Inflammation Injury In Mice Research Articles

Black cumin seed oil and its nano-form ameliorate lipopolysaccharide-induced brain inflammatory injury in mice

Backgrounds and objectives Microglia play a regulatory role in central nervous system inflammatory diseases, such as Alzheimer’s, Parkinson’s, and multiple sclerosis. Natural remedies like black cumin seeds (Nigella sativa) are rich in bioactive compounds that potentially can modulate inflammatory processes in the brain. In the current work, we studied the protective and anti-inflammatory properties of black cumin seed oil (BCSO) and its nano-form on lipopolysaccharide (LPS)-induced neurotoxicity in mice. Materials and methods Forty-eight mice were divided randomly into eight groups (n=6), three control groups (negative control, BCSO control, nano-BCSO control), LPS group, and four treatment groups [BCSO+LPS, nano-BCSO+LPS, indomethacin (5 mg/kg)+LPS, BCSO+indomethacin(2.5 mg/kg)+LPS]. At the end of the experiment, the brain tissues were removed for histopathological and biochemical assessments. Malondialdehyde and interleukin (IL)-10 were assessed using enzyme-linked immunosorbent assay while the gene expression of IL-6, toll-like receptor-4, brain-derived neurotrophic factor, nerve growth factor, cyclooxygenase-2, and B-cell lymphoma-2 were assessed by real-time PCR. IL-1β was quantified immunohistochemically along with the histopathological studies of the cerebral cortex of mice brains. Results and conclusions In our study, BCSO and its nano-form demonstrated a reduction in LPS-induced neurotoxicity, exhibiting comparable or better anti-inflammatory effects to indomethacin. These treatments significantly elevated the gene expression levels of neuroprotective factors brain-derived neurotrophic factor and nerve growth factor in LPS-treated mice. Pretreatment with BCSO and its nano-form reduced the malondialdehyde levels, in addition to gene expressions of cyclooxygenase-2, toll-like receptor-4, IL-6, and B-cell lymphoma-2. Immunohistochemical analysis indicated a decrease in IL-1β with BCSO and the lowering effect of the nano-form was superior. The histopathological studies corroborated with biochemical and molecular findings, suggesting that BCSO and its nano-form attenuated the inflammation and enhanced the microglial antioxidative and anti-inflammatory status. BCSO could enhance the anti-inflammatory activity of indomethacin, so lower doses of indomethacin with BCSO may be suggested for protecting against the adverse effects of high doses of NSAIDs as gastritis. Consequently, BCSO can serve a potential stimulatory supplement of the immunity for neurodegenerative conditions.

Gypenoside XLIX alleviates acute liver injury: Emphasis on NF-κB/PPAR-α/NLRP3 pathways

Liver is one of the vital organs in the human body and liver injury will have a very serious impact on human damage. Gypenoside XLIX is a PPAR-α activator that inhibits the activation of the NF-κB signaling pathway. The components of XLIX have pharmacological effects such as cardiovascular protection, antihypoxia, anti-tumor and anti-aging. In this study, we used cecum ligation and puncture (CLP) was used to induce in vivo mice hepatic injury, and lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells, evaluated whether Gypenoside XLIX could have a palliative effect on sepsis-induced acute liver injury via NF-κB/PPAR-α/NLRP3. In order to gain insight into these mechanisms, six groups were created in vivo: the Contol group, the Sham group, the CLP group, the CLP + XLIX group (40 mg/kg) and the Sham + XLIX (40 mg/kg) group, and the CLP + DEX (2 mg/kg) group. Three groups were created in vitro: Control, LPS, LPS + XLIX (40 μM). The analytical methods used included H&E staining, qPCR, reactive oxygen species (ROS), oil red O staining, and Western Blot. The results showed that XLIX attenuated hepatic inflammatory injury in mice with toxic liver disease through inhibition of the TLR4-mediated NF-κB pathway, attenuated lipid accumulation through activation of PPAR-α, and attenuated hepatic pyroptosis by inhibiting NLRP3 production. Regarding the imbalance between oxidative and antioxidant defenses due to septic liver injury, XLIX reduced liver oxidative stress-related biomarkers (ALT, AST), reduced ROS accumulation, decreased the amount of malondialdehyde (MDA) produced by lipid peroxidation, and increased the levels of antioxidant enzymes such as glutathione (GSH) and catalase (CAT). Our results demonstrate that XLIX can indeed attenuate septic liver injury. This is extremely important for future studies on XLIX and sepsis, and provides a potential pathway for the treatment of acute liver injury.

