Ethanol extract of Evodia lepta Merr. ameliorates cognitive impairment through inhibiting NLRP3 inflammasome in scopolamine-treated mice.

Evodia lepta Merr. (Evodia lepta) is a well-known traditional Chinese medicine, which has been widely used in herbal tea. We previously reported that the coumarin compounds from the root of Evodia lepta exhibited neuroprotective effects. However, whether Evodia lepta could inhibit NLRP3 inflammasome in dementia was still unknown. In this study, the components of the Evodia lepta extract were identified by HPLC-Q-TOF HRMS. We employed a scopolamine-treated mouse model. Evodia lepta extract (10 or 20 mg/kg) and donepezil were treated by gavage once a day for 14 consecutive days. Following the behavioral tests, oxidative stress levels were measured. Then, Western blot and immunofluorescence analysis were used to evaluate the expressions of NLRP3 inflammasome. 14 major components of the Evodia lepta extract were identified by HPLC-Q-TOF HRMS. The results of Morris water maze, object recognition task and open field test indicated that Evodia lepta extract could ameliorate cognitive impairment in scopolamine-treated mice. Evodia lepta extract improved cholinergic system. Moreover, Evodia lepta extract improved the expressions of PSD95 and BDNF. Evodia lepta extract suppressed neuronal oxidative stress and apoptosis. In addition, Evodia lepta extract inhibited NLRP3 inflammasome in the hippocampus of scopolamine-treated mice. Evodia lepta extract could protect against cognitive impairment by inhibiting NLRP3 inflammasome in scopolamine-treated mice.

Effect of Bacillus amyloliquefaciens LSG2-8 on the intestinal barrier function of Amur minnow (Rhynchocypris lagowskii).

Probiotics are widely used in aquaculture. This article aims to study the effect of Bacillus amyloliquefaciens LSG2-8 on the intestinal barrier function of Rhynchocypris lagowskii. B. amyloliquefaciens LSG2-8 were added to R. lagowskii basal diets (CK) as additives at four concentrations: 1.0 × 106 (D-6), 1.0 × 107 (D-7), 1.0 × 108 (D-8) and 1.0 × 109 (D-9) CFU g-1 by dry weight of basal diet. After a 56-day feeding experiment, the activities of intestinal digestive enzymes and immunity-related enzymes of R. lagowskii on group D-6, D-7, D-8 and D-9 diet were significantly higher than the control (P < 0.05). In molecular experiments, the authors found that the levels of TGF-ß mRNA, IL-10 mRNA, ZO-1 mRNA and claudin-3 mRNA in group D-8 R. lagowskii were significantly higher (P < 0.05) than those of the control and other groups. Furthermore, the levels of IL-1ß and IL-8 mRNA of R. lagowskii on group D-6, D-7, D-8 and D-9 diet were significantly lower than those of the control (P < 0.05). In addition, the authors found that B. amyloliquefaciens LSG2-8 can regulate the intestinal flora balance and improve the intestinal structure of R. lagowskii. In conclusion, B. amyloliquefaciens LSG2-8 can improve the intestinal barrier function of R. lagowskii and can be used as a feed additive in aquaculture.

Effect of Selective Dorsal Neurectomy on Erectile Function in Rats.

OBJECTIVE: This study aimed to investigate the effect of penile selective dorsal neurectomy (SDN) on erectile function in rats. METHODS: Twelve adult male Sprague-Dawley rats (15 weeks old) were divided into three groups (n=4 per group): in control group, rats received no treatment; in sham group, rats underwent a sham operation; in SDN group, rats underwent SDN with half of the dorsal penile nerve severed. The mating test was performed, and the intracavernous pressure (ICP) assessed six weeks after the surgical treatment. RESULTS: At postoperative six weeks, the mating test revealed no significant difference in mounting latency and mounting frequency among the three groups (P>0.05), while the ejaculation latency (EL) was significantly longer and ejaculation frequency (EF) lower in the SDN group than in the control and sham groups (P<0.05). There were no significant differences in preoperative and postoperative ICP and ICP/mean arterial blood pressure (MAP) among the three groups (P>0.05). CONCLUSION: SDN does not adversely affect the erectile function and sexual desire of rats, and at the same time it can reduce EL and EF, providing an application basis for SDN in the clinical treatment of premature ejaculation.

