Infection status and molecular detection of pathogens carried by ectoparasites of Miniopterus fuliginosus bats in Yunnan, China.

Bats serve as natural hosts for various infectious agents that can affect both humans and animals, and they are geographically widespread. In recent years, the prevalence of bat-associated pathogens has surged on a global scale, consequently generating significant interest in bats and their ectoparasites. In this study, we specifically selected the Miniopterus fuliginosus as the host and conducted bat captures in Nanjian Yi Autonomous County, Dali Bai Autonomous Prefecture, and the other in Mouding Township, Chuxiong Yi Autonomous Prefecture, located in Yunnan Province, China. Ectoparasites were meticulously collected from the bat body surface, alongside blood samples for subsequent analyses. Following collection, the ectoparasites were methodically identified and subjected to comprehensive ecological analysis. Additionally, DNA was extracted from both the bat blood and bat flies, with conventional PCR techniques utilized for molecular screening of four pathogens: Anaplasma sp., Babesia sp., Hepatozoon sp., and Bartonella sp. The capture efforts yielded a total of 37 M. fuliginosus, from which 388 ectoparasites were recovered, including 197 gamasid mites (Cr = 50.77%, PM = 94.59%, MA = 5.32, MI = 5.63) and 191 bat flies (Cr = 49.23%, PM = 75.68%, MA = 5.16, MI = 6.82). Notably, Steatonyssus nyctali (Y = 0.28, m*/m = 2.44) and Nycteribia allotopa (Y = 0.23,m*/m = 1.54) predominated among different individuals of M. fuliginosus, exhibiting an aggregated distribution pattern. The infection rates of Bartonella sp. were identified to be 18.92% (7/37) among bats and 37.17% (71/191) among bat flies, based on the testing of 37 bats and 191 bat flies. Phylogenetic analysis demonstrated that the Bartonella sequences exhibited similarity to those found in bats and bat flies within China and South Korea. This study not only contributes to our comprehension of ectoparasite infection in M. fuliginosus but also establishes a foundation for potential exploration of their role as vectors.

The complete mitochondrial genome of Nycteribia formosana (Diptera, Nycteribiidae).

The family Hippoboscidae is an ectoparasite that primarily inhabits bats and relies on the host's blood for sustenance. This research provides the first complete mitochondrial genome of Nycteribia formosana, which shares similar characteristics with other dipteran insects. The circularized mitochondrial genome, spanning 15,107 bp, encompasses 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNA), two ribosomal RNA genes, and a control region. The nucleotide composition of A, C, G, and T is 40.4%, 10.9%, 6.7%, and 42.0%, respectively. The findings from the phylogenetic analysis suggest that the species under investigation forms a cluster with other species belonging to the family Nycteribiidae. Consequently, this study provides valuable insights for the identification of N. formosana.

Complete mitogenome of Nycteribia allotopa Speiser, 1901 (Diptera, Hippoboscoidea, Nycteribiidae) and comparative analysis of mitochondrial genomes of Nycteribiidae.

In recent years, the global pandemic of bat-associated pathogens has led to increasing attention on bat ectoparasites. Numerous studies have identified human-associated pathogens in Nycteribiidae, indicating their potential as vectors. In this study, the first complete sequencing of the mitochondrial genome of Nycteribia allotopa Speiser, 1901 was sequenced and analyzed. We also compared the mitochondrial sequences of N. allotopa with those available in the database for other Nycteribiidae species. The complete mitochondrial genome of N. allotopa was found to be 15,161 bp in size with an A + T content of 82.49%. Nucleotide polymorphism analysis of 13 protein-coding genes from five species of Nycteribiidae showed that nad6 exhibited the most significant variation, while cox1 was the most conserved. Furthermore, selection pressure analysis revealed cox1 to exhibit the strongest purifying selection, while atp8, nad2, nad4L, and nad5 showed slightly looser purifying selection. Pairwise genetic distances indicated that cox1 and cox2 were evolving comparatively slowly, whereas atp8, nad2, and nad6 were evolving comparatively quickly. Phylogenetic trees constructed using Bayesian inference and maximum likelihood methods demonstrated that all four families within the superfamily Hippoboscoidea clustered into one branch each, indicating their monophyly. N. allotopa was found to be most closely related to the same genus N. parvula. This study significantly enriches the molecular database for Nycteribiidae and provides invaluable reference data for future species identification, phylogenetic analysis, and exploration of their potential as vectors for human-associated pathogens.

