Methylglyoxal-induced neuroinflammatory response in in vitro astrocytic cultures and hippocampus of experimental animals.

Diabetes mellitus is characterized by chronic hyperglycemia and its diverse complications. Hyperglycemia is associated with inflammatory responses in different organs and diabetic patients have a higher risk of developing neurodegenerative disorders. Methylglyoxal is a reactive advanced glycation end product precursor that accumulates in diabetic patients. It induces various stress responses in the central nervous system and causes neuronal dysfunction. Astrocytes are actively involved in maintaining neuronal homeostasis and possibly play a role in protecting the brain against neurodegeneration. However it is not clear whether methylglyoxal exerts any adverse effects towards these astrocytes. In the present study we investigated the effects of methylglyoxal in astrocytic cultures and hippocampi of experimental animals. The cells from the astrocytic line DITNC1 were treated with methylglyoxal for 1 to 24 h. For the in vivo model, 3 months old C57BL/6 mice were treated with methylglyoxal solution for 6 weeks by intraperitoneal injection. Following the treatment, both astrocytes and hippocampi were harvested for MTT assay, Western blot and real time PCR analyses. We found that methylglyoxal induced astrogliosis in DITNC1 astrocytic cultures and C57BL/6 mice. Further, activation of the pro-inflammatory JNK signaling pathway and its downstream effectors c-Jun were observed. Furthermore, increased gene expression of pro-inflammatory cytokines and astrocytic markers were observed from real time PCR analyses. In addition, inhibition of JNK activities resulted in down-regulation of TNF-α gene expression in methylglyoxal treated astrocytes. Our results suggest that methylglyoxal may contribute to the progression of diabetes related neurodegeneration through JNK pathway activation in astrocytes and the subsequent neuroinflammatory responses in the central nervous system.

Brij-functionalized chitosan nanocarrier system enhances the intestinal permeability of P-glycoprotein substrate-like drugs.

The highly expressed P-glycoprotein (Pgp) in the intestine plays a key role in preventing drugs across the intestinal epithelium, which linked by tight junctions (TJs). Thus increasing the oral bioavailability of Pgp substrate-like drugs (PSLDs) remains a great challenge. Herein, we construct a nanocarrier system derived from Brij-grafted-chitosan (BC) to enhance the oral bioavailability and therapeutic effect of berberine (BBR, a typical PLSD) against diabetic kidney disease. The developed BC nanoparticles (BC-NPs) are demonstrated to improve the intestinal permeability of BBR via transiently and reversibly modulating the intercellular TJs (paracellular pathway) and Pgp-mediated drug efflux (transcellular pathway). As compared to free BBR and chitosan nanoparticles, the BC-NPs enhanced the relative oral bioavailability of BBR in rats (4.4- and 2.7-fold, respectively), and the therapeutic potency of BBR in renal function and histopathology. In summary, such strategy may provide an effective nanocarrier system for oral delivery of BBR and PSLDs.

Brij-grafted-chitosan copolymers with function of P-glycoprotein modulation: Synthesis, characterization and in vitro investigations.

Chitosan (CS), a nature-derived polysaccharide, is a promising nano-carrier material with good biocompatibility and biodegradability. However, the biomedical applications of CS are hindered because of its poor aqueous solubility. To circumvent this drawback, a series of Brij-grafted-chitosan copolymers (BCs) with various grafting degree of Brij-S20 were first developed and reported. The results indicated that the water solubility of BCs (from 9.13 to 9.54 mg/mL) were much higher than that of CS (0.32 mg/mL), due to the broken intra- and/or inter-molecular hydrogen bonds and the decreased initial crystallinity in BCs. The amphiphilic structure of BCs presented lower critical micelle concentration (0.116-0.376 mg/mL) thus facilitating its self-aggregation into micelles for drug encapsulation. Moreover, BCs markedly enhanced the intracellular uptake of rhodamine-123 in MDCK-MDR1 cells. This inhibition on Pgp-mediated efflux was also confirmed by confocal microscopy. In conclusion, BCs could be further developed as polymeric nano-carriers for those drugs with Pgp-mediated efflux.

Ginsenosides attenuate methylglyoxal-induced impairment of insulin signaling and subsequent apoptosis in primary astrocytes.

