Effect of Bugu granules in a drug-containing serum on chondrocyte apoptosis and the Trx2 signaling pathway.

Traditional Chinese medicine for invigorating the kidney and promoting blood circulation is commonly prescribed for the treatment of osteoarthritis associated with kidney deficiency and blood stasis. However, the specific mechanisms of these medicines are still unclear. The present study aimed to evaluate the protective effects of Bugu granules against sodium nitroprusside-induced chondrocyte apoptosis and elucidate the underlying molecular mechanisms. Drug-containing serum was prepared by administering rats with Bugu granules and harvesting the serum. Chondrocytes were exposed to different dilutions of serum, and apoptosis assessed by flow cytometry after staining with annexin V­FITC/PI. Flow cytometry showed that chondrocyte apoptosis increased significantly after incubation with 2 mol/L sodium nitroprusside for 24 h (t = -48.221, P = 0.000), and the apoptotic rate of chondrocytes decreased with increasing concentrations of drug-containing serum (F = 33.965, P = 0.000). Cellular levels of Trx2, ASK1, caspase­3, and reactive oxygen species (ROS) were detected by enzyme-linked immunosorbent assay. The cellular content of Trx2 increased gradually with increasing concentrations of drug-containing serum (F = 2610.593, P = 0.000), while that of ASK1 (F = 2473.545, P = 0.000), caspase­3 (F = 209.921, P = 0.000), and ROS (F = 1666.435, P = 0.000) all decreased significantly. The mRNA expression levels were analyzed by RT-qPCR, which revealed that expression levels of Trx2 and caspase­3 mRNA increased and decreased significantly, respectively, following exposure to Bugu granules in the drug-containing serum (F = 6.974, P = 0.003 and F = 3.691, P = 0.191; respectively), but the expression of ASK1 mRNA was not significantly different between treatment groups (F = 1.784, P = 0.191). Taken together, these results support the hypothesis that the Trx2 signaling pathway is activated by Bugu granules, which in turn inhibits chondrocyte apoptosis. This may play a role in preventing the development of osteoarthritis.

Two new coumarins from the seeds of Solanum indicum.

Two new coumarins, (E)-2-(4-hydroxy-3-methoxybenzylidene)-5-methoxy-2H-[1,4]dioxino[2,3-h]chromene-3,9-dione (indicumin E, 1) and 7-hydroxy-6,8-dimethoxy-3-(4'-hydroxy-3'-methoxyphenyl)-coumarin (2), together with two known coumarins isofraxidin (3) and fraxetin (4), were isolated from the Solanum indicum seeds. Their structures were established on the basis of 1D and 2D spectroscopic data. Compound 1 was the rarest coumarinolignoid known to date.

Neutrophil-to-Lymphocyte Ratio (NLR) and Monocyte-to-Lymphocyte Ratio (MLR) Predict Clinical Outcome in Patients with Stage IIB Cervical Cancer.

INTRODUCTION: Stage IIB cervical cancer (CC) is an advanced stage CC with poor prognosis. Inflammatory response plays a crucial role in the development of CC, and systemic inflammatory indexes were related to the prognosis in several cancers. The objective of the study was to determine the prognostic value of platelet-to-lymphocyte ratio (PLR), neutrophil-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio (MLR), basophil-to-lymphocyte ratio (BLR), and systemic inflammation response index (SIRI) as inflammatory indexes in patients with stage IIB CC. MATERIALS AND METHODS: A retrospective study was performed in 260 patients with stage IIB CC. PLR, NLR, MLR, BLR, and SIRI were obtained from routine blood tests. Prognosis information of the patients was acquired from regular clinical follow-up. Recurrence and response to therapy were determined through electronic medical records (EMRs). Correlations of the inflammatory indexes with overall survival (OS), progression-free survival (PFS), recurrence, and response to therapy were analyzed using SPSS version 26.0 software. RESULTS: Receiver operating characteristic (ROC) curve analyses suggested that NLR, MLR, and SIRI had better predictive value than PLR as well as BLR in the prognosis and recurrence risk. Both univariate and multivariate survival analyses showed that higher NLR and MLR were significantly associated with shorter OS as well as PFS, whereas SIRI was not an independent predictive factor of PFS. Chi-square test results revealed that increased NLR was significantly correlated with higher recurrence rate (P=0.046), and increased MLR showed significant correlation with elevated recurrence risk (P=0.002). Univariate and binary logistic regression analyses for response to therapy indicated that elevated NLR was associated with decreased complete remission (CR) rate (P=0.031), and the P value lost statistical significance while being adjusted by tumor size (P=0.108). CONCLUSIONS: For patients with stage IIB CC, both NLR and MLR are independent prognostic factors as well as risk factors for recurrence; NLR serves as a potential marker for therapeutic response.

