Jian Fei

Anti-inflammatory activity of curcumin-loaded solid lipid nanoparticles in IL-1β transgenic mice subjected to the lipopolysaccharide-induced sepsis

Sepsis is a significant public healthcare problem, affecting millions of people worldwide each year, killing one in four, and increasing in incidence. Thus, advanced therapeutic strategies are required to treat sepsis patients. Curcumin (Cur) is a promising anti-inflammatory agent for various inflammatory disorders. However, the therapeutic efficacy of Cur is limited due to poor aqueous solubility, rapid degradation, and low bioavailability. The aims of this study were to evaluate the therapeutic potential of Cur-loaded solid lipid nanoparticles (Cur-SLNs) for sepsis treatment. A firefly luciferase transgenic mouse was used to monitor real time interleukin 1β (IL-1β) expression in lipopolysaccharide (LPS)-induced sepsis model to examine the protective effect of Cur-SLNs, and to elucidate its underlying molecular mechanisms. Mice (female or male) were intraperitoneally administered with free Cur or Cur-SLNs (30xa0mg/kg) before the intraperitoneal delivery of LPS (3xa0mg/kg). Our results indicated that Cur-SLNs can effectively reduced levels of IL-1β expression compared to free Cur, especially at 3xa0h after LPS injection. Also, Cur-SLNs significantly decreased the expression of serum pro-inflammatory cytokines, including IL-6, TNF-α, and IL-1β as compared with free Cur, but augmented anti-inflammatory cytokine IL-10 by ELISA assay. Further, marked alleviation of the sepsis-induced damage to organs, including kidney, liver, and heart was observed with Cur-SLNs treatment as determined by hematoxylin/eosin-staining. Western blot analyses revealed that Cur-SLNs can significantly lower the expression levels of TLR4, TLR2, and TNF-α in lymph node tissues. Meanwhile, it showed suppressions of NF-κB activation and IκBα degradation levels. In conclusion, we suggested that Cur-SLNs may be used as an effective and safe therapeutic agent in treating sepsis in high-risk patient groups.

Biocompatibility of silk-tropoelastin protein polymers

Blended polymers are used extensively in many critical medical conditions as components of permanently implanted devices. Hybrid protein polymers containing recombinant human tropoelastin and silk fibroin have favorable characteristics as implantable scaffolds in terms of mechanical and biological properties. A firefly luciferase transgenic mouse model was used to monitor real-time IL-1β production localized to the site of biomaterial implantation, to observe the acute immune response (up to 5 days) to these materials. Significantly reduced levels of IL-1β were observed in silk/tropoelastin implants compared to control silk only implants at 1, 2 and 3 days post-surgery. Subsequently, mice (n = 9) were euthanized at 10 days (10D) and 3 weeks (3W) post-surgery to assess inflammatory cell infiltration and collagen deposition, using histopathology and immunohistochemistry. Compared to control silk only implants, fewer total inflammatory cells were found in silk/tropoelastin (∼29% at 10D and ∼47% at 3W). Also fewer ingrowth cells (∼42% at 10D and ∼63% at 3W) were observed within the silk/tropoelastin implants compared to silk only. Lower IL-6 (∼52%) and MMP-2 (∼84%) (pro-inflammatory) were also detected for silk/tropoelastin at 10 days. After 3 weeks implantation, reduced neovascularization (vWF ∼43%), fewer proliferating cells (Ki67 ∼58% and PCNA ∼41%), macrophages (F4/80 ∼64%), lower IL-10 (∼47%) and MMP-9 (∼55%) were also observed in silk/tropoelastin materials compared to silk only. Together, these results suggest that incorporation of tropoelastin improves on the established biocompatibility of silk fibroin, uniquely measured here as a reduced foreign body inflammatory response.

Lyzl4, a novel mouse sperm-related protein, is involved in fertilization

The role of Chicken-type (c-type) lysozyme, a prototype lysozyme, in immunity has been characterized in many organisms. In this study, we cloned a novel c-type lysozyme-like gene, Lyzl4, which was located on mouse chromosome 9F4 and encoded 145 amino acids with a putative signal peptide and a protease cleavage site. The mature recombinant Lyzl4 protein expressed in yeast did not show the bacteriolytic activity. Sequence alignment analysis demonstrated that 3 of the 20 invariant residues in c-type lysozymes were changed in Lyzl4. One of the changed amino acids (D52G) is located in the catalytic domain. Lyzl4 mRNA was selectively expressed in testis and epididymis in adult mice, with varying expression level across different developmental stages. High level of Lyzl4 protein was found on the spermatozoa of acrosomal region and principal piece of tail. Immuno-neutralization of Lyzl4 protein in spermatozoa with its specific antibody significantly decreased in vitro fertilization percentage in a dose-dependent manner, suggesting that Lyzl4 might be important for fertilization.

