INFLUENCES OF HEAT STRESS ON GLUTAMATE TRANSMISSION-DEPENDENT EXPRESSION LEVELS OF IL-1ß and IL-18 IN BV-2 MICROGLIAL CELLS.

ABSTRACT: Objective: This study aimed to explore the impact of heat stress (HS) on glutamate transmission-dependent expression levels of interleukin-1ß (IL-1ß) and IL-18 in BV-2 microglial cells. Methods: BV-2 microglial cells were cultured in vitro , with cells maintained at 37°C serving as the control. The HS group experienced incubation at 40°C for 1 h, followed by further culturing at 37°C for 6 or 12 h. The experimental group was preincubated with glutamate, the glutamate antagonist riluzole, or the mGluR5 agonist, 2-chloro-5-hydroxyphenylglycine (CHPG), before HS. Glutamate content in BV-2 culture supernatant was assessed using colorimetric assay. Moreover, mRNA expression levels of EAAT3 and/or mGluR5 in BV-2 cells were determined via quantitative polymerase chain reaction. Interleukins (IL-1ß and IL-18) in cell culture supernatant were measured using enzyme-linked immunosorbent assay. Western blot analysis was employed to assess protein levels of IL-1ß and IL-18 in BV-2 cells. Results: HS induced a significant release of glutamate and increased the expression levels of mGluR5 and EAAT3 in BV-2 cells. It also triggered the expression levels and release of proinflammatory factors, such as IL-1ß and IL-18, synergizing with the effects of glutamate treatment. Preincubation with both riluzole and CHPG significantly reduced HS-induced glutamate release and mitigated the increased expression levels and release of IL-1ß and IL-18 induced by HS. Conclusion: The findings confirmed that microglia could be involved in HS primarily through glutamate metabolisms, influencing the expression levels and release of IL-1ß and IL-18.

BV2 Membrane-Coated PEGylated-Liposomes Delivered hFGF21 to Cortical and Hippocampal Microglia for Alzheimer's Disease Therapy.

Microglia-mediated inflammation is involved in the pathogenesis of Alzheimer's disease (AD), whereas human fibroblast growth factor 21 (hFGF21) has demonstrated the ability to regulate microglia activation in Parkinson's disease, indicating a potential therapeutic role in AD. However, challenges such as aggregation, rapid inactivation, and the blood-brain barrier hinder its effectiveness in treating AD. This study develops targeted delivery of hFGF21 to activated microglia using BV2 cell membrane-coated PEGylated liposomes (hFGF21@BCM-LIP), preserving the bioactivity of hFGF21. In vitro, hFGF21@BCM-LIP specifically targets Aß1-42-induced BV2 cells, with uptake hindered by anti-VCAM-1 antibody, indicating the importance of VCAM-1 and integrin α4/ß1 interaction in targeted delivery to BV2 cells. In vivo, following subcutaneous injection near the lymph nodes of the neck, hFGF21@BCM-LIP diffuses into lymph nodes and distributes along the meningeal lymphatic vasculature and brain parenchyma in amyloid-beta (Aß1-42)-induced mice. Furthermore, the administration of hFGF21@BCM-LIP to activated microglia improves cognitive deficits caused by Aß1-42 and reduces levels of tau, p-Tau, and BACE1. It also decreases interleukin-6  (IL-6) and tumor necrosis factor-α (TNF-α) release while increasing interleukin-10 (IL-10) release both in vivo and in vitro. These results indicate that hFGF21@BCM-LIP can be a promising treatment for AD, by effectively crossing the blood-brain barrier and targeting delivery to brain microglia via the neck-meningeal lymphatic vasculature-brain parenchyma pathways.

Intradermal injection of icariin-HP-ß-cyclodextrin improved traumatic brain injury via the trigeminal epineurium-brain dura pathway.

