Protective effects and regulatory mechanisms of melatonin in a neonatal mouse model of LPS-induced inflammation.

This experiment mainly explored the protective effect and regulatory mechanism of melatonin (MEL) through its receptor on central nervous system (CNS) inflammation induced by lipopolysaccharide (LPS). The experiment was first divided into the following four groups: control group (CTRL group), LPS-induced inflammation model group (LPS group), LPS-treated MEL group (LPS + MEL group), and MEL administration group (MEL group). Later, a luzindole-antagonized LPS-MEL cotreatment group (LPS + MEL + LUZ group) was added to clarify the experimental results. ELISA was used to determine the inflammatory factor levels IL-6, IL-1ß, and IL-10 in brain slices. Western blotting was used to determine the expression levels of the microglia-specific protein CD11b and melatonin receptors MT1 and MT2 in brain slices. A large amount of IL-6 release and increased expression of CD11b protein were detected 24 h after inflammatory stimulation, while pretreatment with MEL inhibited the release of IL-6 and increased the expression of CD11b. At the same time, LPS induction downregulated the relative protein expression levels of MT1 and MT2. In addition, compared with the CTRL group and the LPS + MEL group, the administration of LUZ inhibited the protein expression of MT1. It increased the release of IL-1ß and IL-10, further indicating that MEL can alleviate LPS-induced neuroinflammation through the MT1 response. In short, MEL can reduce the neuroinflammatory response induced by LPS and exhibit related protective effects through MT1.

Protective Role of Grape Seed Proanthocyanidins Against Ccl4 Induced Acute Liver Injury in Mice.

BACKGROUND: We investigated the effect of grape seed proanthocyanidins (GSPs) on carbon tetrachloride (CCl4)-induced acute liver injury. MATERIAL/METHODS: Sixty SPF KM mice were randomly divided into 6 groups: the control group, CCl4-model group, bifendate group (DDB group), and low-, moderate-, and high-dose GSP groups. The following parameters were measured: serum levels of alanine aminotransferase (ALT); aspartate aminotransferase (AST); tumor necrosis factor (TNF)-α; interleukin-6 (IL-6); high-mobility group box (HMGB)-1; body weight; liver, spleen, and thymus indexes; superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity; HMGB1 mRNA; malondialdehyde (MDA) content; hepatocyte proliferation; and changes in liver histology. RESULTS: Compared to the CCl4-model group, decreases in liver index and increases in thymus index significantly increased SOD and GSH-Px activities and reduced MDA content, and higher hepatocyte proliferative activity was found in all GSP dose groups and the DDB group (all P<0.001). Compared with the CCl4-model group, serum TNF-α and IL-6 levels and HMGB 1 mRNA and protein expressions decreased significantly in the high GSP dose group (all P<0.05). CONCLUSIONS: Our results provide strong evidence that administration of GSPs might confer significant protection against CCl4-induced acute liver injury in mice.

Construction and functional activity of a recombinant vector expressing rat glutamic acid decarboxylase 65.

OBJECTIVE: Glutamic acid decarboxylase 2 (GAD65) is a gamma-aminobutyric acid (GABA) synthetase. This study aimed to construct a recombinant lentivirus-rGAD65 (rLV-rGAD65) vector containing the cDNA of rat GAD65 (rGAD65) and assess its functional activity in vitro and in vivo. METHODS: cDNA of rGAD65 was amplified by RT-PCR and subcloned into the LV vector, forming the rLV-GFP-rGAD65 plasmid. The recombinant lentivirus particles (rLV-rGAD65) were packaged by the LV Helper-Free System and the titer was measured. Primary rat lung fibroblasts were transfected with rLV-rGAD65. The expression of rGAD65 in fibroblasts was detected by immunocytochemistry and western blot and the level of GABA in the medium was assessed by high-performance liquid chromatograph (HPLC). In vivo, rLV-rGAD65 was injected into the subthalamic nucleus (STN) of Sprague-Dawley rats using stereotaxic methods, and rGAD65 protein levels in the STN were assessed by immunohistochemistry and Western blot, while the GABA concentration in the substantia nigra pars reticulata (SNr) was assayed by HPLC. RESULTS: The sequence of rGAD65 cDNA was in accord with that in GenBank. The amino-acid sequence of rGAD65 had no mutations and the titer of rLV-rGAD65 reached 6.8 × 108/mL. The efficiency of infection of fibroblasts was 80%, and the concentration of GABA in the medium was (48.14 +/- 9.35) nmol/L. In vivo, rGAD65 expression was detected in the STN, and the concentration of GABA in the SNr increased from (5.95 +/- 1.09) to (12.44 +/- 3.79) nmol/g tissue. CONCLUSION: The recombinant LV-GFP-rGAD65 vector was successfully constructed. rLV-rGAD65-infected primary fibroblasts in vitro and the expressed rGAD65 catalyzed the formation of GABA from glutamic acid. In vivo, the concentration of GABA in the SNr was increased after rLV-rGAD65 injection into the STN.