An updated meta-analysis evaluating limb management after total knee arthroplasty-what is the optimal method?

PURPOSE: Postoperative knee flexion protocol has been widely recognized as a highly attractive, simple, and cost-effective tactic to improve patient's outcomes after primary total knee arthroplasty (TKA). However, optimal knee position and duration of knee flexion are still controversial. The purpose of this meta-analysis was to compare the effectiveness of different postoperative knee flexion protocols, as an aid to find out optimal limb management strategy following TKA. METHODS: We conducted a meta-analysis to identify the available and relevant randomized controlled trials (RCTs) with regard to the influence of different postoperative knee positions on clinical outcomes after primary TKA in electronic databases, including PubMed, EMBASE, the Cochrane Library, Web of Science, CNKI, Wanfang Med Online, and VIP, up to May 2018. In this meta-analysis, three major subgroups based on diverse postoperative knee flexion protocols were considered: long-term (≥ 24 h) high flexion (> 30°), short term (< 24 h) high flexion (> 30°), and long-term (≥ 24 h) mild flexion (≤ 30°). The statistical analysis was performed using the Review Manager (RevMan) version 5.3 software. RESULTS: A total of 16 trials were finally included in this meta-analysis. The result of subgroup analysis indicated that keeping the knee in high flexion (> 30°) postoperatively for a long time (≥ 24 h) significantly reduced total blood loss (P < 0.00001), hidden blood loss (P < 0.00001), and transfusion requirements (P = 0.003) and led to a significant improvement in range of motion (ROM) at 1 week after operation (P < 0.00001); keeping the knee in high flexion (> 30°) postoperatively for a short time (< 24 h) significantly reduced total blood loss (P = 0.006) and hidden blood loss (P < 0.00001) but not significantly improved ROM at 1 week after operation (P = 0.34) and reduced transfusion requirements (P = 0.62); and keeping the knee in mild flexion (≤ 30°) postoperatively for a long time (≥ 24 h) significantly reduced total blood loss (P = 0.02) and transfusion requirements (P = 0.02) and improved ROM at 1 week after operation (P < 0.00001) but not significantly reduced hidden blood loss (P = 0.11). Furthermore, there was no significant difference with respect to the rates of wound-related infection and DVT between the three knee flexion subgroups. CONCLUSIONS: This meta-analysis showed that the long-term (≥ 24 h) high flexion (> 30°) protocol could be an optimal limb management to reduce blood loss and blood transfusion requirements and facilitate early postoperative rehabilitation exercises in patients after primary TKA without increasing in complication rate.

Four new coumarinolignoids from seeds of Solanum indicum.

Activity-guided fractionation of seeds of Solanum indicum for anti-HBV activity led to the isolation of two novel coumarinolignoid alkaloids (indicumines A-B, 1-2) and two new coumarinolignoids (indicumines C-D, 3-4), together with four known coumarins (5-8). Their structures were established on the basis of spectroscopic data. The two novel coumarinolignoid alkaloids shows anti-HBV activities through specifically inhibiting the secretion of HBsAg in HepG2.2.15.

Correlation between single nucleotide polymorphisms in CYP4F2 and warfarin dosing in Chinese valve replacement patients.

