小蛋白质富集法鉴定酿酒酵母“漏检蛋白”

小蛋白质(Smallproteins,SPs)是由小开放阅读框(Shortopenreadingframes,sORFs)编码长度小于100个氨基酸的多肽。研究发现小蛋白质参与了基因表达调控、细胞信号转导和代谢等重要生物学过程。然而,生命体中大多数的已注释小蛋白质尚缺少蛋白水平存在的实验证据,被称为漏检蛋白(Missing proteins,MPs)。小蛋白质的高效鉴定是其功能研究的前提,也有助于挖掘"漏检蛋白"。文中采用小蛋白质富集策略鉴定到72个酵母小蛋白质,验证9个"漏检蛋白",发现低分子量、高疏水性、膜结合、弱密码子使用偏性及不稳定性是蛋白漏检的主要原因,对进一步的技术优化具有指导意义。

小蛋白质鉴定研究进展

小蛋白质是指可读框编码长度小于100个氨基酸的多肽.近年来研究发现,小蛋白质参与了细胞信号转导、代谢和生长等重要的生物学过程.然而,由于在基因组注释和生化检测上存在技术挑战,小蛋白质研究进展较为缓慢.小蛋白质高效鉴定技术的发展是功能研究的前提,也是完善基因组注释的必然要求.本文系统综述了小蛋白质鉴定的难点、原因和解决方法.

miR-133b靶向抑制SGTB对oxLDL诱导的血管内皮细胞损伤的影响

目的:探讨微小RNA-133b(miR-133b)靶向抑制富含谷氨酰胺三十四肽重复序列的小蛋白质分子(SGTB)对氧化低密度脂蛋白(oxLDL)诱导的血管内皮细胞损伤的影响。方法:采用100μg/ml的oxLDL诱导人脐静脉血管内皮细胞(EVC-304)24 h构建血管内皮细胞损伤模型。将EVC-304细胞分为对照组、oxLDL组(oxLDL处理)、oxLDL+miR-NC组(转染20 nmol/L miR-NC+oxLDL处理)、oxLDL+miR-133b组(转染20 nmol/L miR-133b mimics+oxLDL处理)、oxLDL+si-NC组(转染20 nmol/L si-NC+oxLDL处理)、oxLDL+si-SGTB组(转染20 nmol/L si-SGTB+oxLDL处理)、oxLDL+miR-133b+pcDNA组(转染20 nmol/L si-SGTB和pcDNA+oxLDL处理)、oxLDL+miR-133b+pcDNA-SGTB组(转染20 nmol/L si-SGTB和pcDNA-SGTB处理)。实时荧光定量PCR(qRT-PCR)和蛋白质印记(Western blot)检测miR-133b和SGTB的表达水平;流式细胞术检测细胞凋亡;试剂盒检测丙二醛(MDA)含量、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)的活性;Western blot检测B细胞淋巴瘤/白血病-2(Bcl-2)和Bcl-2相关X蛋白(Bax)的表达水平。双荧光素酶报告基因实验和Western blot验证miR-133b对SGTB的靶向调控关系。结果:与对照组比较,oxLDL诱导后EVC-304细胞miR-133b、Bcl-2的表达水平显著降低(P<0.05),SGTB、Bax的表达水平显著升高(P<0.05),MDA含量和细胞凋亡率显著增加(P<0.05),SOD和GSH-Px活性显著降低(P<0.05)。过表达miR-133b或干扰SGTB均可抑制oxLDL诱导的EVC-304细胞凋亡和氧化应激损伤(P<0.05)。miR-133b与SGTB直接结合,过表达miR-133b显著下调SGTB表达(P<0.05),抑制miR-133b显著上调SGTB表达(P<0.05)。过表达SGTB可逆转过表达miR-133b对oxLDL诱导的血管内皮细胞损伤的影响(P<0.05)。结论:miR-133b通过靶向抑制SGTB的表达,可减轻oxLDL诱导的血管内皮细胞氧化应激损伤和细胞凋亡。

Variations in Phytic Acid Content and Their Relationship with Protein Content and Kernel Morphological Characters of Chinese Winter Wheat Cultivars