The lower bioavailability after oral administration limited icariin applications in central nervous system. Icariin/HP-ß-cyclodextrin (HP-ß-CD) inclusion complex was prepared for acute severe opening traumatic brain injury (TBI) via facial intradermal (i.d.) in the mystacial pad. After fluid percussion-induced TBI, icariin/HP-ß-CD at 0.4 mg/kg i.d. preserved more neurons and oligodendrocytes than intranasal injection (i.n.) or intravenous injection via tail vein (i.v.) and decreased microglia and astrocyte activation. Icariin/HP-ß-CD i.d. reduced apoptosis in cortical penumbra while i.n. and i.v. showed weak or no effects. Icariin/HP-ß-CD i.d. reduced Evans blue leakage and altered CD34, ZO-1, Claudin-5, and beta-catenin expression after TBI. Moreover, icariin/HP-ß-CD promoted human umbilical vein endothelial cells proliferation. Thus, Icariin/HP-ß-CD i.d. improved TBI, including blood-brain barrier opening. Fluorescein 5-isothiocyanate (FITC) and 3,3'-Dioctadecyloxacarbocyanine perchlorate (DiOC18(3)) mimic HP-ß-CD and icariin respectively. FITC and DiOC18(3) were similarly delivered to trigeminal epineurium, perineurium and perivascular spaces or tissues, caudal dura mater, and scattered in trigeminal fasciculus, indicating that icariin/HP-ß-CD was delivered to the brain via trigeminal nerve-dura mater-brain pathways. In sum, intradermal injection in mystacial pad might deliver icariin/HP-ß-CD to the brain and icariin/HP-ß-CD improved acute severe opening TBI.

The involvement of perivascular spaces or tissues in the facial intradermal brain-targeted delivery.

Our previous work indicates the lymphatic network and perivascular spaces or tissues might be involved in the facial intradermal brain-targeted delivery of Evans blue (EB). In this article, we presented the detailed involvement of both, and the linkage between lymphatic network and perivascular spaces or tissues. The in-vivo imaging, the trigeminal transection and immunohistochemistry were used. In-vivo imaging indicated intradermal injection in the mystacial pad (i.d.) delivered EB into the brain at 2-, 6- and 24 h, while intranasal injection (i.n.) delivered EB into the rostral head and intravenous injection (i.v.) diffused EB weakly into the brain. Trigeminal perineurial and epineurial EB occurred along the perivascular spaces or tissues and along brain vessels. EB diffused into the lymphatic vessels and submandibular lymph nodes. Moreover, perineurial and epineurial EB co-located or overlaid with Lyve1 immuno-reactivity and VEGF antibody, and lymphatic network connected with perivascular spaces or tissues, suggesting lymphatic system-perivascular spaces might involve in the EB delivery with i.d. The trigeminal transection reduced the trigeminal epineurial and perineurial EB and brain EB along vessels. EB diffused in the fasciculus and the perineurium, blood and lymphatic vessels in the mystacial pad, mystacial EB overlaid VEGF or Lyve1 antibody. In summary, the dermal-trigeminal-brain perivascular spaces or tissues and the linkage to the lymphatic network mediated the intradermal brain-targeted delivery.

A strategy for bypassing the blood-brain barrier: Facial intradermal brain-targeted delivery via the trigeminal nerve.

Although intranasal delivery bypasses the blood-brain barrier (BBB), the anatomical location of the olfactory mucosa and respiratory airflow interference lead to less brain-targeted drug delivery. In addition to intranasal delivery, evidence indicates that facial intradermal injection might be a novel strategy for bypassing the BBB via the trigeminal nerve (TN). The hypothesis was verified by pharmacokinetic evaluation, nasal injury, lymphatic vessels inhibition and immunohistochemistry. Intradermal injection into the rat mystacial pad (i.d.) elevated the brain sub-areas and trigeminal Evans Blue (EB) concentrations, Cmax and AUC(0-t). I.d. also increased them in brain sub-areas beyond those of intranasal (i.n.) and intravenous injection (i.v.), especially the pons varolii and the medulla oblongata (sub-areas associated with TN). I.d. injection increased the brain drug targeting efficiency, brain direct transport percentage and brain bioavailability of EB while i.n. injection altered them slightly. Trigeminal transection and nasal injury reduced trigeminal EB with i.d. administration. Trigeminal perineurium, epineurium, perivascular spaces, neurons and Schwann cells were involved in the EB brain-targeted delivery. The lymphatic system mediated EB diffusion from the mystacial pad to the nasal mucosa and the brain. Thus, facial intradermal injection might be a promising strategy for brain-targeting delivery, bypassing the BBB via the trigeminal substructures.

Regulation of Caveolin-1 and Junction Proteins by bFGF Contributes to the Integrity of Blood-Spinal Cord Barrier and Functional Recovery.