Chimonanthus nitens var. salicifolius Aqueous Extract Protects against 5-Fluorouracil Induced Gastrointestinal Mucositis in a Mouse Model.

Gastrointestinal mucositis is a major side effect of chemotherapy, leading to life quality reduction in patients and interrupting the therapy of cancer. Chimonanthus nitens var. salicifolius (CS) is a traditional Chinese herb for enteral disease. Considering the protective effect of CS on intestine, we hypothesize that the aqueous extract of CS could be benefcial to gastrointestinal mucositis. To verify this, a mouse mucositis model was induced by 5-Fluorouracil (5-Fu). Male Balb/C mice were treated with CS aqueous extract (5, 10, and 20u2009g/kg) or loperamide (0.2u2009mg/kg) intragastrically for 11 days, and the severity of mucositis was evaluated. Furthermore, the chemical compounds of CS aqueous extract were also analysed by high-performance liquid chromatography (HPLC). Our results demonstrated that CS aqueous extract improved mice body weight, diarrhoea, and faecal blood, maintained the liver function and intestinal length, alleviated villus shortening, and suppressed the apoptosis and inflammation in small intestine. We concluded that CS could protect mice against 5-Fu induced mucositis by inhibiting apoptosis and inflammation, and this protective effect might be associated with the 3 flavonoids (rutin, quercetin, and kaempferol) identified in CS aqueous extract.

Neuroprotective Effect of Coptis chinensis in MPP+ and MPTP-Induced Parkinson’s Disease Models

The rhizome of Coptis chinensis is commonly used in traditional Chinese medicine alone or in combination with other herbs to treat diseases characterized by causing oxidative stress including inflammatory diseases, diabetes mellitus and neurodegenerative diseases. In particular, there is emerging evidence that Coptis chinensis is effective in the treatment of neurodegenerative diseases associated with oxidative stress. Hence, the aim of this study was to investigate the neuroprotective effect of Coptis chinensis in vitro and in vivo using MPP[Formula: see text] and MPTP models of Parkinsons disease. MPP[Formula: see text] treated human SH-SY5Y neuroblastoma cells were used as a cell model of Parkinsons disease. A 24[Formula: see text]h pre-treatment of the cells with the watery extract of Coptis chinensis significantly increased cell viability, as well as the intracellular ATP concentration and attenuated apoptosis compared to the MPP[Formula: see text] control. Further experiments with the main alkaloids of Coptidis chinensis, berberine, coptisine, jaterorrhizine and palmatine revealed that berberine and coptisine were the main active compounds responsible for the observed neuroprotective effect. However, the full extract of Coptis chinensis was more effective than the tested single alkaloids. In the MPTP-induced animal model of Parkinsons disease, Coptis chinensis dose-dependently improved motor functions and increased tyrosine hydroxylase-positive neurons in the substantia nigra compared to the MPTP control. Based on the results of this work, Coptis chinensis and its main alkaloids could be considered potential candidates for the development of new treatment options for Parkinsons disease.

Inducible and reversible regulation of endogenous gene in mouse

Methods for generating loss-of-function mutations, such as conventional or conditional gene knockout, are widely used in deciphering gene function in vivo. By contrast, inducible and reversible regulation of endogenous gene expression has not been well established. Using a mouse model, we demonstrate that a chimeric transcriptional repressor molecule (tTS) can reversibly inhibit the expression of an endogenous gene, Nmyc. In this system, a tetracycline response element (TRE) artificially inserted near the target gene’s promoter region turns the gene on and off in a tetracycline-inducible manner. NmycTRE mice were generated by inserting a TRE into the first intron of Nmyc by the knockin technique. NmycTRE mice were crossed to tTS transgenic mice to produce NmycTRE/TRE: tTS embryos. In these embryos, tTS blocked Nmyc expression, and embryonic lethality was observed at E11.5d. When the dam was exposed to drinking water containing doxycycline (dox), normal endogenous Nmyc expression was rescued, and the embryo survived to birth. This novel genetic modification strategy based on the tTS–dox system for inducible and reversible regulation of endogenous mouse genes will be a powerful tool to investigate target genes that cause embryonic lethality or other defects where reversible regulation or temporary shutdown of the target gene is needed.