[Objective] The aim of this study was to determine the variation of phytic acid content （PAC） and its frequency distribution, as well as the relationship between PAC and protein content, kernel characteristics in 161 Chinese winter wheat cultivars from four regions. [Method] One hundred and sixty-one winter wheat varieties from China Wheat Zones I, II, III and IV （Table 1） were grown in a randomized block de- sign, in the 2009-2011 cropping season; and then the indexes for describing the grain morphological characteristics such as the thousand kernel weight （TKW）, kernel length （KL）, kernel width （KW） and kernel thickness （KT） were measured; the phytic acid content （PAC）, protein content and sedimentation value were also determined; finally, the relationship between PAC and protein content, kernel characteristics were analyzed. [Result] The PAC in the cultivars tested ranged from 0.92% to 1.95% with a mean value of 1.41%. Protein content ranged from 12.60% to 19.20%, with a mean of 15.24%. Most （53.4%） of the wheat genotypes had a PAC value in the range of 1.25% to 1.55%. No significant correlation was found between PAC and protein content, sedimentation value, while protein content and SDS sedimentation value was significant correlated, which suggested the possibility of breeding wheat cultivars that have a low PAC but a high protein content and good gluten quality. There was a high correlation between TKW and KW （,.=0.79）, KL （r=0.50） and KT （r=0.64）. PAC was found having no significant correlation with TKW, KW, KL and KT. [Conclusion] The result suggests the possibility of breeding wheat cultivars that have a low PAC but high kernel weight.

Correlation analysis between major nutritional components and resistant starch content in wheat

With 94 spring wheat cultivars as experimental materials, the correlations between the content of resistant starch （RS） in uncooked flour and cooked flour, and the apparent amylose content （AAC）, protein, lipid were investigated. The results showed that RS contents in both the uncooked flour and cooked flour assumed significantly positive correlation with AAC, and significantly negative cor- relation with protein content; and they were proved to be not significantly correlated with lipid content. RS content in uncooked flour was significantly correlated with that in cooked flour. These results provided references for the genetic improvement of wheat cultivars.

Molecular Cloning the Gene of Small Heat Shock Protein in the Mitochondria and Endoplasmic Reticulum of Tomato

A cDNA Library was constructed with the heat shocked tomato ( Lycopersicon esculentum Mill.) flowers and then was screened with the probes of mitochondrial and endoplasmic reticulum conservative regions that were cloned by using RT-PCR. The complete cDNAs of mitochondrial and endoplasmic reticulum small heat shock protein ( shsp) were selected out from the cDNA library. Furthermore, the temperature responses of these shsp genes were determined. Northern hybridization showed that the heat response temperatures of both genes in tomato flower were lower than that in leaf and that mitochondria shsp in leaf was cold-inducible. In this paper, the molecular features of the cloned genes, the causes of the uncommon heat response temperatures of sHSP in newer and the cold inducible character of mitochondria shsp gene in leaf were discussed.

Predicting Grain Yield and Protein Content in Winter Wheat at Different N Supply Levels Using Canopy Reflectance Spectra

A field experiment using a split-plot randomized complete block design with three replications was carried out to determine relationships between spectral indices and wheat grain yield （GY）, to compare the performance of four vegetation indices （VIs） for GY prediction, and to study the feasibility of VI to estimate grain protein content （GPC） in winter wheat. Two typical winter wheat （Triticum aestivum L.） cultivars ＇Xuzhou 26＇ （high protein content） and ＇Huaimai 18＇ （low protein content） were used as the main plot treatments and four N rates, i.e., 0, 120, 210, and 300 kg N ha^-1, as the sub-plot treatments. Increasing soil N supply significantly increased GY and GPC （P ≤ 0.05）. For the two cultivars combined, significant and positive correlations were found between four VIs and GY, with the strongest relationship observed when using the green ratio vegetation index （GRVI） at mid-filling. Cumulative VI estimates improved yield predictions substantially, with the best interval being heading to maturity stage. Similar results were found between VI and grain protein yield. However, when using cumulative VI, GPC showed no significant improvement. The strong relationship between leaf N status and GPC （R2 =0.9144 for ＇Xuzhou 26＇ and R2 = 0.8285 for ＇Huaimai 18＇） indicated that canopy spectra could be used to predict GPC. The strong fit between estimated and observed GPC （R2 = 0.7939） indicated that remote sensing techniques were potentially useful predictors of grain protein content and quality in wheat.