The blood-spinal cord barrier (BSCB) plays important roles in the recovery of spinal cord injury (SCI), and caveolin-1 is essential for the integrity and permeability of barriers. Basic fibroblast growth factor (bFGF) is an important neuroprotective protein and contributes to the survival of neuronal cells. This study was designed to investigate whether bFGF is beneficial for the maintenance of junction proteins and the integrity of the BSCB to identify the relations with caveolin-1 regulation. We examined the integrity of the BSCB with Evans blue dye and fluorescein isothiocyanate-dextran extravasation, measured the junction proteins and matrix metalloproteinases, and evaluated the locomotor function recovery. Our data indicated that bFGF treatment improved the recovery of BSCB and functional locomotion in contusive SCI model rats, reduced the expression and activation of matrix metalloproteinase-9, and increased the expressions of caveolin-1 and junction proteins, including occludin, claudin-5, p120-catenin, and ß-catenin. In the brain, in microvascular endothelial cells, bFGF treatment increased the levels of junction proteins, caveolin-1 small interfering RNA abolished the protective effect of bFGF under oxygen-glucose deprivation conditions, and the expression of fibroblast growth factor receptor 1 and co-localization with caveolin-1 decreased significantly, which could not be reversed by bFGF treatment. These findings provide a novel mechanism underlying the beneficial effects of bFGF on the BSCB and recovery of SCI, especially the regulation of caveolin-1.

Intranasal delivery of bFGF with nanoliposomes enhances in vivo neuroprotection and neural injury recovery in a rodent stroke model.

Basic fibroblast growth factor (bFGF) may protect stroke patients from cerebral ischemia-reperfusion (I/R) injury. In this study, we report the intranasal use of novel nanoliposomes for the brain delivery of bFGF in a rat model of cerebral I/R. Compared with free bFGF, nanoliposomal therapy was able to significantly improve bFGF accumulation in brain tissues (p<0.05) including the most affected ischemic penumbra regions (e.g. hippocampus, pallium). After intranasal bFGF-nanoliposomal treatment for 3 consecutive days, functional recovery as indicated by improved neurologic deficit score and spontaneous locomotor activity was observed, and the stroke infarct volume was nearly halved (p<0.001) which persisted after 21days. These neuroprotective effects could be blocked by the PI3-K/Akt inhibitor LY294002, indicating the involvement of PI3-K/Akt activation in the therapeutic action. Overall, our results support the intranasal use of nanoliposomal bFGF as an efficient, non-invasive means to bypass the blood-brain barrier for ischemic stroke treatment.

Theanine enhanced both the toxicity of strychnine and anticonvulsion of pentobarbital sodium.

CONTEXT: Theanine, an additive, holds several effects on the central nervous system without toxicity and affects CNS drugs. Theanine bilaterally alters ß wave of the EEG with or without caffeine and pentobarbital-induced locomotor activity. Theanine also enhances hypnosis of pentobarbital sodium (PB) and antidepression of midazolam, suggesting there are complicated interactions between theanine and CNS drugs. On the other side, theanine induces glycine release. Glycine potentiates the strychnine toxicity via NMDA receptor activation. Moreover, PB facilitates GABAA receptor activation by GABA, and it is commonly prescribed for strychnine poison. However, what the role that theanine plays in the anticonvulsion of PB against strychnine poison is still unknown. MATERIALS AND METHODS: Theanine, pentobarbital sodium or strychnine was injected intraperitoneally. EEG was monitored by BIOPAC 16 EEG amplifiers. LD50 of strychnine and hypnotic ED50 of pentobarbital sodium with or without theanine for mice were tested according to Bliss' case. RESULTS: (1) Theanine enhanced the strychnine toxicity. Both theanine and strychnine 1.0 mg/kg increased the power of the ß wave. Theanine aggravated that of strychnine 1.0 mg/kg. Theanine attenuated the LD50 of strychnine. (2) Theanine enhanced the anticonvulsion of PB. Theanine increased the power of α, ß wave and decreased hypnotic ED50 of PB; PB attenuated strychnine-induced EEG excitation and mortality with or without theanine, and theanine enhanced the effects of PB. Further, theanine enhanced the anticonvulsion of PB dose-dependently against the strychnine toxicity but not the lethal toxicity of strychnine. DISCUSSION AND CONCLUSIONS: These results indicated theanine interacted with PB and strychnine. Theanine enhanced both the strychnine toxicity and anticonvulsion of PB against strychnine poison.

bFGF Protects Against Blood-Brain Barrier Damage Through Junction Protein Regulation via PI3K-Akt-Rac1 Pathway Following Traumatic Brain Injury.