The Mechanism of Astragaloside IV in NOD-like Receptor Family Pyrin Domain Containing 3 Inflammasome-mediated Pyroptosis after Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) is one of the most common subtypes of stroke. This study aimed to investigate the mechanism of Astragaloside IV (AS-IV) on inflammatory injury after ICH. The ICH model was established by the injection of collagenase and treated with ASIV (20 mg/kg or 40 mg/kg). The neurological function, water content of the bilateral cerebral hemisphere and cerebellum, and pathological changes in brain tissue were assessed. The levels of interleukin-1 beta (IL-1β), IL-18, tumor necrosis factor-alpha, interferon-gamma, and IL-10 were detected by enzyme-linked immunosorbent assay. The levels of Kruppel-like factor 2 (KLF2), NOD-like receptor family pyrin domain containing 3 (NLRP3), GSDMD-N, and cleaved-caspase-1 were detected by reverse transcription-quantitative polymerase chain reaction and Western blot assay. The binding relationship between KLF2 and NLRP3 was verified by chromatin-immunoprecipitation and dual-luciferase assays. KLF2 inhibition or NLRP3 overexpression was achieved in mice to observe pathological changes. The decreased neurological function, increased water content, severe pathological damage, and inflammatory response were observed in mice after ICH, with increased levels of NLRP3/GSDMD-N/cleaved-caspase-1/IL-1β/IL-18 and poorly-expressed KLF2 in brain tissue. After AS-IV treatment, the neurological dysfunction, high brain water content, inflammatory response, and pyroptosis were alleviated, while KLF2 expression was increased. KLF2 bonded to the NLRP3 promoter region and inhibited its transcription. Down-regulation of KLF2 or upregulation of NLRP3 reversed the effect of AS-IV on inhibiting pyroptosis and reducing inflammatory injury in mice after ICH. AS-IV inhibited NLRP3-mediated pyroptosis by promoting KLF2 expression and alleviated inflammatory injury in mice after ICH.

Enterococcus faecium inhibits NF-κB/NLRP3/IL-1β signaling pathway andantagonizes Salmonella-mediated inflammatory response.

Aim: This study explored the protective effect of Enterococcus faecium as a probiotic against Salmonella typhimurium infection. Materials & methods: The protective role of E. faecium against tissue damage by S. typhimurium infection and the expression of inflammatory cytokines and tight junction proteins were detected by histological observation, real-time quantitative PCR and immunohistochemical methods. Results: E. faecium demonstrateda regulatory function that affected the expression of Claudin-1 and enhanced tight junctions, suppressed theNF-κB/NLRP3/IL-1β signaling pathway andreduced the release of IL-6, TNF-α, IFN-γ, TLR4 and MYD88 and inflammatory damage to tissues by S. typhimurium in the duodenum, cecum and colon of mice. Conclusion: E. faecium antagonized S. Typhimurium alleviating inflammatory injury in mice through the NF-κB/NLRP3/IL-1β signaling pathway.

Mechanism of Zhenwu Decoction in improving renal inflammatory injury in mice with DN of spleen-kidney Yang deficiency syndrome by regulating ROCK/IKK/NF-κB pathway

