Yinghong Li, Li Hu, Zhengzhi Wu, , Zhiling Yu, Meiqun Cao, Kehuan Sun, Yu Jin,Anmin Wu, Andrew CJ Huang

1First Affiliated Hospital of Shenzhen University, Shenzhen Second People’s Hospital, Shenzhen 518035, Guangdong Province, China

2Second Clinical Medical College of Jinan University, Shenzhen Institute of Geriatrics, Shenzhen 518020, Guangdong Province, China

3School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China

4Larry Hillblom Islet Research Center, David Geffen School of Medicine, UCLA, Los Angeles, CA 90024, USA

lNTRODUCTlON

The pathogenesis of Alzheimer’s disease(AD) is closely associated with endoplasmic reticulum stress (ERS)[1-3].Appropriate levels of ERS can upregulate the expression of endoplasmic reticulum chaperones such as GRP78/Bip, GRP94, calreticulin and protein-folding enzymes, and enhance the stress tolerance of cells[4-7].However, sustained serious ERS triggers the endoplasmic reticulum apoptosis signaling pathway, inducing the expression and activation of pro-apoptotic factors such as caspase-12,CHOP/GADD153 and PERK, which can lead to apoptosis and tissue damage[8-10].Immunoglobulin-binding protein (BiP; glucose-regulated protein 78) acts as a key chaperone in the endoplasmic reticulum and plays an important role in ERS.In addition,BiP can inhibit caspase12-induced apoptosis[11-13].PKR-like ER kinase (PERK) is a type I transmembrane serine threonine kinase and is expressed in most cells.PERK is a non-activated monomer when combined with BiP.If the combination is disrupted,PERK forms a dimer, induces self-phosphorylation, then activates the eukaryotic initiation factor 2α kinase and phosphorylates eukaryotic initiation factor 2α, ultimately inhibiting protein translation[14-15].C/EBP homologous protein(CHOP), also known as growth arrest and DNA damage 153, is a member of the transcription factor C/EBP family and is expressed at low levels in normal non-stress conditions.Both CHOP and caspase-12 are involved in the endoplasmic reticulum- induced apoptosis signaling pathways, and CHOP expression levels significantly increase after ERS occurs[16-18].

Fructus Broussonetiae, a traditional Chinese medicinal herb, is the dried fruit ofBroussonetia papyrifera (L.) Vent.In Chinese medicine,Fructus Broussonetiaeis used to nourishyinand tonify the blood, replenish liver and kidney, improve eyesight and promote urination, and can be used for treating soreness and weakness of waist and knees, fatigue, severe fever and dizziness[19].The chemical composition ofFructus Broussonetiaeincludes flavonoids, alkaloids (divided into broussonpapyrine, nitidine, oxyavicine and liriodenine), fatty oil and red pigment, as well as a variety of essential trace elements and acids with important pharmacological roles in humans[20-21].

Fructus Broussonetiaeextract[20]solution has been found to significantly promote memory, enhance immunity,lower blood fat, and exert anti- oxidation and anti-tumor effects.The treatment has also been reported to improve learning and memory function in animal models in which cognitive deficits are induced by scopolamine, sodium nitrite and chloramphenicol[22].Tests of searching food in a complex maze have revealed thatFructus Broussonetiaeextract significantly promoted learning and memory function in normal mice, shortened the time required for feeding in the maze and reduced the number of errors[23-24].Saponins are one of the pharmacologically active ingredients inFructus Broussonetiaeextract,along with fatty oil and red pigment[20-21].However, few studies have reported the role of this extract in ERS in AD pathological models.

The current study sought to elucidate the effects ofFructus Broussonetaeextract on cognitive function in a pathological rat model and to determine its impact on the expression of ERS-related marker proteins.This experiment is part of a broader attempt to investigate the molecular mechanisms ofFructus Broussonetaeextract and provide experimental evidence for clinical applications.

RESULTS

Quantitative analysis of experimental animals

Sixty Sprague-Dawley rats were included in pre-testing and water maze learning training.Rats exhibiting a poor physical state and/or mental abnormalities were excluded from the analysis.Consequently, a total of 32 high-performing rats were included in the experiment.These rats were randomly divided into four groups;normal control, sham-surgery, model, and extract groups.Each group contained eight rats.AD models were established with injection of beta-amyloid peptide 25-35(Aβ25-35) and D-galactose in the model and extract groups, while the sham-surgery group was only injected with D-galactose and the normal control rats were injected with normal saline.Rats in the extract group were intragastrically administeredFructus Broussonetaeextract prior to the experiment.All 32 rats were included in the final analysis.

