Libin Zhan

The effects of the Chinese medicine ZiBu PiYin recipe on the hippocampus in a rat model of diabetes-associated cognitive decline: a proteomic analysis

Aims/hypothesisIncreasing evidence suggests that diabetes is associated with an enhanced risk of cognitive decline. The precise mechanisms underlying diabetes-associated cognitive decline (DACD) remain unclear. Here we investigated the molecular changes associated with DACD using a comparative proteomics study of hippocampus in a rat model of type 2 diabetes. In addition, we tested the effects of the Chinese medicine ZiBu PiYin recipe (ZBPYR) on DACD.MethodsThe hippocampus was dissected from control, diabetic and diabetic rats treated with ZBPYR (DM/ZBPYR). Soluble proteins were separated using fluorescence-based difference gel electrophoresis. Protein spots were visualised with fluorescent dyes and spot density was compared between each pair of groups. Proteins of interest were identified using mass spectrometry. Proteins of specific interest were also tested by western blot and real-time PCR analysis.ResultsWe found 13 spots that were altered between control and diabetes groups, and 12 spots that were changed between diabetes and DM/ZBPYR groups. The identities of nine proteins were determined by mass spectrometry. The identified proteins were largely involved in energy metabolism, cytoskeleton regulation and oxidative stress. The protein alterations observed in the diabetes group were ameliorated to varying degrees following ZBPYR treatment.Conclusions/interpretationThe protein changes identified in hippocampus from a rat model of type 2 diabetes suggest that specific cellular alterations contribute to DACD. The Chinese medicine ZBPYR was found to affect multiple targets and partially repaired the original cellular balance. This study may provide important insights into the molecular events underlying DACD and allow the identification of novel therapeutic targets.

Biotransformation of imperatorin by Penicillium janthinellum. Anti-osteoporosis activities of its metabolites

Imperatorin (IMP) is a major constituent of many herbal medicines and possesses anti-osteoporosis activity. The present research work aimed to study the biotransformation processes of IMP and evaluated the anti-osteoporosis activity of the transformed metabolites. Among 18 strains of filamentous fungi screened, Penicillium janthinellum AS 3.510 exhibited good capability to metabolise IMP to the new derivatives. Ten transformed products were isolated and purified, and their structures were identified accurately based on spectroscopic data. Eight metabolites (2-8 and 10) were novel and previously unreported. The major biotransformation reactions involved hydroxylation of the prenyloxy side-chain and the lactone ring-opening reaction of furocoumarin skeleton. In addition, anti-osteoporosis activities of all products (1-10) were evaluated using MC3T3-E1 cells. The results showed that products 5 and 8 had the best bioactivities in increasing MC3T3-E1 cell growth. These products could be used in future therapeutic regimens for treating osteoporosis.

Biotransformation of 11-keto-β-boswellic acid by Cunninghamella blakesleana.

11-Keto-β-boswellic acid (KBA), as one of the active constituents in the gum resin of Boswellia serrata, possesses significant biological activities including anti-inflammatory activity. However, its extensive metabolism and low polarity has limited the systemic availability of KBA. The present research was aimed to obtain and explore the various possible derivatives of KBA through biotransformation by Cunninghamella blakesleana AS 3.970. A total of ten transformed compounds were isolated and purified, and their chemical structures were characterized as 7β-hydroxy-11-keto-β-boswellic acid; 7β, 15α-dihydroxy-11-keto-β-boswellic acid ; 7β, 16β-dihydroxy-11-keto-β-boswellic acid; 7β, 16α-dihydroxy-11-keto-β-boswellic acid; 7β, 22β-dihydroxy-11-keto-β-boswellic acid; 7β, 21β-dihydroxy-11-keto-β-boswellic acid; 7β, 20β-dihydroxy-11-keto-β-boswellic acid; 7β, 30-dihydroxy-11-keto-β-boswellic acid; 3α, 7β-dihydroxy-11-oxours-12-ene-24, 30-dioic acid and 3α, 7β-dihydroxy-30-(2-hydroxypropanoyloxy)-11-oxours-12-en-24-oic acid by various spectroscopic methods. The biotransformation processes include hydroxylation, oxidation and esterification. Primary structure-activity relationships (SAR) of inhibitory effects on NO production in RAW 264.7 macrophage cells are discussed.

ZiBuPiYin recipe protects db/db mice from diabetes-associated cognitive decline through improving multiple pathological changes.