To investigate the intervention effect and mechanism of Zhenwu Decoction on diabetic nephropathy(DN) mice of spleen-kidney Yang deficiency syndrome based on the Rho-associated coiled-coil kinase(ROCK)/IκB kinase(IKK)/nuclear factor-κB(NF-κB) pathway. Ninety-five 7-week-old db/db male mice and 25 7-week-old db/m male mice were fed adaptively for one week. The DN model of spleen-kidney Yang deficiency syndrome was induced by Dahuang Decoction combined with hydrocortisone by gavage, and then the model was evaluated. After modeling, they were randomly divided into a model group, high-dose, medium-dose, and low-dose Zhenwu Decoction groups(33.8, 16.9, and 8.45 g·kg~(-1)·d~(-1)), and an irbesartan group(25 mg·kg~(-1)·d~(-1)), with at least 15 animals in each group. The intervention lasted for eight weeks. After the intervention, body weight and food intake were measured. Serum crea-tinine(Scr), blood urea nitrogen(BUN), fasting blood glucose(FBG), urinary albumin(uALb), and urine creatinine(Ucr) were determined. The uALb/Ucr ratio(ACR) and 24 h urinary protein(UTP) were calculated. Renal pathological morphology was evaluated by HE staining and Masson staining. The levels of key molecular proteins in the ROCK/IKK/NF-κB pathway were detected by Western blot. Enzyme-linked immunosorbent assay(ELISA) was used to detect interleukin-1β(IL-1β), interleukin-6(IL-6), interleukin-8(IL-8), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Compared with the blank group, the model group showed increased content of BUN, uALb, and SCr, increased values of 24 h UTP and ACR, decreased content of Ucr(P<0.05), enlarged glomeruli, thickened basement membrane, mesangial matrix proliferation, inflammatory cell infiltration, and collagen fiber deposition. The protein expression of ROCK1, ROCK2, IKK, NF-κB, phosphorylated IKK(p-IKK), phosphorylated NF-κB(p-NF-κB), and phosphorylated inhibitor of NF-κB(p-IκB) increased(P<0.05), while the protein expression of inhibitor of NF-κB(IκB) decreased(P<0.05). The levels of inflammatory factors IL-1β, IL-6, IL-8, and TNF-α increased(P<0.05), while the level of IL-10 decreased(P<0.05). Compared with the model group, the groups with drug treatment showed decreased levels of BUN, uALb, SCr, 24 h UTP, and ACR, increased level of Ucr(P<0.05), and improved renal pathological status to varying degrees. The high-and medium-dose Zhenwu Decoction groups and the irbesartan group showed reduced protein expression of ROCK1, ROCK2, IKK, NF-κB, p-IKK, p-NF-κB, and p-IκB in the kidneys(P<0.05), increased protein expression of IκB(P<0.05), decreased levels of serum inflammatory factors IL-1β, IL-6, IL-8, and TNF-α(P<0.05), and increased level of IL-10(P<0.05). Zhenwu Decoction can significantly improve renal function and renal pathological damage in DN mice of spleen-kidney Yang deficiency syndrome, and its specific mechanism may be related to the inhibition of inflammatory response by down-regulating the expression of key molecules in the ROCK/IKK/NF-κB pathway in the kidney.

GAT107-mediated α7 nicotinic acetylcholine receptor signaling attenuates inflammatory lung injury and mortality in a mouse model of ventilator-associated pneumonia by alleviating macrophage mitochondrial oxidative stress via reducing MnSOD-S-glutathionylation

Supraphysiological concentrations of oxygen (hyperoxia) can compromise host defense and increase susceptibility to bacterial and viral infections, causing ventilator-associated pneumonia (VAP). Compromised host defense and inflammatory lung injury are mediated, in part, by high extracellular concentrations of HMGB1, which can be decreased by GTS-21, a partial agonist of α7 nicotinic acetylcholine receptor (α7nAChR). Here, we report that a novel α7nAChR agonistic positive allosteric modulator (ago-PAM), GAT107, at 3.3 mg/kg, i.p., significantly decreased animal mortality and markers of inflammatory injury in mice exposed to hyperoxia and subsequently infected with Pseudomonas aeruginosa. The incubation of macrophages with 3.3 μM of GAT107 significantly decreased hyperoxia-induced extracellular HMGB1 accumulation and HMGB1-induced macrophage phagocytic dysfunction. Hyperoxia-compromised macrophage function was correlated with impaired mitochondrial membrane integrity, increased superoxide levels, and decreased manganese superoxide dismutase (MnSOD) activity. This compromised MnSOD activity is due to a significant increase in its level of glutathionylation. The incubation of hyperoxic macrophages with 3.3 μM of GAT107 significantly decreases the levels of glutathionylated MnSOD, and restores MnSOD activity and mitochondrial membrane integrity. Thus, GAT107 restored hyperoxia-compromised phagocytic functions by decreasing HMGB1 release, most likely via a mitochondrial-directed pathway. Overall, our results suggest that GAT107 may be a potential treatment to decrease acute inflammatory lung injury by increasing host defense in patients with VAP.