Effect of Fructus Broussonetae extract on hippocampus BiP, PERK and CHOP protein expression in AD models

Immunohistochemical assay for BiP, PERK and CHOP protein expression levels in rat hippocampus revealed that the protein expression levels of BiP, PERK and CHOP were similar between the sham-surgery and normal control groups, while significantly increased expression levels were found in the model group (P＜0.01).Fructus Broussonetaeextract treatment upregulated BiP protein expression, while downregulating Perk and CHOP protein expression (P＜0.01; Figure 1,Table 1).

Figure 1 BiP, PERK and CHOP protein expression in rat hippocampus (immunohistochemistry, × 400).BiP protein (arrows)was stained as light and dark brown, localized in the cytoplasm, minimally expressed in normal cells and highly expressed in stressed cells.PERK protein (arrows) was stained as brown, localized in the cytoplasm and the nucleus, was absent or minimally expressed in normal cells and highly expressed in the stressed cells.CHOP protein (arrows) was stained as brown or dark brown, localized in the nucleus, exhibited no expression in normal cells and was highly expressed in apoptotic cells.BiP:Immunoglobulin-binding protein; PERK: PKR-like endoplasmic reticulum kinase; CHOP: C/EBP homologous protein.

Table 1 BiP, PERK, CHOP protein expression (mean gray value) in rat hippocampus

Effect of Fructus Broussonetae extract on the behavior of AD models

Navigation test

The navigation test in the Morris water maze revealed no significant difference in escape latency and search distance between the sham-surgery group and the normal control group.However, these two indices were significantly prolonged in the model rats (P＜0.05).Fructus Broussonetaeextract treatment significantly shortened the escape latency and search distance compared with the model group (P＜0.05), indicating enhanced learning and memory capacities in the extract-treated rats (Table 2).

Table 2 Effect of Fructus Broussonetae extract on escape latency and search distance in Alzheimer’s disease model rats in navigation test

Spatial exploration test

The spatial exploration test in the Morris water maze revealed that the time spent searching for the platform quadrant and the search distance were significantly reduced in the normal control and sham-surgery groups (P＜0.01).Fructus Broussonetaeextract significantly improved performance on the above two indices compared with the model group (P＜0.01), indicating that learning and memory capacities in rats were enhanced by treatment (Table 3).

DlSCUSSlON

A large body of evidence suggests that Aβ neurotoxic effects lead to senile plaques, intracellular neurofibrillary tangles, cell death, vascular disease, and eventually dementia[25-28].D-galactose is a type of aldose andin vivoinjections may damage organs.In the current study, the AD model was fabricated with bilateral hippocampal injection of (Aβ25-35) and intraperitoneal injection of D-galactose.Since this model exhibits many AD-like pathological changes, such as aging-like effects, Aβ deposition and memory impairment in animals within a short period, it is considered a suitable complex pathological model of AD[29-30].The water maze test also verified the impairment of learning and memory ability in the model rats in the current study, in accordance with the clinical features of AD.

Table 3 Effect of Fructus Broussonetae extract on the percentage of the time spent searching the platform quadrant (%) and the percentage of search distance (%) of Alzheimer’s disease model rats in spatial exploration test

ERR-induced cell death contributes to nerve cell decay in AD, Parkinson’s disease and other neurodegenerative diseases[33-36].The present results also revealed that key factors in the ERS signaling pathway, including BiP,PERK, and CHOP, were minimally expressed in the non-stress conditions, but that expression levels increased under ERS conditions.Fructus Broussonetaeextract was found to upregulate BiP expression in rat hippocampus, while downregulating CHOP and PERK expression compared with the model group.These results indicate that the extracts can effectively block the ERD-induced apoptosis pathway.The improvement in spatial learning and memory capacity in AD rats is associated with the up-regulation of GRP78/BiP expression and the down-regulation of CHOP and PERK expression.

In Chinese medicine,Fructus Broussonetaeextract is used to nourishyin, tonify the marrow, and replenish liver and kidney.This study sought to elucidate the effect ofFructus Broussonetaeextract on spatial learning and memory capacities in Aβ25-35and D-galactose-induced AD model rats, as well as ERS.The results revealed that the escape latency and search distance were significantly reduced in the treatment group, while the percentage of the time spent searching for the platform quadrant and the percentage of search distance were significantly improved afterFructus Broussonetaeextract treatment compared with model group.These findings indicate thatFructus Broussonetaeextract exerts an antagonistic action against Aβ25-35and D-galactose-induced damage.Tests of key factors in ERS in rat hippocampus revealed thatFructus Broussonetiaecan significantly upregulate BiP protein expression in the hippocampus, while downregulating PERK and CHOP protein expression.Importantly,Fructus Broussonetiaeimproved ERS in Aβ25-35and D-galactose-induced rats, and reduced the neuronal apoptosis caused by ERS.The current findings thus provide experimental evidence for traditional Chinese medical theory regarding the liver-marrow correlation and the notion that treating the liver can improve AD.In conclusion, ERS molecular pathological changes were examined in hippocampal neurons of model rats in which AD was induced by Aβ25-35and D-galactose.Fructus Broussonetiaesignificantly improves cognitive impairment in AD pathological models, significantly antagonizes ERS-induced cellular apoptosis, and accordingly protects neurons.However, further studies are required to conclusively determine the critical pharmacologically active component in the extract, and its specific targets and applications.