Characteristics of activation of monocyte-derived macrophages versus microglia after mouse experimental intracerebral hemorrhage.

Both monocyte-derived macrophages (MDMs) and brain resident microglia participate in hematoma resolution after intracerebral hemorrhage (ICH). Here, we utilized a transgenic mouse line with enhanced green fluorescent protein (EGFP) labeled microglia (Tmem119-EGFP mice) combined with a F4/80 immunohistochemistry (a pan-macrophage marker) to visualize changes in MDMs and microglia after ICH. A murine model of ICH was used in which autologous blood was stereotactically injected into the right basal ganglia. The autologous blood was co-injected with CD47 blocking antibodies to enhance phagocytosis or clodronate liposomes for phagocyte depletion. In addition, Tmem119-EGFP mice were injected with the blood components peroxiredoxin 2 (Prx2) or thrombin. MDMs entered the brain and formed a peri-hematoma cell layer by day 3 after ICH and giant phagocytes engulfed red blood cells were found. CD47 blocking antibody increased the number of MDMs around and inside the hematoma and extended MDM phagocytic activity to day 7. Both MDMs and microglia could be diminished by clodronate liposomes. Intracerebral injection of Prx2 but not thrombin attracted MDMs into brain parenchyma. In conclusion, MDMs play an important role in phagocytosis after ICH which can be enhanced by CD47 blocking antibody, suggesting the modulation of MDMs after ICH could be a future therapeutic target.

The complete mitochondrial genome of Nycteribia parvula Speiser, 1901 (Diptera, Nycteribiidae).

Species of the family Nycteribiidae are blood-sucking ectoparasites that parasitize bats. To further enrich the molecular data of species in the family Nycteribiidae, the complete mitochondrial genome of Nycteribia parvula was sequenced for the first time in this study. The complete mitochondrial genome of N. parvula is 16,060 base pairs (bp) in size, including 13 protein-coding genes (PCGs), 22 transfer RNA genes, two ribosomal RNA genes, and a control region. The nucleotide contents of A, T, G, and C are respectively 40.86%, 42.19%, 6.51%, and 10.44%. The phylogenetic analysis based on 13 PCGs supports the monophyly of the family Nycteribiidae, and N. parvula is the closest relative to Phthiridium szechuanum.

Complete mitochondrial genome of Penicillidia jenynsii (Diptera: Hippoboscoidea: Nycteribiidae) and phylogenetic relationship.

In recent years, bat-associated pathogens, such as 2019 novel coronavirus, have been ravaging the world, and ectoparasites of bats have received increasing attention. Penicillidia jenynsii is a member of the family Nycteribiidae which is a group of specialized ectoparasites of bats. In this study, the complete mitochondrial genome of P. jenynsii was sequenced for the first time and a comprehensive phylogenetic analysis of the superfamily Hippoboscoidea was conducted. The complete mitochondrial genome of P. jenynsii is 16 165 base pairs (bp) in size, including 13 protein-coding genes (PCGs), 22 transfer RNA genes, 2 ribosomal RNA genes and 1 control region. The phylogenetic analysis based on 13 PCGs of the superfamily Hippoboscoidea known from the NCBI supported the monophyly of the family Nycteribiidae, and the family Nycteribiidae was a sister group with the family Streblidae. This study not only provided molecular data for the identification of P. jenynsii, but also provided a reference for the phylogenetic analysis of the superfamily Hippoboscoidea.