Tanshinone IIA alleviates brain damage in a mouse model of neuromyelitis optica spectrum disorder by inducing neutrophil apoptosis.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD), an autoimmune astrocytopathic disease associated with the anti-aquaporin-4 (AQP4) antibody, is characterized by extensive necrotic lesions primarily located on the optic nerves and spinal cord. Tanshinone IIA (TSA), an active natural compound extracted from Salvia miltiorrhiza Bunge, has profound immunosuppressive effects on neutrophils. OBJECTIVE: The present study aimed to evaluate the effect of TSA on NMOSD mice and explore the underlying mechanisms. Mice were initially administered TSA (pre-TSA group, n = 20) or vehicle (vehicle group, n = 20) every 8 h for 3 days, and then NMOSD model was induced by intracerebral injection of NMOSD-immunoglobulin G (NMO-IgG) and human complement (hC). In addition, post-TSA mice (n = 10) were administered equal dose of TSA at 8 h and 16 h after model induction. At 24 h after intracerebral injection, histological analysis was performed to assess the inhibitory effects of TSA on astrocyte damage, demyelination, and neuroinflammation in NMOSD mice, and western blotting was conducted to clarify the effect of TSA on the NF-κB and MAPK signaling pathways. Furthermore, flow cytometry and western blotting were conducted to verify the proapoptotic effects of TSA on neutrophils in vitro. RESULTS: There was a profound reduction in astrocyte damage and demyelination in the pre-TSA group and post-TSA group. However, prophylactic administration of TSA induced a better effect than therapeutic treatment. The number of infiltrated neutrophils was also decreased in the lesions of NMOSD mice that were pretreated with TSA. We confirmed that prophylactic administration of TSA significantly promoted neutrophil apoptosis in NMOSD lesions in vivo, and this proapoptotic effect was mediated by modulating the caspase pathway in the presence of inflammatory stimuli in vitro. In addition, TSA restricted activation of the NF-κB signaling pathway in vivo. CONCLUSION: Our data provide evidence that TSA can act as a prophylactic agent that reduces NMO-IgG-induced damage in the mouse brain by enhancing the resolution of inflammation by inducing neutrophil apoptosis, and TSA may serve as a promising therapeutic agent for neutrophil-associated inflammatory disorders, such as NMOSD.

Spermidine and spermine delay brain aging by inducing autophagy in SAMP8 mice.

The natural polyamine spermidine and spermine have been reported to ameliorate aging and aging-induced dementia. However, the mechanism is still confused. An aging model, the senescence accelerated mouse-8 (SAMP8), was used in this study. Novel object recognition and the open field test results showed that oral administration of spermidine, spermine and rapamycin increased discrimination index, modified number, inner squares distance and times. Spermidine and spermine increased the activity of SOD, and decreased the level of MDA in the aging brain. Spermidine and spermine phosphorylate AMPK and regulate autophagy proteins (LC3, Beclin 1 and p62). Spermidine and spermine balanced mitochondrial and maintain energy for neuron, with the regulation of MFN1, MFN2, DRP1, COX IV and ATP. In addition, western blot results (Bcl-2, Bax and Caspase-3, NLRP3, IL-18, IL-1ß) showed that spermidine and spermine prevented apoptosis and inflammation, and elevate the expression of neurotrophic factors, including NGF, PSD95and PSD93 and BDNF in neurons of SAMP8 mice. These results indicated that the effect of spermidine and spermine on anti-aging is related with improving autophagy and mitochondrial function.

Carnosine ameliorates age-related dementia via improving mitochondrial dysfunction in SAMP8 mice.

Dementia is a kind of age-related neurodegenerative disease. Carnosine, an endogenous dipeptide consisting of ß-alanine and l-histidine, has been shown to have neuroprotective effects. However, the exact mechanism is still obscure. In this study, senescence-accelerated mouse prone 8 (SAMP8) mice, an age-related animal model, were used. Carnosine (100 and 200 mg kg-1 day-1) was orally administered to the mice once daily for six weeks. Behavioral tests, western blotting, and detection kits were used to evaluate the potential effects of carnosine on SAMP8 mice. Open-field and new object recognition experiments have shown that carnosine improved cognitive deficits in SAMP8 mice. Carnosine decreased the levels of malondialdehyde (MDA) and reactive oxygen species (ROS), increased the activity of superoxide dismutase (SOD) and the level of adenosine triphosphate (ATP) in SAMP8 mice. Concomitantly, western blotting results proved that carnosine increased the protein expressions of Mitofusin-1, Mitofusin-2, and Bcl-2 and reduced the protein expressions of P-Drp1, Bax, cleaved Caspase-3 and NLRP3 inflammasomes in the hippocampus of SAMP8 mice. The present data provided evidence that carnosine might improve cognitive impairment in SAMP8 mice through modulating mitochondrial dysfunction.

