pathway involving Akt and extracellular-signal-regulated kinase (ERK) and inhibited neutrophil infiltration, which was compromised by CD39 deficiency. Finally, the peripheral blood mononuclear cells of acute myocardial infarction (AMI) patients after primary percutaneous coronary intervention (PCI) revealed a decrease in CD4+CD25+CD127low Tregs and a relative increase in CD39+ cells within the Treg population. In conclusion, our data validated a protective role for Tregs in MIRI. Moreover, in vitro-activated Tregs ameliorated MIRI via a CD39-dependent mechanism, representing a putative therapeutic strategy.

Osteosarcoma cells promote the production of pro-tumor cytokines in mesenchymal stem cells by inhibiting their osteogenic differentiation through the TGF-ß/Smad2/3 pathway.

Mesenchymal stem cells (MSCs) are among the most important components of the osteosarcoma microenvironment and are reported to promote tumor progression. However, the means by which osteosarcoma cells modulate MSC behavior remains unclear. The aim of this study was to determine the effects of osteosarcoma cells on both the production of pro-tumor cytokines by mesenchymal stem cells (MSCs) and the osteogenic differentiation of MSCs. High level of transforming growth factor-ß (TGF-ß) was detected in three osteosarcoma cell lines. Conditioned media (CM) from the osteosarcoma cell lines Saos-2 and U2-OS were used to stimulate the cultured MSCs. We found that osteosarcoma cells promoted the production of IL-6 and VEGF in MSCs by inhibiting their osteogenic differentiation. Furthermore, TGF-ß in tumor CM was proved to be an important factor. The TGF-ß neutralizing antibody antagonized the effects induced by osteosarcoma CM. The inhibition of Smad2/3 by siRNA significantly decreased the production of IL-6 and VEGF in MSCs and induced their osteogenic differentiation. We also found that Smad2/3 enhanced the expression of ß-catenin in MSCs by decreasing the level of Dickkopf-1 (DKK1). Although the inhibition of ß-catenin did not affect the production of IL-6 or VEGF, or the gene expression of the early osteogenic markers Runx2 and ALP, it did enhance the gene expression of osteocalcin. Taken together, our data indicate that osteosarcoma cells secrete TGF-ß to maintain the stemness of MSCs and promote the production of pro-tumor cytokines by these cells.

Synergistic suppression of human breast cancer cells by combination of plumbagin and zoledronic acid In vitro.

AIM: Zoledronic acid (ZA), a bisphosphonate, is currently used in combination with chemotherapeutic agents to suppress breast cancer cell proliferation or breast cancer-induced osteolysis. The aim of this study was to investigate the effects of ZA combined with a natural anticancer compound plumbagin (PL) against human breast cancer cells in vitro. METHODS: Human breast cancer MDA-MB-231SArfp cells were treated with ZA, PL or a combination of ZA and PL. The cell growth, apoptosis and migration were evaluated using CCK-8 assay, flow cytometry and transwell assay, respectively. The expression of apoptosis-related proteins was measured using real-time PCR and Western blotting. Synergism was evaluated using Compusyn software, and the combination index (CI) and drug reduction index (DRI) values were determined. RESULTS: PL or ZA alone caused mild cytotoxicity (the IC50 value at 24 h was 12.18 and above 100 µmol/L, respectively). However, the combination of ZA and PL caused a synergistic cytotoxicity (CI=0.26). The DRI values also showed a synergistic effect between PL and ZA, with actual values of 5.52 and 3.59, respectively. Furthermore, PL and ZA synergistically induced apoptosis and inhibited migration of the breast cancer cells. Moreover, the combination of ZA and PL decreased the expression of Notch-1, cleaved PARP, Bcl-2 and Bcl-xl, and increased the expression of cleaved caspase-3, CDKN1A and ID1. When the breast cancer cells were transfected with specific siRNA against Notch-1, the combination of ZA and PL markedly increased the expression of Bcl-2. CONCLUSION: Combination of ZA and PL synergistically suppresses human breast cancer MDA-MB-231SArfp cells in vitro. PL can inhibit ZA-induced activation of the Notch-1 signaling pathway and subsequently reduce the expression of Bcl-2, thus potentiating cancer cell apoptosis.