Sestrin2 prevents neonatal incision pain and re-incision enhanced hyperalgesia in adult rats.

BACKGROUND: Improving the methods for recognizing pain is important for infants admitted to the neonatal intensive care unit. Sestrin2 is a novel stress-inducible protein with a neuroprotective role that functions as a molecular mediator of hormesis. Nevertheless, the role of sestrin2 in the pain process is still unclear. The following study examined the role of sestrin2 on mechanical hypersensitivity after pups incision, as well as enhanced pain hyperalgesia after adulthood re-incision in rats. METHODS: The experiment was divided into two parts: (1) studying the effect of sestrin2 in the neonatal incision; (2) studying the priming effect in adulthood re-incision. An animal model was established in seven-day-old rat pups with a right hind paw incision. Pups were intrathecally administrated rh-sestrin2 (exogenous sestrin2). Paw withdrawal threshold testing was performed to assay mechanical allodynia; tissue was analyzed in ex vivo using Western blot and immunofluorescence. SB203580 was further used to inhibit microglial function and evaluate the sex-dependent effect in adulthood. RESULTS: Sestrin2 expression increased transitorily in the spinal dorsal horn in pups after incision. Administration of rh-sestrin2 improved pups' mechanical hypersensitivity by regulating the AMPK/ERK pathway and alleviated re-incision-induced enhanced hyperalgesia in male and female adult rats. After administration of SB203580 in pups, the mechanical hyperalgesia following re-incision in adult rats was prevented in males but not females; however, the protective effect of SB203580 in males was counteracted by silencing sestrin2. CONCLUSIONS: These data suggest that sestrin2 prevents neonatal incision pain and re-incision enhanced hyperalgesia in adult rats. Moreover, microglia inhibition affects enhanced hyperalgesia only in adult males, which may be regulated through the sestrin2 mechanism. To sum up, these sestrin2 data may be a potential common molecular target for treating re-incision hyperalgesia in different sexes.

The complete mitochondrial genome of Phthiridium szechuanum (Nycteribiidae, Diptera).

Phthiridium szechuanum is a bat surface parasite under the family Nycteribiidae that prefers to roost in the hair of bats to feed on their blood. In this study, the complete mitochondrial genome of P. szechuanum was studied for the first time using Illumina sequencing technology. The mitochondrial genome was 14,896 bp in size and was predicted to encode 37 genes including 13 protein-coding genes, 22 transfer RNA genes, and 2 ribosomal RNA genes. Phylogenetic trees were constructed using the IQ-TREE web server and phylogenetic analysis was performed using the maximum likelihood method, and P. szechuanum was found to be phylogenetically closest to Basilia ansifera. These data will provide a molecular biological approach to the species identification of P. szechuanum and provide a new reference for further studies on the population genetics and phylogeny of the family Nycteribiidae.

Novel Approach to Visualize Microglia Death and Proliferation After Intracerebral Hemorrhage in Mice.

BACKGROUND: Microglia are important brain immune cells. However, it is difficult to differentiate microglia from monocyte-derived macrophages. To visualize microglia changes following intracerebral hemorrhage (ICH), we utilized a genetic knock-in mouse line, Tmem119 (transmembrane protein 119)-EGFP (enhanced green fluorescent protein), which expresses EGFP specifically in microglia. METHODS: There were 2 parts in this study. First, autologous blood was injected into the right basal ganglia to model ICH in Tmem119-EGFP mice. Mice were euthanized at 4 hours, days 1, 3, and 7 after ICH. Sham animals were used as controls. Second, Tmem119-EGFP mice were injected with iron or thrombin, factors involved in ICH-induced injury, and were euthanized at 4 hours. Naïve mice were controls. Brains were harvested for histology. RESULTS: The number of perihematomal microglia significantly decreased 1 day after ICH, but markedly increased by days 3 and 7. Microglia death was also induced by intracerebral iron injection while microglia proliferation was found with intracerebral thrombin injection. CONCLUSIONS: Perihematomal microglia death and proliferation after ICH are visualized in vivo with a Tmem119-EGFP transgenic mouse line. Iron and thrombin may contribute to ICH-induced microglia death and proliferation, respectively.