BACKGROUND: Individuals with implanted mechanical valve prostheses require lifelong anticoagulation therapy with warfarin. The narrow therapeutic index of warfarin makes it difficult to dose and maintain proper anticoagulation. A number of single nucleotide polymorphisms (SNPs) affecting vitamin K or warfarin metabolism have been shown to affect warfarin dosing. Our aim was to study the effect of the CYP4F2 rs2108622-1347 (C > T) variant on warfarin dosing in Chinese patients. METHODS: We studied 352 patients after heart valve replacement surgery. Warfarin dosing for patients was adjusted to achieve 1.8 ≤ INR ≤ 2.5. We determined the presence of SNPs in CYP4F2 in these patients and investigated their association with warfarin dosing. RESULTS: We found the frequency of the CYP4F2 rs2108622 C allele was 79.5% and T-allele frequency was 20.5%. The warfarin dose requirement for CC individuals was significantly lower than that for CT or TT individuals (P < 0.05). TT-homozygous individuals required a 0.56 mg/day higher dose of warfarin than their CC counterparts. CONCLUSIONS: This study demonstrates that CYP4F2 rs2108622 significantly affects the warfarin dose requirement to achieve adequate anticoagulant activity in Chinese individuals. Genotyping of this SNP may allow clinicians to determine the initiation dose for patients following valve-replacement surgery in China.

The cathepsin B death pathway contributes to TNF plus IFN-gamma-mediated human endothelial injury.

Vascular endothelial cells are primary targets of cytokine-induced cell death leading to tissue injury. We previously reported that TNF in combination with LY294002, a PI3K inhibitor, activates caspase-independent cell death initiated by cathepsin B (Cat B) in HUVEC. We report that TNF in the presence of IFN-gamma activates Cat B as well as a caspase death pathway in both HUVEC and human dermal microvascular endothelial cells, but only activates caspase-mediated death in HeLa cells and human embryonic kidney (HEK)293 cells. Like LY294002, IFN-gamma triggers Cat B release from lysosomes in HUVEC. Cat B-triggered death involves mitochondria, indicated by release of cytochrome c, loss of mitochondrial membrane potential and inhibition of death by overexpressed Bcl-2. Cat B effects on mitochondria do not depend upon Bid cleavage. Unexpectedly, overexpression of a dominant negative mutated form of Fas-associated death domain protein (FADD), which blocks caspase activation by TNF, potentiates TNF activation of Cat B and cell death in HUVEC. Similarly, mutant Jurkat cells lacking FADD also show increased susceptibility to TNF-induced Cat B-dependent cell death. These observations suggest that the Cat B death pathway is cell type-specific and may contribute to cytokine-mediated human tissue injury and to the embryonic lethality of FADD gene disruption in mice.

Immune privilege and FasL: two ways to inactivate effector cytotoxic T lymphocytes by FasL-expressing cells.

The theory that Fas ligand (FasL)-expressing tumours are immune-privileged and can directly counterattack Fas-expressing effector T lymphocytes has recently been questioned and several alternative mechanisms have been proposed. To address this controversial issue, we analysed the impact of FasL-expressing tumours on in vivo-primed cytotoxic T lymphocytes (CTLs) and the mechanisms involved. CTLs were obtained from the peritoneal cavity (PEL) after in vivo priming with syngeneic or allogeneic murine tumour cells. We have found that PEL populations undergo Fas-based apoptotic cell death when co-cultured with FasL-expressing tumour cells and that PEL destruction of cognate targets in a 51Cr-release assay was markedly inhibited by the pre-exposure to either cognate or non-cognate tumour cells expressing FasL. Furthermore, cytocidal function of PEL was markedly inhibited by preincubation with FasL-negative tumour cells, if and only if they were the cognate targets of the CTL; this CTL inhibition involved FasL-Fas interactions. The killing function of 'bystander' PELs, reactive to a third-party target cell, was inhibited by co-cultivation with PELs mixed with their cognate target. This activation-induced CTL fratricide was not influenced by the expression of FasL on the cognate target cells. These studies demonstrate the existence of two distinct pathways whereby FasL-expressing cells inhibit in vivo-primed FasL- and Fas-expressing CTLs: first, by FasL-based direct tumour counterattack, and second, by FasL-mediated activation-induced cell death of the CTLs, which is consistent with the concept that FasL expression in vivo could play a role in inducing immune privilege.

TRAIL induces apoptosis and inflammatory gene expression in human endothelial cells.