MATERlALS AND METHODS

Design

A randomized controlled animal experiment.

Time and setting

Experiments were conducted from July 2008 to June 2010 in the Laboratory Animal Laboratory, the Second Clinical Medical College of Jinan University (Shenzhen People’s Hospital), and the Central Laboratory in the First Affiliated Hospital of Shenzhen University(Shenzhen Second People’s Hospital), China.

Materials

Animals

Eighty Sprague-Dawley male rats of SPF level, weighing 260±20 g, were purchased from Guangdong Medical Experimental Animal Center, China, with license No.of SCXK (Guangdong) 2003-0002, Guangdong Monitoring and Certificate No.2008A020.All rats were adaptively fed for 3 days prior to enrollment in this experiment.Experimental protocols were in accordance with theGuidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of the People’s Republic of China[37].

Drugs

Fructus Broussonetiaeextract was prepared according to the operating method described in the Pharmacopoeia of the People’s Republic of China, formulated by the China Pharmacopoeia Committee[19].Briefly, the raw drug was identified and added to eight times the volume of water, extracted three times, then combined three times, followed by centrifugation, concentration, drying and sterilization.The extract was stored at 4°C for further use.The concentration ofFructus Broussonetiaeextract was 20%.Thus, each 1 g of extract was equivalent to 20 g of crude drug.Extract was dissolved in saline before use, and stored at 4°C.An increasing number of studies have reported that the active components ofFructus Broussonetiaeextract are flavonoids and alkaloids[20-23].The chemical structure of the extract is shown in Figures 2-3.

Figure 2 Chemical structures of part of flavonoid compounds in Broussonetia[21].

Methods

Animal selection

Rats were adaptively fed for 3 days, then subjected to the water maze test to assess learning and memory.According to previously described methods[26], rats were placed in the platform of the Morris water maze apparatus (Panlab Co., Barcelona, Spain) for 15 seconds, facing toward the maze wall.Rats were positioned in the pool through the center of four quadrants (northeast, southeast, southwest, northwest), swimming for 120 seconds then resting on the platform for 30 seconds.The platform was located in the southwest quadrant.One training session involved swimming in all four quadrants in the morning.Each rat was tested after three sessions of training.Rats that could not find the platform in more than two quadrants within 120 seconds were considered of abnormal intelligence, and were excluded from the study.

Establishment of AD animal models

Rats were intraperitoneally anesthetized with 45 mg/kg of 3% sodium pentobarbital.According to theRat Brain in Stereotaxic Coordinates[38], a hallmark on the skull surface was labeled, at the bregma as the base point,2.5 mm lateral and 3.6 mm posterior.Two holes were drilled on the left and right sides with a dental drill,3.0 mm below the surface of the skull, to produce an injection site into the dorsal hippocampus.Condensed Aβ25-35(Sigma, St.Louis, MO, USA) 1 μL was slowly injected through a micro-syringe (Hamilton Co.,Bonaduz, Switzerland) for 5 minutes.The needles were retained for 10 minutes, and then slowly removed.The holes were blocked with a small amount of bone wax,the muscle and skin were sutured layer by layer, and the wound was disinfected with povidone-iodine before final suturing (supplementary Figures 1–2 online).Rats were then intramuscularly injected with penicillin 5 × 104U for 3 consecutive days.The sham-surgery group was injected with artificial cerebrospinal fluid (pH 7.4; containing NaCl 147 mmol/L, KCl 2.7 mmol/L, CaCl21.2 mmol/L and MgCl20.85 mmol/L)[29]into the hippocampus.Model rats and sham-operated rats were subcutaneously injected daily with 25% D-galactose solution(Amresco Co., Solon, Ohio, USA) 150 mg/kg per day into the dorsal neck, for a continuous injection period of 30 days.Normal control rats received normal salineviasubcutaneous injection.

Figure 3 Chemical structures of broussonetine A-X in Broussonetia[21].