A timeline of oligodendrocyte death and proliferation following experimental subarachnoid hemorrhage.

AIMS: White matter (WM) injury is a critical factor associated with worse outcomes following subarachnoid hemorrhage (SAH). However, the detailed pathological changes are not completely understood. This study investigates temporal changes in the corpus callosum (CC), including WM edema and oligodendrocyte death after SAH, and the role of lipocalin-2 (LCN2) in those changes. METHODS: Subarachnoid hemorrhage was induced in adult wild-type or LCN2 knockout mice via endovascular perforation. Magnetic resonance imaging was performed 4 hours, 1 day, and 8 days after SAH, and T2 hyperintensity changes within the CC were quantified to represent WM edema. Immunofluorescence staining was performed to evaluate oligodendrocyte death and proliferation. RESULTS: Subarachnoid hemorrhage induced significant CC T2 hyperintensity at 4 hours and 1 day that diminished significantly by 8 days post-procedure. Comparing changes between the 4 hours and 1 day, each individual mouse had an increase in CC T2 hyperintensity volume. Oligodendrocyte death was observed at 4 hours, 1 day, and 8 days after SAH induction, and there was progressive loss of mature oligodendrocytes, while immature oligodendrocytes/oligodendrocyte precursor cells (OPCs) proliferated back to baseline by Day 8 after SAH. Moreover, LCN2 knockout attenuated WM edema and oligodendrocyte death at 24 hours after SAH. CONCLUSIONS: Subarachnoid hemorrhage leads to T2 hyperintensity change within the CC, which indicates WM edema. Oligodendrocyte death was observed in the CC within 1 day of SAH, with a partial recovery by Day 8. SAH-induced WM injury was alleviated in an LCN2 knockout mouse model.

Sevoflurane preconditioning promotes mesenchymal stem cells to relieve myocardial ischemia/reperfusion injury via TRPC6-induced angiogenesis.

BACKGROUND: Ischemic heart diseases is one of the leading causes of death worldwide. Although revascularization timely is an effective therapeutic intervention to salvage the ischemic myocardium, reperfusion itself causes additional myocardial injury called ischemia/reperfusion (I/R) injury. Bone marrow-derived mesenchymal stem cells (MSCs) is one of the promising cells to alleviate ischemic myocardial injury. However, this cell therapy is limited by poor MSCs survival after transplantation. Here, we investigated whether sevoflurane preconditioning could promote MSCs to attenuate myocardial I/R injury via transient receptor potential canonical channel 6 (TRPC6)-induced angiogenesis. METHODS: The anti-apoptotic effect of sevoflurane preconditioning on MSCs was determined by Annexin V-FITC/propidium iodide staining. TRPC6, hypoxia-inducible factor-1α (HIF-1α), Chemokine receptor 4 (CXCR4) and vascular endothelial growth factor (VEGF) protein expressions and VEGF release from MSCs were determined after hypoxia and reoxygenation (H/R). Small interfering RNA (siRNA) was used to knock down TRPC6 gene expression in MSCs. The angiogenesis of human umbilical vein endothelial cells (HUVECs) co-cultured with MSCs was determined by Matrigel tube formation. Myocardial I/R mouse model was induced by occluding left anterior descending coronary artery for 30 min and then reperfusion. MSCs or sevoflurane preconditioned MSCs were injected around the ligature border zone 5 min before reperfusion. Left ventricle systolic function, infarction size, serum LDH, cTnI and inflammatory cytokines were determined after reperfusion. RESULTS: Sevoflurane preconditioning up-regulated TRPC6, HIF-1α, CXCR4 and VEGF expressions in MSCs and VEGF release from MSCs under H/R, which were reversed by knockdown of TRPC6 gene using siRNA in MSCs. Furthermore, sevoflurane preconditioning promoted the angiogenic and anti-inflammatory effect of HUVECs co-cultured with MSCs. Sevoflurane preconditioned MSCs improved left ventricle systolic function and alleviated myocardial infarction and inflammation in mice subjected to I/R insult. CONCLUSION: The current findings reveal that sevoflurane preconditioned MSCs boost angiogenesis in HUVECs subjected to H/R insult and attenuate myocardial I/R injury, which may be mediated by TRPC6 up-regulated HIF-1α, CXCR4 and VEGF.