Polyphyllin I alleviates inflammatory injury in mice with gestational diabetes through AMPK pathway

Our study aimed to detect the effects of polyphyllin I (PPI) on relieving gestational diabetes mellitus (GDM), and the possible mechanism. A mouse model of GDM was constructed. The effects of PPI on GDM mice were evaluated by detecting blood glucose, insulin level, glucose tolerance test, and insulin tolerance test. The inflammation response in GDM and GDM+PPI group were evaluated by enzyme-linked immunosorbent assay (ELISA). The effect of PPI on the offspring of GDM mice was analyzed. In addition, immunoblot assays were performed to investigate the effects of PPI on the AMPK pathway. We found that PPI improved diabetes-related symptoms and decreased serum inflammatory response in GDM mice. In addition, we also found that PPI reduced the tissue damage of GDM mice. We noticed that PPI alleviated inflammatory injury in GDM mice through targeting AMPK pathway. Our findings showed that PPI has the potential to be explored as the drug for GDM treatment.

Aluminum activates NLRP3 inflammasome-mediated pyroptosis via reactive oxygen species to induce liver injury in mice

Aluminum (Al) exposure can lead to oxidative stress and liver inflammatory injury in mice. The overproduction of reactive oxygen species (ROS) activates the NLRP3 inflammasome and further induces liver pyroptosis. However, the participation of pyroptosis in inducing Al-mediated liver injury and the underlying regulatory mechanisms remain largely unclear. Herein, a mice model of subchronic Al exposure was established to investigate the role of pyroptosis in Al-induced liver injury. Then, MCC950 and N-acetylcysteine were used to inhibit NLRP3 inflammasome-mediated pyroptosis and ROS production for exploring the role and the underlying mechanisms of pyroptosis in determining Al-induced liver injury. It was confirmed that Al induced hepatocyte pyroptosis in mice, and that NLRP3 inflammasome-mediated pyroptosis plays a damaging role in Al-induced liver injury. ROS promotes pyroptosis in an Al-induced liver injury model by activating the NLRP3 inflammasome. Collectively, it was shown that ROS promotes pyroptosis to aggravate Al-induced liver injury by activating the NLRP3 inflammasome.

7-Methoxyisoflavone suppresses vascular endothelial inflammation by inhibiting the expression of endothelial adhesion molecules

Endothelial cells (ECs) are vital regulators of inflammatory processes, there is the potential for inhibition of EC inflammation to be a therapeutic target in chronic inflammatory diseases. This study aimed to investigate the effect of 7-methoxyisoflavone (7-Mif) on endothelial inflammation. Our results showed that 7-Mif have no cytotoxicity on HUVECs. Pretreatment with 5 μM, 10 μM and 50 μM 7-Mif significantly reduced IL-1β-induced ICAM-1 (28.1% ± 4.1%, 25.9 ± 2.5% and 32.0% ± 3.2%, respectively) and VCAM-1 (48.0% ± 5.6%, 40.1 ± 3.1% and 39.6% ± 3.1%, respectively) mRNA expression. And pretreatment with 10 μM and 50 μM 7-Mif significantly reduced IL-1β-induced ICAM-1 (45.1% ± 4.4% and 33.6 ± 4.4%, respectively) and VCAM-1 (53.0% ± 3.7% and 53.7 ± 5.1%, respectively) protein levels. Furthermore, pretreatment with 50 μM 7-Mif inhibited monocyte-endothelial cell adhesion (50.2% ± 4.2%). Mechanistically, our results showed that 7-Mif reversed IL-1β-induced NF-κB activation and p65 translocation to the nucleus, therefore inhibiting endothelial cell inflammation. In addition, we confirmed that 7-Mif 10 mg/kg and 20 mg/kg reduced LPS-induced ICAM-1 (47.3% ± 1.3% and 39.0% ± 3.2%, respectively) and VCAM-1 (56.5 ± 2.8% and 47.8 ± 4.3%, respectively) expression and attenuated inflammatory injury in mice. In conclusion, we showed the inhibitory effect of 7-Mif on endothelial inflammation by suppressing the expression of endothelial adhesion molecules and monocyte adhesion. Our data illustrated that 7-Mif could positively regulate the process of endothelial inflammation.