Ligustilide improves aging-induced memory deficit by regulating mitochondrial related inflammation in SAMP8 mice.

Alzheimer's disease (AD) is an age-related neurodegenerative disease. The main active component in Angelica sinensis, ligustilide, has been reported to have the protective effect on AD. Whether ligustilide could protect against age-induced dementia is still unknown. In this study, we used an aging model, SAMP8 mice to investigate the neuroprotective effect of ligustilide. The behavioral tests (Morris water maze, object recognition task, open field test and elevated plus maze) results showed that ligustilide could improve the memory deficit in SAMP8 mice. For mechanism study, we found that the protein level of P-Drp1 (fission) was decreased and the levels of Mfn1 and Mfn2 (fusion) were increased after ligustilide treatment in animals and cells. Ligustilide increased P-AMPK and ATP levels. Malondialdehyde and superoxide dismutase activity results indicated that ligustilide exerts antioxidant effects by reducing the level of oxidative stress markers. In addition, ligustilide improved neural function and alieved apoptosis and neuroinflammation. These findings have shown that ligustilide treatment improves mitochondrial function in SAMP8 mice, and improves memory loss.

Sodium tanshinone IIA sulfonate protects against Aß1-42-induced cellular toxicity by modulating Aß-degrading enzymes in HT22 cells.

ß-Amyloid (Aß) plays an important role in the pathogenesis of Alzheimer's disease (AD). However, there is still no effective Aß-targeting drugs for AD treatment. In this study, we explored the effect and mechanism of Sodium Tanshinone IIA Sulfonate (STS) on AD. Aß-treated HT22 cells, an immortalized mouse hippocampal neuronal cell line, were employed. Different dosages of STS (0.1, 1 and 10 µM) were selected. STS improved cell viability and protected against Aß-induced apoptosis in a dose-dependent manner. Furthermore, the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) were decreased, while the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were significantly increased after STS treatment. STS decreased the levels of phosphorylate PKR-like (p-PERK), phosphorylate eukaryotic initiation factor 2 (p-eIF2α), phosphorylate inositol-requiring enzyme (p-IRE1α), X-box binding protein 1 (XBP1) and binding immunoglobulin heavy chain protein (Bip), while increased protein disulfide isomerase (PDI) levels in Aß-treated HT22 cells. In addition, the levels of insulin degrading enzymes (IDE) and Nepterrilysin (NEP) (or call it CD10) were significantly increased after STS treatment. Taken together, these results indicated that STS might be effective in treating AD via increasing the levels of Aß-degrading enzymes.

Induction of apomixis by dimethyl sulfoxide (DMSO) and genetic identification of apomictic plants in cassava.

Apomixis, or asexual seed formation, is of great value for plant breeding and seed production, and is desirable in modern agriculture, but natural apomixis occurs in cassava at very low frequency. In present study, apomixis was induced by the treatments of female flower buds with 1%, 1.5% and 2% (v/v) dimethyl sulfoxide (DMSO) and the results showed that 1.5% DMSO treatment was most effective for the induction of apomictic seed formation in cassava cultivar SC5 with the highest percentages of fruit set and true apomictic seeds. The germinated seedlings resembled their parents and displayed no morphological characteristics of cassava polyploid. Flow cytometry and chromosome counting showed that these plants were uniform diploids. Analysis of 34 DMSO-induced cassava progenies by the expressed sequence tag-simple sequence repeat (EST-SSR) and sequence-related amplified polymorphism (SRAP) markers showed that three true apomictic seeds were obtained from the group of SC5 treated with 1.5% DMSO.

Effect of Smo SiRNA-mediated Hedgehog Signaling Pathway Inhibition on Palatal Fusion.

We used Smo siRNA to inhibit hedgehog signaling pathway in embryonic day (E) 13 palatal shelves in organ culture. SiRNA 4 was chosen as the most efficient from four synthesized Smo siRNAs. Palatal shelf fusion rate of 4 µg/mL cyclopamine group was the lowest and significantly lower than that of blank control group (P<0.05), and that of siRNA 4 group was also lower than that of blank control group (P=0.183). At 48 h after transfection, Smo protein level of siRNA 4 group was 64.8% lower than that of blank control group (P<0.05), and Gli1 protein level of 4 µg/mL cyclopamine group was 68.9% lower than that of blank control group (P<0.05). Hedgehog signaling pathway inhibition decreased palatal fusion in organ culture, probably owing to downregulation of Smo and Gli1 proteins.