Diabetes mellitus (DM), which is characterized by chronic hyperglycemia, is known to increase the risk of neurodegeneration. In type 2 diabetes, hyperglycemia could cause insulin resistance and neurodegeneration in various cells including neurons and astrocytes. Hyperglycemia is also known to result in the formation of advanced glycation end-products (AGE) Methylglyoxal (MG) is one of the most reactive AGE precursors in which its abnormal accumulation is usually found in diabetic patients and induces neuronal cell death in central nervous system. Ginseng is a herb that has been widely used to treat various diseases in traditional Chinese medicine. Ginsenosides, the pharmacologically active component isolated from ginseng, have been shown to have cryoprotective effects in different neural cells. In the present study we investigated the effects of MG in disturbing insulin signaling and leading to further cellular apoptosis in rat primary astrocytes. Furthermore, the protective effects of different subtypes of ginsenosides were studied. From the results, impairment of insulin signaling was found in astrocytes under MG treatment. Moreover, cleavage of caspase and Poly ADP ribose polymerase (PARP) was observed in line with insulin signaling disruption, showing the neurotoxic effects of MG towards astrocytes. The effects of ginsenosides in MG treated astrocytes were also investigated. After treatment, ginsenosides Rd and R-Rh2 were shown to ameliorate the cell viability of MG-treated astrocytes. In addition, Rd and R-Rh2 could improve insulin signaling and inhibit apoptosis, indicating that Rd, R-Rh2 and related compounds may have therapeutic potential in treating diabetes-induced neurodegeneration.

Modulation of endoplasmic reticulum chaperone GRP78 by high glucose in hippocampus of streptozotocin-induced diabetic mice and C6 astrocytic cells.

Diabetes mellitus is known to increase the risk of neurodegeneration, and both diseases are reported to be linked to dysfunction of endoplasmic reticulum (ER). Astrocytes are important in the defense mechanism of central nervous system (CNS), with great ability of tolerating accumulation of toxic substances and sensitivity in Ca(2+) homeostasis which are two key functions of ER. Here, we investigated the modulation of the glucose-regulated protein 78 (GRP78) in streptozotocin (STZ)-induced diabetic mice and C6 cells cultured in high glucose condition. Our results showed that more reactive astrocytes were presented in the hippocampus of STZ-induced diabetic mice. Simultaneously, decrease of GRP78 expression was found in the astrocytes of diabetic mice hippocampus. In in vitro study, C6 cells were treated with high glucose to investigate the role of high glucose in GRP78 modulation in astrocytic cells. GRP78 as well as other chaperones like GRP94, calreticulin and calnexin, transcription levels were down-regulated after high glucose treatment. Also C6 cells challenged with 48h high glucose were activated, as indicated by increased level of glial fibrillary acidic protein (GFAP). Activated C6 cells simultaneously exhibited significant decrease of GRP78 level and was followed by reduced phosphorylation of Akt. Moreover, unfolded protein response was induced as an early event, which was marked by the induction of CHOP with high glucose treatment, followed by the reduction of GRP78 after 48h. Finally, the upsurge of ROS production was found in high glucose treated C6 cells and chelation of ROS could partially restore the GRP78 expression. Taken together, these data provide evidences that high glucose induced astrocytic activation in both in vivo and in vitro diabetic models, in which modulation of GRP78 would be an important event in this activation.

Neuroprotective effects of neurokinin receptor one in dopaminergic neurons are mediated through Akt/PKB cell signaling pathway.

Neurokinin one (NK1) receptor is Substance P (SP) receptor and it is abundantly distributed in the basal ganglia. Growing evidences were shown on their possible roles in the pathogenesis and treatment of Parkinson's disease (PD). NK1 receptor is a kind of G-protein-coupled-receptor (GPCR) and it links to various downstream survival signaling pathways. In the present study, treatment of NK1 receptor agonist septide [(Pyr6, Pro9)-SP (6-11)] was found to ameliorate the motor deficit in 6-hydroxydopamine (6-OHDA) lesioned rats in apomorphine rotation test. Septide treatments were also demonstrated to provide neuroprotection. In 6-OHDA lesioned rats, protection of TH immunoreactive neurons and terminals in substantia nigra (SN) and striatum was found after septide treatment. In SH-SY5Y cultures, cytotoxicity of 6-OHDA was reduced by septide pretreatment. In addition, up-regulations of phosphorylated serine-threonine kinase Akt and phosphorylated mitochondrial apoptotic protein BAD were observed in both in vivo and in vitro models, indicating the inhibition of apoptotic pathway by septide. In conclusion, septide could trigger the pro-survival Akt/PKB signaling pathway and protect dopaminergic neurons in in vivo and in vitro models against 6-OHDA toxicity. Therefore septide treatment may have therapeutic implications in treatment of PD.