ROS antagonizes the protection of Parkin-mediated mitophagy against aluminum-induced liver inflammatory injury in mice

Aluminum (Al) is a food pollutant that has extensive deleterious effects on the liver. Our previous research proposed that E3 ubiquitin ligase PARK2 knockout (Parkin−/-) could aggravate Al-induced liver damage by inhibiting mitophagy, during which the reactive oxygen species (ROS) content increases. Inhibition of mitophagy can activate inflammasome. But the link between Parkin-mediated mitophagy and liver inflammatory injury caused by Al, and the role of ROS in it remain unclear. In this study, we applied Al, Parkin−/- and N-acetyl-L-cysteine (NAC) to act on C57BL/6N mice to investigate them. We found that Al could induce liver inflammatory injury and Parkin−/- could aggravate it. Meanwhile, inhibition of ROS alleviated oxidative stress, mitochondrial damage, mitophagy and inflammatory injury caused by Al in Parkin−/- mice liver. These results indicated that ROS antagonized the protection of Parkin-mediated mitophagy against Al-induced liver inflammatory damage in mice.

Picroside II prevents inflammation injury in mice with diabetic nephropathy via TLR4/NF-κB pathway

The purpose of this study was to investigate the therapeutic effects of picroside II on diabetic nephropathy and reveal the involved underlying signal pathway. Male Sprague–Dawley (SD) mice were used to construct an animal model of streptozotocin (STZ)-induced diabetic nephropathy. Body weight and fasting blood glucose values were recorded. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of proteinuria, blood urea nitrogen (BUN), serum creatinine (Scr), interleukin (IL)-1β, IL-6, monocyte chemoattractant protein-1 (MCP-1) and necrosis factor alpha (TNF-α). Protein expression was determined using Western blotting test. Hematoxylin and eosin (H&E) staining was used to examine the morphological changes in kidney tissues. Treatment with picroside II (10 and 20 mg/kg) increased the STZ-induced reduction in body weight of diabetic mice. It also reversed the elevation of fasting blood glucose in STZ-induced diabetic mice. The levels of proteinuria, BUN and Scr were significantly increased in STZ-induced diabetic mice and these increments were prevented by picroside II. The serum levels of MCP-1, IL-1β, IL-6 and TNF-α were reduced, and the morphological damage was lessened by Picroside II in mice with diabetic nephropathy. Besides, picroside II prevented the activation of TLR4/NF-κB pathway. This study proved that picroside II inhibited inflammatory response and prevented kidney injury in mice with diabetic nephropathy through modulation of TLR4/NF-κB pathway, indicating beneficial effect of picroside II on diabetic nephropathy.

The endogenous ligand for guanylate cyclase-C activation reliefs intestinal inflammation in the DSS colitis model.

Ulcerative colitis (UC) is a major type of inflammatory bowel disease (IBD) and significantly impacts patient quality of life. Previous research revealed that the guanylate cyclase-C (GC-C) signaling pathway is associated with the severity of UC. We aimed to investigate the effect of the GC-C agonist, guanylin (Gn), on inflammatory injury in mice with colitis. An experimental UC model was established in Balb/c mice. Mesalamine served as a positive control. The Gn overexpression vector was administered once per day for 1 week. Intestinal permeability of the mice was measured using fluorescein isothiocyanate-dextran after the treatment. Histopathologic grading was estimated to assess the inflammatory injury of the colon. The expression level of crucial mediators of the GC-C signaling pathway (Gn, Ugn and GC-C) and tight junction proteins (occludin, claudin-1 and ZO-1) was measured in the colon. Additionally, the level of pro-inflammatory cytokines (IL-8 and TNF-α) in serum was measured. After injecting the UC mice with the Gn overexpression vector, the body weight increased, and the frequency of loose stools and bloody stools was decreased. Intestinal permeability and histopathologic score were significantly reduced (P<0.05). The expression level of GC-C, Gn, Ugn, claudin-1 and ZO-1 was significantly increased (P<0.05). The level of IL-8 and TNF-α in the serum was significantly decreased (P<0.01). Therefore, the application of Gn overexpression vector can ameliorate the intestinal inflammatory injury and repair the mucosal barrier in colitis mice, which further suggests the clinical therapeutic potential of GC-C agonists in IBD.