Sestrin2 is involved in the Nrf2-regulated antioxidative signaling pathway in luteolin-induced prevention of the diabetic rat heart from ischemia/reperfusion injury.

Luteolin attenuates myocardial ischemia/reperfusion (I/R) injury in diabetes through activating the nuclear factor erythroid 2-related factor 2 (Nrf2)-related antioxidative response. Though sestrin2, a highly conserved stress-inducible protein, is regarded as a modulator of Nrf2 and reduces I/R injury, the effect of sestrin2 on luteolin-induced prevention of the diabetic heart from I/R injury remains unclear. We hypothesized that luteolin could relieve myocardial I/R injury in diabetes by activating the sestrin2-modulated Nrf2 antioxidative response. Diabetes was induced in rats using a single dose of streptozotocin (65 mg kg-1, i.p.) for 6 weeks, and then luteolin (100 mg kg-1 d-1, i.g.), Nrf2 inhibitor brusatol, or sestrin2 blocker leucine was administered for 2 consecutive weeks. After that, the hearts were isolated and exposed to global I/R (30 min/120 min). Luteolin markedly improved cardiac function, myocardial viability and expressions of Nrf2-regulated antioxidative genes, and reduced lactate dehydrogenase release, malondialdehyde, and 8-hydroxydeoxyguanosine in the diabetic I/R hearts. Ca2+-induced mitochondrial permeability transition and membrane potential disruption were markedly inhibited in luteolin-treated diabetic ventricular myocytes. All these effects of luteolin were significantly reversed by Nrf2 inhibitor brusatol or sestrin2 inhibitor leucine. Luteolin-induced diminished Keap1 and augmented nuclear translocation and ARE binding activity of Nrf2 were hampered by leucine in the diabetic I/R heart. In addition, luteolin-induced augmented transcription of sestrin2 was markedly blocked by brusatol in the diabetic I/R heart. These data suggest that sestrin2 and Nrf2 positively interact to promote antioxidative actions and attenuate mitochondrial damage, by which luteolin relieves diabetic myocardial I/R injury.

Luteolin Attenuates Cardiac Ischemia/Reperfusion Injury in Diabetic Rats by Modulating Nrf2 Antioxidative Function.

Luteolin has been reported to attenuate ischemia/reperfusion (I/R) injury in the diabetic heart through endothelial nitric oxide synthase- (eNOS-) related antioxidative response. Though the nuclear factor erythroid 2-related factor 2 (Nrf2) is regarded as a key endogenous factor to reduce diabetic oxidative stress, whether luteolin reduces cardiac I/R injury in the diabetic heart via enhancing Nrf2 function needs to be clarified. We hypothesized that pretreatment with luteolin could alleviate cardiac I/R injury in the diabetic heart by affecting the eNOS/Nrf2 signaling pathway. The diabetic rat was produced by a single injection of streptozotocin (65 mg/kg, i.p.) for 6 weeks, and then, luteolin (100 mg/kg/day, i.g.), eNOS inhibitor L-NAME, or Nrf2 inhibitor brusatol was administered for the succedent 2 weeks. After that, the isolated rat heart was exposed to 30 min of global ischemia and 120 min of reperfusion to establish I/R injury. Luteolin markedly ameliorated cardiac function and myocardial viability; upregulated expressions of heme oxygenase-1, superoxide dismutase, glutathione peroxidase, and catalase; and reduced myocardial lactate dehydrogenase release, malondialdehyde, and 8-hydroxydeoxyguanosine in the diabetic I/R heart. All these ameliorating effects of luteolin were significantly reversed by L-NAME or brusatol. Luteolin also markedly reduced S-nitrosylation of Kelch-like ECH-associated protein 1 (Keap1) and upregulated Nrf2 and its transcriptional activity. This effect of luteolin on Keap1/Nrf2 signaling was attenuated by L-NAME. These data reveal that luteolin protects the diabetic heart against I/R injury by enhancing eNOS-mediated S-nitrosylation of Keap1, with subsequent upregulation of Nrf2 and the Nrf2-related antioxidative signaling pathway.