Many traumatic brain injury (TBI) survivors sustain neurological disability and cognitive impairments due to the lack of defined therapies to reduce TBI-induced blood-brain barrier (BBB) breakdown. Exogenous basic fibroblast growth factor (bFGF) has been shown to have neuroprotective function in brain injury. The present study therefore investigates the beneficial effects of bFGF on the BBB after TBI and the underlying mechanisms. In this study, we demonstrate that bFGF reduces neurofunctional deficits and preserves BBB integrity in a mouse model of TBI. bFGF suppresses RhoA and upregulates tight junction proteins, thereby mitigating BBB breakdown. In vitro, bFGF exerts a protective effect on BBB by upregulating tight junction proteins claudin-5, occludin, zonula occludens-1, p120-catenin, and ß-catenin under oxygen glucose deprivation/reoxygenation (OGD) in human brain microvascular endothelial cells (HBMECs). Both the in vivo and in vitro effects are related to the activation of the downstream signaling pathway, PI3K/Akt/Rac-1. Inhibition of the PI3K/Akt or Rac-1 by specific inhibitors LY294002 or si-Rac-1, respectively, partially reduces the protective effect of bFGF on BBB integrity. Overall, our results indicate that the protective role of bFGF on BBB involves the regulation of tight junction proteins and RhoA in the TBI model and OGD-induced HBMECs injury, and that activation of the PI3K/Akt /Rac-1 signaling pathway underlies these effects.

Prevent diabetic cardiomyopathy in diabetic rats by combined therapy of aFGF-loaded nanoparticles and ultrasound-targeted microbubble destruction technique.

Acidic fibroblast growth factor (aFGF) has shown the great potential to prevent the structural and functional injuries caused by diabetic cardiomyopathy (DCM). The present study sought to investigate the preclinical performance and mechanism of the combination therapy of aFGF-nanoparticles (aFGF-NP) and ultrasound-targeted microbubble destruction (UTMD) technique for DCM prevention. From Mason staining and TUNEL staining, aFGF-NP+UTMD group showed significant differences from the diabetes group and other groups treated with aFGF or aFGF-NP. The cardiac collagen volume fraction (CVF) and cardiac myocyte apoptosis index in aFGF-NP+UTMD group reduced to 4.15% and 2.31% respectively, compared with those in the diabetes group (20.5% and 11.3% respectively). Myocardial microvascular density (MCD) in aFGF-NP+UTMD group was up to 35n/hpf, much higher than that in the diabetes group (14n/hpf). The diabetes group showed similar results (MCD, CVF and cardiac myocyte apoptosis index) to other aFGF treatment groups (free aFGF±UTMD or aFGF-NP). Indexes from transthoracic echocardiography and hemodynamic evaluation also proved the same conclusion. These results confirmed that the abnormalities including diastolic dysfunctions, myocardial fibrosis and metabolic could be suppressed by the different extents of twice weekly aFGF treatments for 12 consecutive weeks (free aFGF or aFGF-NP±UTMD), with the strongest improvements observed in the aFGF-NP+UTMD group. Western blot and immunohistochemical analyses of heart tissue samples further revealed the high efficiency of heart-targeted delivery and effective cardioprotection with this combination approach. Overall, this study has generated supportive data that are critical for the translation of a promising DCM prevention strategy.

Pharmacokinetics and tissue distribution profile of icariin propylene glycol-liposome intraperitoneal injection in mice.

OBJECTIVES: The pharmacokinetics and tissue distribution of icariin propylene glycol-liposome suspension (ICA-PG-liposomes) have been investigated. METHODS: ICA-PG-liposomes or ICA-PG-solution were prepared and intraperitoneally injected to mice. Morphology and size distribution of ICA-PG-liposomes were observed by transmission electron microscopy (TEM) and laser particle sizer. Plasma and tissues were collected at different times after intraperitoneal injection and icariin concentrations were determined by HPLC. KEY FINDINGS: From TEM, ICA-PG-liposomes showed spherical vesicles with a mean particle size of 182.4 nm. The encapsulation efficiency of ICA-PG-liposomes reached 92.6%. Pharmacokinetics of ICA-PG-liposomes displayed the three open compartments model. ICA-PG-liposomes enhanced icariin absorption from the abdominal cavity, prolonged mean retention time (MRT((0-t))), increased area under curve (AUC((0-t))) and maximum concentration in plasma. Compared with ICA-PG-solution, ICA-PG-liposomes resulted in larger amounts of icariin being distributed into spleen (60.38% total icariin), liver (16.68%), lung (6.21%), kidney (4.64%), heart (1.43%) and brain (1.83%). AUC((0-t)) values in most tissues (except lung) of mice administered ICA-PG-liposomes were higher than those administered ICA-PG-solution, while Clearance in most tissues (except brain and lung) decreased. The MRT((0-t)) values of ICA-PG-liposomes in all tissues and half lives of most tissues (except brain) were prolonged. From Targeted efficiency and relative uptake data, the spleen was the target tissue of the ICA-PG-liposomes. CONCLUSIONS: ICA-PG-liposomes changed the pharmacokinetic behaviour and enhanced icariin distribution in tissues. With nanometer size, high encapsulation efficiency and improved pharmacokinetics, ICA-PG-liposomes might be developed as promising carriers for icariin injection.