Multiple organ systems, including the brain, which undergoes changes that may increase the risk of cognitive decline, are adversely affected by diabetes mellitus (DM). Here, we demonstrate that type 2 diabetes mellitus (T2DM) db/db mice exhibited hippocampus-dependent memory impairment, which might associate with a reduction in dendritic spine density in the pyramidal neurons of brain, Aβ1-42 deposition in the prefrontal cortex (PFC) and hippocampus, and a decreased expression of neurostructural proteins including microtubule-associated protein (MAP2), a marker of dendrites, and postsynaptic density 95 (PSD95), a marker of excitatory synapses. To investigate the effects of the ZiBuPiYin recipe (ZBPYR), a traditional Chinese medicine recipe, on diabetes-related cognitive decline (DACD), db/db mice received daily administration of ZBPYR over an experimental period of 6 weeks. We then confirmed that ZBPYR rescued learning and memory performance impairments, reversed dendritic spine loss, reduced Aβ1-42 deposition and restored the expression levels of MAP2 and PSD95. The present study also revealed that ZBPYR strengthened brain leptin and insulin signaling and inhibited GSK3β overactivity, which may be the potential mechanism or underlying targets of ZBPYR. These findings conclude that ZBPYR prevents DACD, most likely by improving dendritic spine density and attenuating brain leptin and insulin signaling pathway injury. Our findings provide further evidence for the effects of ZBPYR on DACD.

Efficient protocol for purification of diosgenin and two fatty acids from Rhizoma dioscoreae by SFE coupled with high‐speed counter‐current chromatography and evaporative light scattering detection

Supercritical fluid extraction (SFE) was used to extract diosgenin, linoleic acid, and linolenic acid following acid hydrolysis from Rhizoma dioscoreae, a famous traditional Chinese medicine. The process was performed using a preparative SFE system under 35 MPa of pressure, 65 degrees C of temperature, and modified CO(2) with 95% ethanol for 180 min dynamic extraction. Then, the crude extract was successfully isolated and separated by high-speed counter-current chromatography (HSCCC) with evaporative light scattering detection (ELSD). A two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water was used for HSCCC separation in a stepwise elution mode. The upper phase of the solvent system at the volume ratio of 1:1:1.4:0.6 by volume was used as the stationary phase, and the mobile phase after 200 min was changed into the lower phase of the solvent system at the volume ratio of 1:1.2:1.4:0.6 by volume. The separation produced a total of 20.8 mg diosgenin, 12.1 mg linoleic acid, and 18.4 mg linolenic acid from 300 mg crude extract in one-step purification. The purities of the products were 98.9, 99.0, and 99.4%, respectively, as determined by HPLC. Their chemical structures were identified by MS, UV, and the standards.

The effects of Zibu Piyin Recipe components on scopolamine-induced learning and memory impairment in the mouse.

ETHNOPHARMACOLOGICAL RELEVANCE The Zibu Piyin Recipe (ZBPYR) is derived from Zicheng Decoction, a traditional Chinese medicine formula recorded in the book of Bujuji, written by Wu Cheng in the Qing dynasty and used for clinical treatment of amnesia. Our aim was to study the effects of Zibu Piyin Recipe (ZBPYR) fractions on scopolamine-induced learning and memory impairment in the mouse. MATERIALS AND METHODS Crude extracts were prepared using various solvents, and individual fractions produced following D101 macroporous resin column chromatography. The passive avoidance task, step down test and Morris water maze test were then performed in mice for the evaluation of learning and memory alterations. The effective fractions were then analyzed using GC-MS and polysaccharide measurement methods, respectively. RESULTS The treatment group latency for the alcohol precipitation from water part (EP) and 95% ethanol part (95%E) following D101 macroporous resin column chromatography was significantly prolonged when compared to that of the scopolamine treated groups for both the passive avoidance task and step down test. In the Morris water maze tests, treatment with EP and 95%E resulted in a significantly shorter escape latency time (from the fourth day and the second day) and swimming distance (on the third day and from the third day) in scopolamine-induced mice. In the memory retention test, treatment with EP and 95%E dramatically shortened the latency to cross platform location and increased the numbers of platform location crosses in the scopolamine-induced mice. The polysaccharide content in EP was determined to be 69.79%. The 95%E was found to mainly contain asarone, α-cadinol, isocalamendiol, 2,4,7,14-tetramethyl-4-vinyl-tricyclo[5.4.3.0(1,8)]tetradecan-6-ol, 3-isopropyl-6,7-dimethyltricyclo[4.4.0.0(2,8)]decane-9,10-diol, 2-methyl-9-(prop-1- -en-3-ol-2-yl)-bicyclo[4.4.0]dec-2-ene-4-ol, diepicedrene-1-oxide, 7-methoxy-6-(3- -methyl-2-oxobutyl)-2H-1-benzopyran-2-one and diisooctyl phthalate when assessed using GC-MS analysis. CONCLUSION These findings suggest that the polysaccharide and volatile oil present in ZBPYR exhibit ameliorating effects on scopolamine-induced memory dysfunction.