Luteolin alleviates cardiac ischemia/reperfusion injury in the hypercholesterolemic rat via activating Akt/Nrf2 signaling.

Myocardial ischemia/reperfusion (I/R) injury in hypercholesterolemia is associated with oxidative stress, while luteolin is known to reduce oxidative stress by activating Akt/nuclear factor erythroid-2-related factor 2 (Nrf2) signaling and alleviate cardiac I/R injury. Here, we investigated whether luteolin pretreatment diminishes myocardial I/R injury in hypercholesterolemic rats by activating Akt/Nrf2 signaling. Hypercholesterolemic rats were produced by 2% cholesterol diet for 8 weeks. Luteolin (100 mg/kg/day, i.g.) or LY294002 was administered for the last 2 weeks. The hearts were then isolated and subjected to 30 min of global ischemia followed by 120 min of reperfusion. Pretreatment with luteolin significantly improved left ventricular function throughout reperfusion, increased cardiac tissue viability, reduced coronary lactate dehydrogenase release and the myocardial malondialdehyde level, upregulated p-Akt and p-GSK3ß expressions, inhibited nuclear translocation of Fyn, and activated Nrf2 function in hypercholesterolemic I/R rat hearts. All these improving effects of luteolin were significantly attenuated by LY294002. Ca2+-induced mitochondrial permeability transition pore (mPTP) opening and mitochondrial inner membrane potential reduction were significantly inhibited in ventricular myocytes isolated from luteolin-treated hypercholesterolemic rats, which were attenuated by LY294002. These results indicate that luteolin protects the hypercholesterolemic heart against I/R injury due to upregulation of Akt-mediated Nrf2 antioxidative function and inhibition of mPTP.

Effect of X-ray irradiation on hepatocarcinoma cells and erythrocytes in salvaged blood.

The broad clinical acceptance of intraoperative blood salvage and its applications in cancer surgery remain controversial. Until now, a method that can safely eliminate cancer cells while preserving erythrocytes does not exist. Here, we investigated whether X-ray generated from linear accelerator irradiation at a certain dose can kill hepatocarcinoma cells while preserving erythrocytes. HepG2, SK-Hep1 or Huh7 cells were mixed into the aliquots of erythrocytes obtained from healthy volunteers. After the mixed cells were exposed to 30 Gy and 50 Gy X-rays irradiation, the viability, clonogenicity, DNA synthesis and tumorigenicity of the tumor cells were determined by the MTT assay, plate colony formation, 5-ethynyl-2'-deoxyuridine incorporation, and subcutaneous xenograft implantation into immunocompromised mice. The ATP, 2,3-DPG, free Hb, osmotic fragility, blood gas variables in erythrocytes and morphology of erythrocytes at 0 h, 12 h, 24 h, 48 h, 72 h after irradiation were analyzed. X-ray irradiation at 30 Gy effectively inhibited the viability, proliferation, and tumorigenicity of HepG2, SK-Hep1 and Huh7 cells without noticeably damaging the ability of oxygen-carrying, membrane integrity and morphology of erythrocytes. Theses results suggest that X-ray at 30 Gy irradiation might be safe to eliminate hepatocarcinoma cells while preserving erythrocytes in salvaged blood.