In vivo effect of quaternized chitosan-loaded polymethylmethacrylate bone cement on methicillin-resistant Staphylococcus epidermidis infection of the tibial metaphysis in a rabbit model.

Infection of open tibial fractures with contamination remains a challenge for orthopedic surgeons. Local use of antibiotic-impregnated polymethylmethacrylate (PMMA) beads and blocks is a widely used procedure to reduce the risk of infection. However, the development of antibiotic-resistant organisms make the management of infection more difficult. Our in vitro study demonstrated that quaternized chitosan (hydroxypropyltrimethyl ammonium chloride chitosan [HACC])-loaded PMMA bone cement exhibits strong antibacterial activity toward antibiotic-resistant bacteria. Therefore, the present study aimed to investigate the in vivo antibacterial activity of quaternized chitosan-loaded PMMA. Twenty-four adult female New Zealand White rabbits were used in this study. The right proximal tibial metaphyseal cavity was prepared, 10(7) CFU of methicillin-resistant Staphylococcus epidermidis was inoculated, and PMMA-only, gentamicin-loaded PMMA (PMMA-G), chitosan-loaded PMMA (PMMA-C), or HACC-loaded PMMA (PMMA-H) bone cement cylinders were inserted. During the follow-up period, the infections were evaluated using X rays on days 21 and 42 and histopathological and microbiological analyses on day 42 after surgery. Radiographic indications of bone infections, including bone lysis, periosteal reactions, cyst formation, and sequestral bone formation, were evident in the PMMA, PMMA-G, and PMMA-C groups but not in the PMMA-H group. The radiographic scores and gross bone pathological and histopathological scores were significantly lower in the PMMA-H group than in the PMMA, PMMA-G, and PMMA-C groups (P < 0.05). Explant cultures also indicated significantly less bacterial growth in the PMMA-H group than in the PMMA, PMMA-G, and PMMA-C groups (P < 0.01). We concluded that PMMA-H bone cement can inhibit the development of bone infections in this animal model inoculated with antibiotic-resistant bacteria, thereby demonstrating its potential application for treatment of local infections in open fractures.

Plumbagin attenuates cancer cell growth and osteoclast formation in the bone microenvironment of mice.

AIM: To investigate the effects of plumbagin, a naphthoquinone derived from the medicinal plant Plumbago zeylanica, on human breast cancer cell growth and the cancer cell-induced osteolysis in the bone microenvironment of mice. METHODS: Human breast cancer cell subline MDA-MB-231SA with the ability to spread and grow in the bone was tested. The cell proliferation was determined using the CCK-8 assay. Apoptosis was detected with Annexin V/PI double-labeled flow cytometry. Red fluorescent protein-labeled MDA-MB-231SArfp cells were injected into the right tibia of female BALB/c-nu/nu mice. Three days after the inoculation, the mice were injected with plumbagin (2, 4, or 6 mg/kg, ip) 5 times per week for 7 weeks. The growth of the tumor cells was monitored using an in vivo imaging system. After the mice were sacrificed, the hind limbs were removed for radiographic and histological analyses. RESULTS: Plumbagin (2.5-20 µmol/L) concentration-dependently inhibited the cell viability and induced apoptosis of MDA-MB-231SA cells in vitro (the IC50 value of inhibition of cell viability was 14.7 µmol/L). Administration of plumbagin to breast cancer bearing mice delayed the tumor growth by 2-3 weeks and reduced the tumor volume by 44%-74%. The in vivo imaging study showed that plumbagin dose-dependently inhibited MDA-MB-231SArfp cell growth in bone microenvironment. Furthermore, X-ray images and micro-CT study demonstrated that plumbagin reduced bone erosion area and prevented a decrease in bone tissue volume. Histological studies showed that plumbagin dose-dependently inhibited the breast cancer cell growth, enhanced the cell apoptosis and reduced the number of TRAcP-positive osteoclasts. CONCLUSION: Plumbagin inhibits the cell growth and induces apoptosis in human breast cancer cells in mice bone microenvironment, leading to significant reduction in osteolytic lesions caused by the tumor cells.