Human TRAIL can efficiently kill tumor cells in vitro and kill human tumor xenografts in mice with little effect on normal mouse cells or tissues. The effects of TRAIL on normal human tissues have not been described. In this study, we report that endothelial cells (EC), isolated from human umbilical veins or human dermal microvessels, express death domain-containing TRAIL-R1 and -R2. Incubation with TRAIL for 15 h causes approximately 30% of cultured EC to die, as assessed by propidium iodide uptake. Death is apoptotic, as assessed by Annexin V staining, 4',6'-diamidino-2-phenylindole staining, and DNA fragment ELISA. EC death is increased by cotreatment with cycloheximide but significantly reduced by caspase inhibitors or transduced dominant-negative Fas-associated death domain protein. In surviving cells, TRAIL activates NF-kappaB, induces expression of E-selectin, ICAM-1, and IL-8, and promotes adhesion of leukocytes. Injection of TRAIL into human skin xenografts promotes focal EC injury accompanied by limited neutrophil infiltration. These data suggest that TRAIL is an inducer of tissue injury in humans, an outcome that may influence antitumor therapy with TRAIL.

Inhibition of phosphatidylinositol 3-kinase sensitizes vascular endothelial cells to cytokine-initiated cathepsin-dependent apoptosis.

In the presence of cycloheximide, tumor necrosis factor or interleukin-1 initiates caspase activation, loss of mitochondrial membrane potential (DeltaPsi), DNA degradation, and nuclear condensation and fragmentation characteristic of apoptotic cell death in human vascular endothelial cells (EC). Inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002, but not inhibition of Akt by dominant-negative mutation, also sensitizes EC to cytokine-initiated apoptosis. Cytokine-initiated caspase activation is slower and comparatively less with LY294002 than with cycloheximide. Cycloheximide but not LY294002 decreases expression of c-FLIP (cellular FLICE inhibitory protein), an inhibitor of caspase-8 activation. The caspase inhibitor zVADfmk completely blocks caspase activation, DNA degradation, and nuclear fragmentation in both cases but only prevents loss of DeltaPsi and cell death for cytokine plus cycloheximide treatment. In contrast, overexpression of Bcl-2 protects EC treated with cytokine plus LY294002 but not EC treated with cytokine plus cycloheximide. The cathepsin B inhibitor CA-074-Me prevents loss of DeltaPsi, caspase activation, and cell death for EC treated with cytokine plus LY294002 but has no effect on EC treated with cytokine plus cycloheximide. Cathepsin B translocates from lysosomes to cytosol following treatment with LY294002 prior to the activation of caspases. These results suggest that inhibition of PI3K allows cytokines to activate a cathepsin-dependent, mitochondrial death pathway in which caspase activation is secondary, is not inhibited by c-FLIP, and is not essential for cell death.

Interferon-gamma augments CD95(APO-1/Fas) and pro-caspase-8 expression and sensitizes human vascular endothelial cells to CD95-mediated apoptosis.

We have examined the effects of interferon (IFN)-gamma on expression and function of CD95 (APO-1/Fas) and associated proteins in cultured human umbilical vein and dermal microvascular endothelial cells (HUVEC and HDMEC, respectively). Unstimulated cells express only low levels of CD95; IFN-gamma produces a time- and concentration-dependent increase of CD95 in both cell types at the mRNA and cell surface protein levels. IFN-gamma also produces an increase in expression of pro-caspase-8 (FLICE/MACH) but does not significantly change expression of either Fas-associated death domain (FADD) protein or cellular FLICE inhibitory protein (cFLIP), other proteins associated with the CD95 death-inducing signaling complex (DISC). Neither resting nor IFN-gamma-treated EC express detectable CD95L mRNA or protein. Untreated HUVEC and HDMEC show minimal apoptosis when transduced to express CD95L. Treatment of CD95L-transduced cells with IFN-gamma causes apoptosis within 24 to 36 hours that can be blocked by antagonistic anti-CD95 antibody or by the caspase-inhibitory peptide zVAD-FMK. The extent of apoptosis is increased by co-treatment with either the protein synthesis inhibitor cycloheximide or the phosphatidylinositol 3-kinase inhibitor LY294002. Untransduced HUVEC treated with IFN-gamma also undergo CD95-initiated apoptosis when mixed with CD95L-transduced HUVEC or when incubated with pharmacologically activated cytolytic T lymphocytes. Overexpression of CD95 in HUVEC confers sensitivity to CD95L in the absence of IFN-gamma-treatment. We conclude that IFN-gamma induces sensitivity of endothelium to CD95L-mediated apoptosis, and that this response may result from increased expression of CD95 and/or pro-caspase-8.