Neuronal conditional knockout of NRSF decreases vulnerability to seizures induced by pentylenetetrazol in mice

Neuron restrictive silencer factor (NRSF), also known as repressor element-1 silencing transcription factor, has been reported to modulate neuronal excitability and acts as endogenous anticonvulsant in kainic acid-induced or kindling-evoked seizure activity. However, whether NRSF functions in pentylenetetrazol (PTZ)-induced seizure activity has never been studied. To investigate the role of endogenous NRSF in the epileptogenesis induced by PTZ, in our experiment, NRSF neuronal conditional knockout mice (NRSF cKO) were adopted, in which NRSF was specifically deleted in neurons by the Cre-loxP system. Seizure threshold for PTZ, including the dose-response convulsions and the threshold dose, was compared between NRSF cKO and control mice. The threshold dose of PTZ that induced clonic and tonic seizures was significantly higher in NRSF cKO mice compared with the control. Similarly, the median lethal dose (LD(50)) of PTZ in NRSF cKO mice was also considerably higher than that of the control mice. These results revealed that NRSF cKO mice are of higher resistance to convulsions induced by PTZ. Our work first demonstrated the function of NRSF in PTZ-induced seizure and provided new evidence for differential pathways in diverse types of seizure.

Bioluminescence imaging for IL-1β expression in experimental colitis

BackgroundInterleukin 1 beta (IL-1β) contributes to the development of inflammatory bowel disease (IBD) and is correlated with the severity of intestinal inflammation. However, the precise source of IL-1β producing cells in DSS colitis is currently not known.MethodsTo determine IL-1β activity during intestinal inflammation in real time, an IL-1β transgenic mouse has been generated by incorporating the firefly luciferase gene driven by a 4.5-kb fragment of human IL-1β gene promoter (named cHS4I-hIL-1βP-Luc transgenic mice). Dextran sodium sulfate (DSS) induced colitis was confirmed with clinical presentation and histopathology.ResultsA substantial increase in luciferase activity (reflecting IL-1β production) in the region of inflamed colon was observed in a time dependent manner, followed by additional activity in the region of the mesenteric lymph node. The up-regulated luciferase activity was suppressed by dexamethasone (steroids) during DSS challenge, consistent with reduced severity of colitis, confirming the specificity of luciferase activity.ConclusionsOur data suggests that bioluminescence is an interesting technology, which may be used to evaluate transcription of various genes in real time in experimental colitis.

Reserpine-induced model of stress suppresses mucosal immunity

Stress contributes significantly to the development of many diseases. In clinical studies, a strong correlation between depression and immune dysfunction has been shown. Our previous studies indicated that sympathetic innervation can regulate intestinal mucosal immunity through sympathetic synapses, but the mechanism in stress/depression‐induced intestinal immune deficiency was unclear. Using a mouse model in which behavioural stress/depression is chemically induced by reserpine, it is found that there is a substantial deficiency of intestinal local humoral and particularly specific antibody response to the antigen stimulation in reserpine‐treated group. No significant difference of CD4+, CD8+ or Mac1+ cells between reserpine‐treated and control groups was detected in the intestine. This deficiency is closely correlated with stress/depression. A possible correlation between stress, cytokine secretion and humoral immunity in vivo is postulated.

TRPV2 in the Development of Experimental Colitis

Colitis is still a significant disease challenge in humans, but its underlying mechanism remains to be fully elucidated. The transient receptor potential vanilloid (TRPV) ion channel plays an important pathological role in host immunity, as deficiency of TRPV compromises host defence in vivo and in vitro. Using a DSS‐induced colitis mouse model, the function of TRPV2 in the development of colitis was investigated, utilizing TRPV2−/− and Wt mice. Less severe colitis was observed in TRPV2−/−, compared to that of Wt mice, at the clinical, histopathological and immunohistochemical levels. Compared to Wt mice, reduced severity of colitis in TRPV2−/− mice may be due to less intestinal inflammation via reduced recruitment of macrophages. The TRPV2 pathway contributes to the development of colitis. These data provide useful information for potential therapeutic intervention in colitis patients.