Fructus Broussonetiae extract intervention

Fructus Broussonetiaeextract (93.1 mg/kg) was given to ratsviaintragastric injection according to the clinical adult dose calculated with body surface area method[39].Administration was given 0.5 hour before each experiment, for 30 days.A normal control group, model group and sham-surgery group were given an equal volume of saline (2.0-3.0 mL).

Behavioral test

Spatial navigation test: Rats underwent training on days 26-30 of intragastric administration[26].Rats were released into a fixed quadrant (e.g., northeast) when the learning and training were over at 30 days.The time taken to find a safe platform was recorded as the escape latency.Rats that could not find the platform within 2 minutes were recorded as taking 120 seconds.The track length (search distance) was simultaneously recorded.Spatial exploration test: After the navigation test was completed, the safe platform was withdrawn from the pool, and the rats were placed into the pool from the northeast quadrant contralateral to the original quadrant(southwest).The track of rat swimming continuously for 120 seconds was recorded, and the time spent and search distance in each quadrant was automatically analyzed with water maze software (Panlab Co.Barcelona, Spain).The percentage of time and distance rats spent swimming in the original platform quadrant in proportion to the total time and search distance was measured.

Immunohistochemical analysis of rat hippocampus BiP, PERK and CHOP protein expression

Rats were decapitated within 2 hours after the last intragastric administration.The parietal bone was quickly opened on ice, the meninges were removed, and the left hemisphere was utilized for examination.Specimens were fixed with 4% paraformaldehyde for 24 hours,sliced into 4 μm thick slices after dehydration and embedded in paraffin.The BiP, PERK and CHOP were immunohistochemically stained with the SP method.Slices were dewaxed and hydrated and incubated with 3% H2O2for 20 minutes at room temperature, to inactivate endogenous peroxidase.Samples were then rinsed with distilled water and soaked in PBS for 5 minutes ×three times; 0.01 mol/L sodium citrate buffer (pH 6.0)was added for antigen heat repair at 95°C for 15 minutes, samples were rinsed with PBS three times; samples were blocked with normal goat serum at 37°C for 30 minutes, excess liquid was removed; diluted mouse anti-rat BiP (1: 100; Cell Signaling Technology, Inc,Boston, USA), PERK (1: 100; Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) and GADD153/CHOP (1: 100;Cell Signaling Technology) were added into the sample at 4°C overnight, and rinsed with PBS for 5 minutes ×three times; biotin-labeled goat anti-mouse IgG (1: 100;Beijing Biosynthesis Biotechnology Co., Ltd., China) was added to incubate samples at room temperature for 45 minutes, and rinsed with PBS for 5 minutes × three times;horseradish peroxidase-labeled streptavidin-biotin working solution (1: 100; Beijing Biosynthesis Biotechnology Co., Ltd.) was added at 37°C for 20 minutes, and samples were rinsed with PBS for 5 minutes × four times;finally, 3, 3’-diaminobenzidine solution coloration and mounting with neutral gum were performed.Specimens were analyzed using the Leica automatic image analysis system (Qwin Pro Software; Leica Co., Solms, Germany)as follows: a total of eight slices were used, and each slice randomly selected four hippocampal fields of vision.The average gray value of each field was calculated.The smaller average gray value indicated a higher degree of immunostaining and more protein content.The average gray value of four fields of vision was calculated as the average gray value of each slice for statistical analysis[40].

Statistical analysis

Data were analyzed with SPSS 13.0 statistical software(SPSS, Chicago, IL, USA) and count data were expressed as mean±SD.Comparison among several groups were conducted with one-way analysis of variance.Dunnett’s T3 method and the Welch method, an alternative to theF-test, were used to handle the heterogeneity of variance.

Author contributions:Yinghong Li implemented the majority of experiments, reviewed the manuscript, provided information and technology, and analyzed data.Li Hu was responsible for data collection and statistical analysis, and wrote the manuscript.Zhengzhi Wu conceived and designed this study, provided the funding and validated the manuscript.Zhiling Yu participated in the study design and revised the final paper.Meiqun Cao, Kehuan Sun, Yu Jin, Anmin Wu, and Andrew CJ Huang provided technology and information.

Conflicts of interest:None declared.

Funding:This study was financially sponsored by the National Natural Science Foundation of China, No.30973779.

Ethical approval:This pilot project has been approved by the Animal Ethics Committee in the First Affiliated Hospital of Shenzhen University (Shenzhen Second People’s Hospital),China.

Supplementary information:Supplementary data associated with this article can be found, in the online version, by visiting www.nrronline.org, and entering Vol.6, No.30, 2011 item after selecting the “NRR Current Issue” button on the page.

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