The selective mGluR5 agonist CHPG attenuates SO2-induced oxidative stress and inflammation through TSG-6/NF-κB pathway in BV2 microglial cells.

Sulfur dioxide (SO2) is a common air pollutant and can cause harmful insults on neurons. Microglial activation has been implicated in the signaling cascades that contribute to neuronal cell death in various neurological disorders. In the present study, we found that SO2 derivatives decreased cell viability via inducing oxidative stress, inflammatory responses and apoptotic cell death in BV2 microglial cells. Pretreatment with (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), an mGluR5 agonist, significantly attenuated the SO2-induced cytotoxicity, which was fully prevented by the mGluR5 antagonist MPEP. CHPG increased the expression of TNF-α stimulated gene/protein 6 (TSG-6), but decreased the activation of nuclear factor-κB (NF-κB) after SO2 derivatives treatment in BV2 cells. In addition, knockdown of TSG-6 expression by specific targeted short interfering RNA (siRNA) partially reversed the protection induced by CHPG. Therefore, our findings reveal a mechanistic basis for exploring the association between SO2 exposure and neurological disorders, and also for opening up therapeutic approaches of ameliorating neuronal injury resulting from exposure in atmospheric polluting environment.

Identification of geometrical isomers and comparison of different isomeric samples of astaxanthin.

A high-performance liquid chromatographic (HPLC) analysis system for isomeric astaxanthin was developed. The separation system consisted of a C(30) column and an elution system of methanol/MTBE/water/dichloromethane (77:13:8:2, v/v/v/v). Using the combination of HPLC diode array detector and HPLC atmospheric pressure chemical ionization mass spectrometry, 11 geometrical isomers and 4 epoxides of astaxanthin were successfully identified. Referred to crystal, only isomerization with different degrees was found for solvent dissolving and iodine catalysis, while melting of astaxanthin caused isomerization, slight oxidation, and more noticeable polymerization confirmed by gel permeation chromatography. Chemical changes in isomeric samples all caused a decrease in UV content. The vibrational spectra (infrared and Raman) showed that epoxide was the only new functional group generated for melting. Changes of several key bands and formations of new bands were found in iodine catalysis and melting samples because of isomerization. Practical Application: Eleven geometrical isomers and 4 epoxides, which were normally generated for solvent dissolving, iodine catalysis, and melting of astaxanthin, have been identified by C(30) -HPLC-MS technology. Furthermore, different samples were measured by gel permeation chromatography, UV, infrared, and Raman, based on the analysis of messages, the effect of each processing was well understood.

[Association between polymorphism of IRF6 rs2235371 locus and nonsyndromic cleft lip with or without cleft palate].

OBJECTIVE: To assess the association between polymorphism of interferon regulatory factor 6 (IRF6) gene rs2235371 locus and nonsyndromic cleft lip with or without cleft palate in Chinese population. METHODS: Blood samples from 106 patients and their parents and 129 controls and their parents were collected. The polymorphism of IRF6 rs2235371 locus was determined with PCR-restriction fragment length polymorphism (PCR-RFLP) method. Case-control analysis, transmission disequilibrium test(TDT), haplotype-based haplotype relative risk analysis (HHRR) and family-based association test (FBAT) were carried out. RESULTS: By case-control analysis, no significant difference was found in the frequencies of GG, GA and AA genotypes of rs2235371 locus between the patient group and control group (P> 0.05), but there was a significant difference in allelic frequencies (P< 0.05). There was also a significant difference in genotype and gene frequencies of rs2235371 variant between family members from cleft lip only group and control group. However, in cleft lip with cleft palate group, no such difference was observed. TDT analysis suggested a linkage in the presence of disequilibrium (chi-square=5.56, P=0.024). Results of HHRR analysis (chi-square=5.115, P=0.024) and FBAT (Z=2.218, P=0.027) also indicated an association between IRF6 rs2235371 variant and the risk of NSCL with or without cleft palate. CONCLUSION: Genetic polymorphism of IRF6 gene rs2235371 locus is associated with NSCL with or without cleft palate.

[Chromosomal location of nonsyndromic cleft lip with or without palates for two multiplex families].