The involvement of DAMPs-mediated inflammation in cyclophosphamide-induced liver injury and the protection of liquiritigenin and liquiritin

Cyclophosphamide (CPA) is a chemotherapeutic drug widely used in the treatment of breast cancer or leukemia in clinic. However, CPA was reported to have hepatotoxicity. This study aims to observe the engaged mechanism of CPA-induced liver injury in mice and the protection of liquiritin (LQ) and liquiritigenin (LG). Liver sinusoidal endothelial injury induced by CPA (20, 40 mg/kg) in mice was evidenced by the elevated hepatic metalloproteinase-9 (MMP-9) expression, and the results from liver histological evaluation and scanning electron microscope observation. CPA increased hepatic infiltration of neutrophils, liver myeloperoxidase (MPO) activity, serum interleukin-6 (IL-6) content, hepatic IL-6 mRNA expression, toll-like receptor-4 (TLR4) expression and nuclear factor κB (NFκB) activation in mice. Elevated serum contents of damage associated molecular patterns (DAMPs) including high mobility group box 1 (HMGB1), heat shock protein 60 (HSP60) and glucose-regulated protein 94 (Grp94) were found in mice treated with CPA. Liver sinusoidal endothelial injury and inflammation induced by CPA were diminished in TLR4 knock-out mice. LG and LQ (40, 80 mg/kg) both ameliorated liver sinusoidal endothelial injury, and reduced the increased hepatic infiltration of neutrophils, MPO activity, hepatic IL-6 mRNA expression and NFκB activation induced by CPA. In summary, these results indicate that TLR4-NFκB-mediated inflammatory injury initiated by DAMPs was critically involved in CPA-induced hepatotoxicity. LG and LQ alleviated CPA-induced liver sinusoidal endothelial injury and inflammatory injury in mice.

Pudilan xiaoyan oral liquid alleviates LPS-induced respiratory injury through decreasing nitroxidative stress and blocking TLR4 activation along with NF-ΚB phosphorylation in mice

Ethnopharmacological relevancePudilan xiaoyan oral liquid (PDL), collected in Chinese Pharmacopoeia, has been used clinically for treating inflammatory diseases such as upper respiratory tract infection diseases. However, its potential anti-inflammation and the mechanism are still unclear. Materials and methodslipopolysaccharide (LPS) was used to induce respiratory inflammation of mice by intratracheal administration. UPLC/MS was performed for components analysis of PDL. Enzyme-linked immune sorbent assay (ELISA) was conducted for determining interleukin-6(IL-6), interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) in serum and supernatant of tracheal tissue while Nitric oxide assay kit for nitric oxide (NO) content. Hematoxylin-Eosin (HE) staining was applied to evaluate pathological lesions. Western blotting analysis (WB) and Immunohistochemistry(IHC) were employed for the determination of Toll-like receptors 4(TLR4), TNF-α, IL-6, inducible nitric oxide synthase(iNOS) and nuclear factor-kappa B p65 (NF-κB p65) protein expressions. ResultsSeven major compounds of PDL were analyzed simultaneously. The treatment of PDL could attenuate LPS-induced histopathological damage of tracheal tissues, followed by reducing pro-inflammation mediators including TNF-α and IL-6 in serum and supernatant of tracheal tissue. LPS-induced nitroxidative stress including NO content and iNOS expression was inhibited significantly by PDL. Furthermore, PDL also down-regulated NF-kB p65 phosphorylation and TLR4 expressions. ConclusionThe results indicated that the PDL had a protective effect on LPS-induced respiratory inflammation injury in mice. Our findings for the first time provide experimental evidence for the application of PDL on respiratory inflammation injury in clinical practice.

Association of EGF Receptor and NLRs signaling with Cardiac Inflammation and Fibrosis in Mice Exposed to Fine Particulate Matter.

ЄAmbient fine particulate matter (PM2.5 ) could induce cardiovascular diseases (CVD), but the mechanism remains unknown. To investigate the roles of epidermal growth factor receptor (EGFR) and NOD-like receptors (NLRs) in PM2.5 -induced cardiac injury, we set up a BALB/c mice model of PM2.5 -induced cardiac inflammation and fibrosis with intratracheal instillation of PM2.5 suspension (4.0mg/kg b.w.) for 5 consecutive days (once per day). After exposure, we found that mRNA levels of CXCL1, interleukin (IL)-6, and IL-18 were elevated, but interestingly, mRNA level of NLRP12 was significant decreased in heart tissue from PM2.5 -induced mice compared with those of saline-treated mice using real-time PCR. Protein levels of phospho-EGFR (Tyr1068), phospho-Akt (Thr308), NLRP3, NF-κB-p52/p100, and NF-κB-p65 in heart tissue of PM2.5 -exposed mice were all significantly increased using immunohistochemistry or Western blotting. Therefore, PM2.5 exposure could induce cardiac inflammatory injury in mice, which may be involved with EGFR/Akt signaling, NLRP3, and NLRP12.