Irradiation Can Selectively Kill Tumor Cells while Preserving Erythrocyte Viability in a Co-Culture System.

An understanding of how to safely apply intraoperative blood salvage (IBS) in cancer surgery has not yet been obtained. Here, we investigated the optimal dose of 137Cs gamma-ray irradiation for killing human hepatocarcinoma (HepG2), gastrocarcinoma (SGC7901), and colonic carcinoma (SW620) tumor cells while preserving co-cultured erythrocytes obtained from 14 healthy adult volunteers. HepG2, SGC7901, or SW620 cells were mixed into the aliquots of erythrocytes. After the mixed cells were treated with 137Cs gamma-ray irradiation (30, 50, and 100 Gy), tumor cells and erythrocytes were separated by density gradient centrifugation in Percoll with a density of 1.063 g/ml. The viability, clonogenicity, DNA synthesis, tumorigenicity, and apoptosis of the tumor cells were determined by MTT assay, plate colony formation, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, subcutaneous xenograft implantation into immunocompromised mice, and annexin V/7-AAD staining, respectively. The ATP concentration, 2,3-DPG level, free Hb concentration, osmotic fragility, membrane phosphatidylserine externalization, blood gas variables, reactive oxygen species levels, and superoxide dismutase levels in erythrocytes were analyzed. We found that 137Cs gamma-ray irradiation at 50 Gy effectively inhibited the viability, proliferation, and tumorigenicity of HepG2, SGC7901, and SW620 cells without markedly damaging the oxygen-carrying ability or membrane integrity or increasing the oxidative stress of erythrocytes in vitro. These results demonstrated that 50 Gy irradiation in a standard 137Cs blood irradiator might be a safe and effective method of inactivating HepG2, SGC7901, and SW620 cells mixed with erythrocytes, which might help to safely allow IBS in cancer surgery.

Cisplatin combined with hyperthermia kills HepG2 cells in intraoperative blood salvage but preserves the function of erythrocytes.

The safe use of intraoperative blood salvage (IBS) in cancer surgery remains controversial. Here, we investigated the killing effect of cisplatin combined with hyperthermia on human hepatocarcinoma (HepG2) cells and erythrocytes from IBS in vitro. HepG2 cells were mixed with concentrated erythrocytes and pretreated with cisplatin (50, 100, and 200 µg/ml) alone at 37 °C for 60 min and cisplatin (25, 50, 100, and 200 µg/ml) combined with hyperthermia at 42 °C for 60 min. After pretreatment, the cell viability, colony formation and DNA metabolism in HepG2 and the Na(+)-K(+)-ATPase activity, 2,3-diphosphoglycerate (2,3-DPG) concentration, free hemoglobin (Hb) level, osmotic fragility, membrane phosphatidylserine externalization, and blood gas variables in erythrocytes were determined. Pretreatment with cisplatin (50, 100, and 200 µg/ml) combined with hyperthermia (42 °C) for 60 min significantly decreased HepG2 cell viability, and completely inhibited colony formation and DNA metabolism when the HepG2 cell concentration was 5×10(4) ml(-1) in the erythrocyte (P<0.01). Erythrocytic Na(+)-K(+)-ATPase activity, 2,3-DPG level, phosphatidylserine externalization, and extra-erythrocytic free Hb were significantly altered by hyperthermia plus high concentrations of cisplatin (100 and 200 µg/ml) (P<0.05), but not by hyperthermia plus 50 µg/ml cisplatin (P>0.05). In conclusion, pretreatment with cisplatin (50 µg/ml) combined with hyperthermia (42 °C) for 60 min effectively eliminated HepG2 cells from IBS but did not significantly affect erythrocytes in vitro.

Cardioprotective effects of luteolin on ischemia/reperfusion injury in diabetic rats are modulated by eNOS and the mitochondrial permeability transition pathway.