OBJECTIVE: To find chromosome region closely linked to nonsyndromic cleft lip with or without palates (NSCL±P) by genome-wide scan and linkage analysis for two multiplex families. METHODS: Whole-genome scan and fine genome scan were used to analyse multiplex families members, and parametric, nonparametric and interaction statistical analysis software to determine which chromosomal section was linked to the genetic disease. RESULTS: Both parametric and nonparametric linkage scores increased by a big margin over the initial linkage scores on 1q32.2-41. Although parametric results were not significant, nonparametric linkage gave a strong evidence for a candidate region on chromosome 2p25.1-24.2. The multiplicative model gave the strongest evidence for interaction in 1q32.2-41 and 2p25.1-24.2. CONCLUSION: Parametric and nonparametric linkage analyses for 2 NSCL±P multiplex families show that there may be candidate regions on chromosome 1q32.2-41 and 2p25.1-24.2.The two regions of 1q32.2-41 and 2p25.1-24.2 may contribute to NSCL±P risks with interaction.

Genome-wide and interaction linkage scan for nonsyndromic cleft lip with or without cleft palate in two multiplex families in Shenyang, China.

OBJECTIVES: To identify the loci involved in nonsyndromic cleft lip with or without cleft palate (NSCL/P) in Northern Chinese people in Shenyang by using genomewide and interaction linkage scan. METHODS: Two multiplex families in Shenyang from North China were ascertained through probands with NSCL/P. Blood of every member was drawn for DNA extraction and analysis. Genotypes were available for 382 autosomal short tandem repeat (STR) markers from the ABI Prism Linkage Mapping Set version 2.5. Linkage between markers and NSCL/P was assessed by 2-point parametric LOD scores, multipoint-heterogeneity parametric LOD scores (HLODs), and multipoint nonparametric linkage score (NPL). RESULTS: The initial scan suggested linkage on Chromosomes 1, 2, and 15. In subsequent fine mapping, 1q32-q42 showed a maximum multipoint LOD score of 1.9(empirical P=0.013) and an NPL score of 2.35 (empirical P=0.053). For 2p24-p25, the multipoint NPL increased to 2.94 (empirical P=0.007). 2-locus interaction analysis obtained a maximum NPL score of 3.73 (P=0.00078) and a maximum LOD score of 3 for Chromosome 1 (at 221 cM) and Chromosome 2 (at 29 cM). CONCLUSION: Both parametric and nonparametric linkage scores greatly increased over the initial linkage scores on 1q32-q42, suggesting a susceptibility locus in this region. Nonparametric linkage gave a strong evidence for a candidate region on chromosome 2p24-p25. The superiority of 2-locus linkage scores compared to single-locus scores gave additional evidence for linkage on 1q32-q42 and 2p24-p25, and suggested that certain genes in the two regions may contribute to NCSL/P risks with interaction.

[Relationship between genetic polymorphisms of MTHFR C677T and nonsyndromic cleft lip with or without palate].

OBJECTIVE: To explore the relationship between genetic polymorphisms of MTHFR C677T and nonsyndromic cleft lip with or without palate in Chinese population. METHODS: There were 97 NSCL/P case-parent triads that were selected as case group. At the same period, 104 healthy subjects were selected together with their biological parents as control group. For all the subjects the polymorphism of MTHFR C677T was examined by PCR-RFLP method. RESULTS: There was no statistical difference in genotype and gene frequencies for MTHFR C677T variants among family members between case group and control group in the offspring, fathers and mothers. The odds ratio(OR) between heterozygotes (CT) versus wild homozygotes (CC) were 1.02 (95% CI 0.47-2.21), 0.62 (95% CI 0.29-1.32) and 0.66 (95% CI 0.31-1.40) in the offspring, fathers and mothers, respectively. The OR between mutant homozygotes(TT) versus wild homozygotes (CC) were 1.10 (95% CI 0.44-2.74), 0.95 (95% CI 0.39-2.32) and 0.68 (95% CI 0.28-1.66) in the offspring, fathers and mothers, respectively. The OR between allele (T) versus allele (C) were 1.07 (95% CI 0.72-1.58), 0.98 (95% CI 0.66-1.46) and 0.83 (95% CI 0.56-1.24) in the offspring, fathers and mothers, respectively. T allele could not increase the risk of NSCL/P. For the MTHFR gene C677T variant, transmission disequilibrium test (TDT) analysis yielded no evidence of linkage in the presence of disequilibrium (chi(2) = 1.817, P > 0.05). Results of haplotype-based haplotype relative risk (HHRR) analysis (chi(2) = 1.76, P > 0.05) and family-based association tests (FBAT) (Z = 1.348, P > 0.05) also showed that there was no association between MTHFR C677T variant and the risk of NSCL/P . CONCLUSION: No association between genetic polymorphism of MTHFR C677T and NSCLP was observed. Our findings suggest that the MTHFR gene variations C677T do not contribute to the development of NSCLP in Chinese population.