Impaired thymic export and increased apoptosis account for regulatory T cell defects in patients with non-ST segment elevation acute coronary syndrome.

Regulatory T (Treg) cells play a protective role against the development of atherosclerosis. Previous studies have revealed Treg cell defects in patients with non-ST elevation acute coronary syndrome (NSTACS), but the mechanisms underlying these defects remain unclear. In this study, we found that the numbers of peripheral blood CD4(+)CD25(+)CD127(low) Treg cells and CD4(+)CD25(+)CD127(low)CD45RA(+)CD45RO(-) naive Treg cells were lower in the NSTACS patients than in the chronic stable angina (CSA) and the chest pain syndrome (CPS) patients. However, the number of CD4(+)CD25(+)CD127(low)CD45RA(-)CD45RO(+) memory Treg cells was comparable in all of the groups. The frequency of CD4(+)CD25(+)CD127(low)CD45RO(-)CD45RA(+)CD31(+) recent thymic emigrant Treg cells and the T cell receptor excision circle content of purified Treg cells were lower in the NSTACS patients than in the CSA patients and the CPS controls. The spontaneous apoptosis of Treg cells (defined as CD4(+)CD25(+)CD127(low)annexin V(+)7-AAD(-)) was increased in the NSTACS patients compared with the CSA and CPS groups. Furthermore, oxidized LDL could induce Treg cell apoptosis, and the oxidized LDL levels were significantly higher in the NSTACS patients than in the CSA and CPS groups. In accordance with the altered Treg cell levels, the concentration of TNF-α was increased in the NSTACS patients, resulting in a decreased IL-10/TNF-α ratio. These findings indicate that the impaired thymic output of Treg cells and their enhanced susceptibility to apoptosis in the periphery were responsible for Treg cell defects observed in the NSTACS patients.

IL-17A induces pro-inflammatory cytokines production in macrophages via MAPKinases, NF-κB and AP-1.

BACKGROUND: Interleukin (IL)-17A, a newly identified cytokine, may participate in the transition of a stable plaque into an unstable plaque. Macrophages play a critical role in the destabilization of atherosclerotic plaque. METHODS: RAW 264.7 cells were stimulated with IL-17A. The mRNA expression of inflammatory cytokines was determined by RT-PCR. The cytokines production in the supernatants was measured by ELISA. Small interfering RNA (siRNA) was used to confirm that IL-17A-induced pro-inflammatory cytokines production via IL-17RA signaling. The western blot assay was used to detect the phosphorylation of MAPKinases including p38 and ERK1/2. The DNA binding activity of nuclear factor NF-κB and AP-1 were detected by EMSA. RESULTS: IL-17A induced the production of pro-inflammatory cytokines in macrophages in a time- and dose-dependent manner, such as tumor necrosis factor (TNF)-α, IL-1ß, and IL-6. Meanwhile, IL-17A resulted in the phosphorylation of p38 and ERK1/2 and increased DNA-binding activity of NF-κB and AP-1. Pharmacological inhibitors of p38 and ERK1/2 partly attenuated IL-17A-induced TNF-α, IL-1ß, and IL-6 production. Either NF-κB inhibitor or AP-1 inhibitor also partly decreased the IL-17A-induced cytokine production. CONCLUSIONS: IL-17A induces pro-inflammatory cytokines production in macrophages via MAPKinases, NF-κB and AP-1 pathway.