Induction of microRNA-21 with exogenous hydrogen sulfide attenuates myocardial ischemic and inflammatory injury in mice.

Maintaining physiological levels of hydrogen sulfide during ischemia is necessary to limit injury to the heart. Because of the anti-inflammatory effects of hydrogen sulfide, we proposed that the hydrogen sulfide donor, sodium sulfide (Na2S), would attenuate myocardial injury through upregulation of protective microRNA-21 (miR-21) and suppression of the inflammasome, a macromolecular structure that amplifies inflammation and mediates further injury. Na2S-induced miR-21 expression was measured by quantitative polymerase chain reaction in adult primary rat cardiomyocytes and in the mouse heart. We measured inflammasome formation and activity in cardiomyocytes challenged with lipopolysaccharide and ATP or simulated ischemia/reoxygenation and in the heart after regional myocardial ischemia/reperfusion, in the presence or absence of Na2S. To assess the direct anti-inflammatory effects of hydrogen sulfide in vivo, we used a peritonitis model by way of intraperitoneal injection of zymosan A. Na2S attenuated inflammasome formation and activity, measured by counting cytoplasmic aggregates of the scaffold protein apoptosis speck-like protein containing a caspase-recruitment domain (-57%) and caspase-1 activity (-50%) in isolated cardiomyocytes and in the mouse heart (all P<0.05). Na2S also inhibited apoptosis (-38%) and necrosis (-43%) in cardiomyocytes in vitro and reduced myocardial infarct size (-63%) after ischemia/reperfusion injury in vivo (all P<0.05). These protective effects were absent in cells treated with the miR-21 eraser, antagomiR-21, and in miR-21 knockout mice. Na2S also limited the severity of inflammasome-dependent inflammation in the model of peritonitis (P<0.05) in wild-type but not in miR-21 knockout mice. Na2S induces cardioprotective effects through miR-21-dependent attenuation of ischemic and inflammatory injury in cardiomyocytes.

MicroRNA‐21 mediates hydrogen sulfide‐induced protection against ischemia/reperfusion injury in diabetic heart (1080.6)

Background: Hydrogen sulfide (H2S) protects against myocardial ischemia/reperfusion (I/R) and inflammatory injury in mice via induction of microRNA (miR)-21. Since the diabetic heart is non-compliant with most known cardioprotective strategies, we tested whether H2S would also induce miR-21 in the diabetic heart and protect against I/R injury. Methods and Results: Adult db/db mice were treated with vehicle (saline) or the H2S donor, Na2S, (100 µg/kg, iv) at the end of 30 min. of regional ischemia prior to reperfusion for 24 h. After reperfusion, left ventricular function was assessed by echocardiography and infarct size (IS) was measured by TTC staining. Mir-21 expression was assessed by qPCR. Na2S increased miR-21 in db/db mice (5.3-Fold, P<0.005 vs. control) and reduced IS (34±2%) as compared to control db/db mice (68±3%). To test the role of miR-21 in infarct reduction, adult C57BL or miR-21 KO mice were challenged with streptozotocin (STZ; 150 mg/kg, ip) to induce diabetes 4 weeks prior to subjecting them to I/R injury. Na2S reduced IS in STZ-C57BL mice, but not in STZ-miR-21 KO mice (Figure). Moreover, Na2S preserved fractional shortening (FS: 26±3%) post myocardial infarction in STZ-C57BL mice as compared to saline-treated STZ-C57BL mice (16±2%, P<0.05), but not in miR-21 KO-STZ mice (FS: 14±2%). Conclusion: Hydrogen sulfide reduces infarct size and preserves function in the post ischemic diabetic heart through miR-21-dependent pathway. We propose that H2S might be a novel strategy to up-regulate miR-21 for its therapeutic use against I/R injury in diabetics. Grant Funding Source: This research was funded by AHA (10SDG3770011) and VCU PRQF to FNS.