Myocardial ischemia/reperfusion (I/R) injury in diabetes is associated with oxidative stress, endothelial nitric oxide synthase (eNOS) dysfunction, and mitochondrial collapse, whereas luteolin is known to protect the cardiovascular system against diabetes and I/R injury. Here, we investigated whether luteolin pretreatment diminishes myocardial I/R injury in diabetic rats by affecting eNOS and the mitochondrial permeability transition pore (mPTP). After diabetic rats were produced by streptozotocin treatment (65 mg/kg) for 3 weeks, luteolin (100 mg·kg·d) or L-NAME (25 mg·kg·d) was administered intragastrically for 2 weeks. Hearts were then isolated and subjected to 30 minutes of global ischemia followed by 120 minutes of reperfusion. Pretreatment with luteolin significantly improved left ventricular function and coronary flow throughout reperfusion, increased cardiac tissue viability and manganese superoxide dismutase (MnSOD) activity, and reduced coronary lactate dehydrogenase release, and the myocardial malonaldehyde level in diabetic I/R rat hearts. All these improving effects of luteolin were significantly attenuated by L-NAME. Luteolin also significantly upregulated eNOS expression in diabetic rat hearts after I/R. Ca-induced mPTP opening and mitochondrial inner membrane potential reduction were significantly inhibited in ventricular myocytes isolated from luteolin-treated diabetic rats, and this effect was attenuated by L-NAME. These findings indicate that luteolin protects the diabetic heart against I/R injury by upregulating the myocardial eNOS pathway, and downstream effects include the enhancement of MnSOD and inhibition of mPTP.

[Pretreatment with cisplatin plus hyperthermia kills different tumor cells but preserves the erythrocyte function in the intra-operative blood salvage in vitro].

OBJECTIVE: To investigate the effects of cisplatin plus hyperthermia on erythrocytes and killing human hepatocarcinoma (HepG2), gastro carcinoma (SGC7901) and colonic carcinoma (SW620) cells in the intra-operative blood salvage from cancer surgery in vitro. METHODS: HepG2, SGC7901 or SW620 cells were mixed into the aliquot of erythrocyte concentrated from each intra-operative blood salvage of 30 patients subjected to gastrointestinal cancer surgery. The mixture cells were divided into the following groups (n = 30): A group (37 °C); B group (42 °C); C, D, E groups (50, 100, or 200 µg/ml DDP); F, G, H, I groups (42 °C, 25, 50, 100, or 200 µg/ml DDP). After treating for 60 min, tumor cells and erythrocytes were separated by density gradient centrifugation. The Na(+)-K(+)-ATPase activity, cell count, osmotic fragility, and blood gas variables were determined in erythrocytes. Cell viability and colony formation were determined in tumor cells. RESULTS: Compared with A [(0.30 ± 0.08) µmol Pi/10(7)/h], the Na(+)-K(+)-ATPase activity was significantly decreased in E, H and I groups [(0.24 ± 0.07), (0.25 ± 0.06) and (0.24 ± 0.07) µmol Pi/10(7)/h] (P < 0.05). Extra-erythrocytic K(+) in E, H and I groups [(2.16 ± 0.37), (2.16 ± 0.38) and (2.56 ± 0.50) mmol/L] were significantly increased compared with A group [(1.53 ± 0.43) mmol/L] (P < 0.05). Compared with A group, osmotic fragility in E, H and I groups was significantly increased (P < 0.05). Among B, C, D, E, F, G groups, only in G group colony formations of HepG2, SGC7901, and SW620 (0% ± 0%, 0% ± 0% and 0.01% ± 0.01%) at 14 d were completely inhibited (P < 0.01) compared with A group (78.54% ± 7.83%, 72.28% ± 6.58% and 66.69% ± 6.69%). CONCLUSION: Pretreatment with cisplatin (50 µg/ml) plus hyperthermia (42 °C) for 60 min in vitro might be an effective strategy to clear tumor cells contamination but preserve erythrocytes, which is worthy to be optimized and used in the intra-operative blood salvage in cancer surgery.