Endoplasmic Reticulum Stress Impairs Insulin Receptor Signaling in the Brains of Obese Rats

The incidence of obesity is increasing worldwide. It was reported that endoplasmic reticulum stress (ERS) could inhibit insulin receptor signaling by activating c-Jun N-terminal kinase (JNK) in the liver. However, the relationship between ERS and insulin receptor signaling in the brain during obesity remains unclear. The aim of the current study was to assess whether ERS alters insulin receptor signaling through the hyper-activation of JNK in the hippocampus and frontal cortex in the brains of obese rats. Obesity was induced using a high fat diet (HFD). The Morris water maze test was then performed to evaluate decreases in cognitive function, and western blot was used to verify whether abnormal insulin receptor signaling was induced by ERS in HFD rats exhibiting cognitive decline. In addition, to determine whether ERS activated JNK and consequently impaired insulin receptor signaling, SH-SY5Y cells were treated with the JNK inhibitor, SP600125, followed by tunicamycin or thapsigargin, and primary rat hippocampal and cortical neurons were transfected with siRNA against IRE1α and JNK. We found that the expression of phosphorylation of PKR-like kinase (PERK), phosphorylation of α subunit of translation initiation factor 2 (eIF2α), and phosphorylation of inositol-requiring kinase-1α (IRE-1α) were increased in the brains of rats with HFD when compared with control rats. The level of serine phosphorylation of insulin receptor substrate-1 (IRS-1) was also increased, while protein kinase B (PKB/Akt) was reduced. ERS was also found to inhibit insulin receptor signaling via the activation of JNK in SH-SY5Y cells, primary rat hippocampal, and cortical neurons. These results indicate that ERS was increased, thereby resulting in impaired insulin receptor signaling in the hippocampus and frontal cortex of obese rats.

Rat hippocampal proteomic alterations following intrahippocampal injection of amyloid beta peptide (1–40)

Amyloid beta peptide 1-40 (Aβ(1-40)) is closely associated with the progressive neuronal loss and cognitive decline observed in Alzheimers disease (AD). This study aimed to establish a proteomic strategy for the profiling of AD tissues for disease-specific changes in protein abundance. Intrahippocampal injection of Aβ(1-40) induced spatial memory and learning decline in rats. Proteomic analysis revealed the changes in protein expression in the rat hippocampus treated with Aβ(1-40). Four proteins of interest which was in abundance was significantly altered in Aβ(1-40)-treated rats were identified by peptide mass fingerprint (PMF). These proteins corresponded to synapsin Ib, protein disulfide-isomerase A3 precursor, tubulin β chain and ATP synthase β subunit. Our results provide new insights into the relationship between Aβ and the pathogenesis of AD, and suggest potential targets for the therapy of AD.

Role of the SNK-SPAR pathway in the development of Alzheimer's disease

Alzheimers disease (AD) is characterized by the presence of senile plaques and neurofibrillary tangles in the brain. The beta‐amyloid peptide (Aβ) is the primary constituent of the senile plaques, and has been proposed to be a key contributor to the neurodegeneration observed in AD. The molecular mechanisms underlying dendritic spine damage that is induced by Aβ toxicity in AD patients remain largely unknown. It has been suggested previously that the SNK‐SPAR signaling pathway is involved in activity‐dependent remodeling of synapses. The relationship between the SNK‐SPAR pathway and Aβ‐induced excitotoxicity, however, is poorly understood. The present study investigated the effects of bilateral intrahippocampal injection of Aβ peptide 1–40 (Aβ1–40) on learning and memory in the rat, and explored the mechanisms underlying the effects of this injection. We reported that bilateral injection of Aβ1–40 in rats resulted in impaired performance in the step‐down passive avoidance and Morris water maze tasks. Then we examined mRNA and protein expression levels in the different brain regions one week after injection with Aβ1–40 and found that the SNK‐SPAR signaling pathway was possibly involved in dendritic spine damage in the different brain regions of Aβ‐treated rats. These results demonstrate that the SNK‐SPAR pathway may possibly play a crucial role in Aβ‐induced excitotoxic damage in the central nervous system by regulating synaptic stability.

Comparison of microwave-assisted and heat reflux extraction techniques for the extraction of ten major compounds from Zibu Piyin Recipe using ultra high performance liquid chromatography with tandem mass spectrometry.

Microwave-assisted extraction and efficient ultra-high performance liquid chromatography with triple quadrupole mass spectrometry were previously used to quickly extract and simultaneously quantify ginsenoside Rf, Ro, and Rd, 20(S)-ginsenoside-Rg2 , 20(R)-ginsenoside-Rg2 , tanshinone IIA, cryptotanshinone, dihydrotanshinone I, lithospermic acid, and osthole from Zibu Piyin Recipe. We here showed that heat reflux extraction provides higher extraction efficiency of these target compounds but is more time consuming. Chromatographic separation was achieved on an Agilent ZORBAX RRHD Eclipse Plus C18 column with a gradient mobile phase consisting of water/0.5% formic acid and acetonitrile at a flow rate of 0.2 mL/min, and detection was performed by positive and negative ion multiple-reaction monitoring mode. All analytes showed good linearity (r, 0.9989-0.9999) within the test range, with a limit of detection of 0.002-0.180 μg/mL. The overall intra- and interday variations of the ten compounds were ≤2.9%, and the accuracy was evaluated using a recovery test at three concentrations and was in the range 97.61-103.18% (RSD ≤ 4.25%). The analytical results showed remarkable differences in the concentrations of the ten compounds extracted from Zibu Piyin Recipe by microwave-assisted extraction and heat reflux extraction. These findings provide important information for determining the quality of Zibu Piyin Recipe.