A novel mono-carbonyl analogue of curcumin induces apoptosis in ovarian carcinoma cells via endoplasmic reticulum stress and reactive oxygen species production.

The aim of the present study was to investigate the apoptosis of human ovarian cancer cell lines, A2780 and CP70, induced by a novel curcumin analogue, B19. The proliferation of cells was detected with methyl thiazolyl tetrazolium (MTT) assay and apoptosis was examined by flow cytometry. Reactive oxygen species (ROS) were assessed by the fluorescent indicator DCF-DA. The protein expression of the endoplasmic reticulum (ER) stress pathways, GRP78, XBP-1, ATF-4 and CHOP, was examined with Western blotting. A growth inhibitory effect was observed after treatment with B19 in a dose-dependent manner and with more potential than curcumin. At 20 µM, B19 induced significant apoptosis in CP70 cells. Furthermore, B19 induced the ER stress response, while curcumin had no effect on ER stress. These results suggest that B19 has more effective antitumor properties than curcumin, and is associated with the activation of ER stress and ROS in ovarian cancer cells.

[The study of effects of pirfenidone on the pulmonary fibrosis induced by paraquat in mice].

OBJECTIVE: To study the curative effects of pirfenidone (PF) on pulmonary fibrosis induced by paraquat (PQ) in mice and to provide the theoretical basis for clinical treatment. METHODS: Ninety adult healthy male ICR mice were randomly divided into six groups: control group, PQ group, 2 mg/kg Dexamethasone group, 25 mg/kg PF group, 50 mg/kg PF group and 100 mg/kg PF group, there were 15 mice in each group. The corresponding volume of normal saline was given to the each mouse in control group according to the weight, after 2 h 0.1% CMC was given to the each mouse of control group one time by intragastric administration, then the CMC was administrated at regular time until sacrifice. All mice for other 5 groups were exposed to 100 mg/kg PQ by intragastric administration. At 2 h after exposure to PQ, 0.02 ml/10 g dexamethasone and 25, 50, 100 mg/kg PF were given to mice for dexamethasone group and for 3 PF groups by intragastric administration each day for 49 days, respectively. The lung coefficient was calculated and pathological changes of lung tissue were observed by HE staining for each mouse. The hydroxyproline (HYP) level in lung tissue was measured for each mouse. The mRNA level of and the protein level of TGF-ß(1) in lung tissue for each mouse were determined, and the protein level of TGF-ß(1) in the bronchus-alveolus lavage fluid (BALF) of each mouse was detected. RESULTS: The survival rates on the 3rd day in PQ group, 3 PF groups and dexamethasone group were 53.33%, 46.67%, 73.33%, 86.67% and 80%, respectively. The survival rates on the 3rd day in dexamethasone group, 50 mg/kg and 100 mg/kg PF groups were significantly higher than those of PQ group and 25 mg/kg PF group (P < 0.05). The lung coefficients of 3 PF groups were significantly lower than that of the PQ group (P < 0.05). The lung tissue HYP levels of dexamethasone group and 3 PF groups were 50.95 ± 11.65, 44.52 ± 9.48, 43.27 ± 6.01 and 40.82 ± 5.90 mg/g respectively, which were significantly lower than that (74.27 ± 3.68) of PQ group (P < 0.01). The TGF-ß(1) protein levels of BALF in dexamethasone group, 50 and 100 mg/kg PF groups were 22.03 ± 7.27, 27.75 ± 5.84 and 21.31 ± 6.82 ng/ml respectively, which were significantly lower than that (52.52 ± 15.51) ng/ml of PQ group (P < 0.01) The expression level of TGF-ß(1) mRNA in 100 mg/kg PF group decreased significantly, as compared with PQ group (P < 0.01). CONCLUSION: PF could reduce the collagen deposition and pulmonary fibrosis induced by PQ in mice lungs.