Effect of safflor yellow injection on inhibiting lipopolysaccharide-induced pulmonary inflammatory injury in mice

To observe the effect of Safflor Yellow (SY) Injection on acute lung injury (ALI) induced by lipopolysaccharide (LPS) in mice. Seventy-two mice were divided into six groups: control (saline + saline); LPS (LPS + saline); SY Injection [LPS + SY (10, 20 or 40 mg/kg, intravenously)] and anisodamine (AD) (LPS + AD). Thirty minutes after SY or AD administration, 15 mg/kg LPS was given intraperitoneally. All animals were sacrificed 4 h after LPS injection. Arterial blood gas and lung water content index (LWCI) were measured. Lung tissue myeloperoxidase (MPO) activity was assayed. mRNA expression of inflammatory cytokines was assayed by reverse-transcription polymerase chain reaction. Lung morphological and nuclear factor (NF)-κB p65-positive cell changes were observed by HE and immunohistochemical staining. p38 mitogen-activated protein kinase (MAPK) phosphorylation was observed by Western blotting. After LPS administration, all animals displayed increased arterial carbon dioxide partial pressure (PaCO2) and decreased arterial oxygen partial pressure (PaO2), arterial oxygen saturation (SO2), HCO3 (-) concentration and pH, and increased LWCI, MPO activity, interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α mRNA expression, NF-κB p65-positive staining and p38 MAPK activation compared with normal controls (all P<0.01). SY Injection significantly mitigated the LPS-induced increase in arterial PaCO and the decreases in arterial PaO2, SO2 and pH, and attenuated increases in LWCI and lung tissue MPO activity (all P<0.01). Moreover, SY Injection inhibited the increases in NF-κB p65 staining and in TNF-α, IL-1β and IL-6 mRNA expression (all P<0.01), and promoted the expression of the antiinflammatory cytokine IL-10 (P<0.05) following LPS injection. LPS-induced pulmonary p38 MAPK phosphorylation was suppressed by pretreatment with SY Injection (P<0.01). SY Injection ameliorates inflammatory ALI induced by LPS in mice.

An oligodeoxynucleotide capable of lessening acute lung inflammatory injury in mice infected by influenza virus

Infection of influenza virus could induce acute lung inflammatory injury (ALII) that was at least partially caused by excessive innate immune responses. To study whether down-regulating Toll-like receptor (TLR)-mediated innate immune response could lessen influenza virus-induced ALII, a microsatellite DNA mimicking oligodeoxynucleotide (MS ODN), named as SAT05f capable of inhibiting TLR7/9-activation in vitro, was used to treat mice infected with FM1 virus. In parallel, two MS ODNs confirmed with less or no in vitro activities, named as MS19 and MS33, were used as controls. Unexpectedly, SAT05f failed to lessen ALII in the mice, whereas MS19 significantly inhibited the weight loss and displayed dramatic effect on lessening the ALII by reducing consolidation, hemorrhage, intra-alveolar edema and neutrophils infiltration in lungs of the mice. Meanwhile, MS19 could decrease the mortality of influenza virus infected mice and down-regulate TNF-α production in their lungs. The data suggest that MS19 might display its therapeutic role on ALII induced by influenza virus by reducing over-production of TNF-α.

The ability of hydroxysafflor yellow a to attenuate lipopolysaccharide‐induced pulmonary inflammatory injury in mice

Hydroxysafflor yellow A (HSYA) is a component of the flower of Carthamus tinctorius L. The present study investigated whether HSYA could attenuate acute lung injury (ALI) induced by lipopolysaccharide (LPS) administration. Male Kunming mice were pretreated with HSYA 0.5 h prior to intraperitoneal application of LPS. Arterial blood gas, lung water content index, lung tissue myeloperoxidase (MPO) activity, mRNA expression of inflammatory cytokines, NF-κBp65, p38 mitogen-activated protein kinase (MAPK) and pathological changes in lung morphology were assessed. After LPS administration, all animals displayed increased arterial carbon dioxide partial pressure (PaCO₂), and decreased arterial oxygen partial pressure (PaO₂), arterial oxygen saturation (SO₂), HCO₃⁻ concentration and pH, which were ameliorated by pretreating the animals with HSYA. HSYA administration significantly attenuated inflammatory cell infiltration and alleviated pulmonary edema induced by LPS. Moreover, HSYA decreased NF-κB p65 nuclear translocation, inhibited proinflammatory cytokine TNF-α, IL-1β and IL-6 mRNA expression and promoted antiinflammatory cytokine IL-10 gene expression following LPS injection. Pulmonary p38 MAPK phosphorylation was upregulated 4 h after LPS treatment, which could be suppressed by pretreatment with HSYA. These findings demonstrated the protective effect of HSYA against LPS-induced acute lung injury, which is suggested to be associated with the inhibition of p38 MAPK, NF-κB p65 activation and alteration of inflammatory cytokine expression.