Generation of the regulatory protein rtTA transgenic mice

AIM: To translate Tet-on system into a conditional mouse model, in which hepatitis B or C virus (HBV or HCV) gene could be spatiotemporally expressed to overcome 'immune tolerance' formed during the embryonic development and 'immune escape' against hepatitis virus antigen(s), an effector mouse, carrying the reverse tetracycline-responsive transcriptional activator (rtTA) gene under the tight control of liver-specific human apoE promoter, is required to be generated. METHODS: To address this end, rtTA fragment amplified by PCR was effectively inserted into the vector of pLiv.7 containing apoE promoter to create the rtTA expressing vector, I.e., pApoE-rtTA. ApoE-rtTA transgenic fragment (-6.9 kb) released from pApoE-rtTA was transferred into mice by pronucleus injection, followed by obtaining one transgene (+) founder animal from microinjection through PCR and Southern blot analysis.RESULTS: rtTA transgene which could be transmitted to subsequent generation (F1) derived from founder was expressed in a liver-specific fashion. CONCLUSION: Taken together, these findings demonstrate that rtTA transgenic mice, in which rtTA expression is appropriately targeted to the murine liver, are successfully produced, which lays a solid foundation to 'off-on-off' regulate expression of target gene (s) (e.g., HBV and/or HCV) in transgenic mice mediated by Tet-on system.

Defective maintenance of intracellular Ca^(2+) homeostasis is linked to increased muscle fatigability in the MG29 null mice

Mitsugumin 29 (MG29) is a transmembrane protein that is normally found in the triad junction of skeletal muscle. Our previous studies have shown that targeted deletion of mg29 from the skeletal muscle resulted in abnormality of the triad junction structure, and also increased susceptibility to muscle fatigue. To elucidate the basis of these effects, we investigated the properties of Ca2+-uptake and -release in toxin-skinned Extensor Digitorium Longus (EDL) muscle fibers from control and mg29 knockout mice. Compared with the control muscle, submaximal Ca2+-uptake into the sarcoplasmic reticulum (SR) was slower and the storage of Ca2+ inside the SR was less in the mutant muscle, due to increased leakage process of Ca2+ movement across the SR. The leakage pathway is associated with the increased sensitivity of Ca2+/caffeine -induced Ca2+ release to myoplasmic Ca2+. Therefore, the increased fatigability of mutant EDL muscles can result from a combination of a slowing of Ca2+ uptake, modification of Ca2+-induced Ca2+ release (CICR), and a reduction in total SR Ca2+ content.

Chromatin-binding in vivo of the erythroid kruppel-like factor,EKLF,in the murine globin loci

EKLF is an erythroid-specific, zinc finger-containing transcription factor essential for the activation of the mammalian beta globin gene in erythroid cells of definitive lineage. We have prepared a polyclonal anti-mouse EKLF antibody suitable for Western blotting and immunoprecipitation （IP） qualities, and used it to define the expression patterns of the EKLF protein during mouse erythroid development. We have also used this antibody for the chromatin-immunoprecipitation （CHIP） assay. EKLF was found to bind in vivo at both the mouse beta-major-globin promoter and the HS2 site of beta-LCR in the mouse erythroleukemia cells （MEL） in a DMSO-inducible manner. The DMSO-induced bindings of EKLF as well as three other proteins, namely, RNA polymerase Ⅱ, acetylated histone H3, and methylated histone H3, were not abolished but significantly lowered in CB3, a MEL-derived cell line with null-expression of p45/NF-E2, an erythroid-enriched factor needed for activation of the mammalian globin loci. Interestingly, binding of EKLF in vivo was also detected in the mouse alpha-like globin locus, at the adult alpha globin promoter and its far upstream regulatory element alpha-MRE （HS26）. This study provides direct evidence for EKLF-binding in vivo at the major regulatory elements of the mouse beta-like globin gene clusters the data also have interesting implications with respect to the role of EKLF-chromatin interaction in mammalian globin gene regulation.

Studies on BN rats model to determine the potential allergenicity of proteins from genetically modified foods

AIM： To develop a Brown Norway （BN） rat model to determine the potential allergenicity of novel proteins in genetically modified food. METHODS： The allergenicity of different proteins were compared, including ovalbumin （OVA）, a potent respiratory and food allergen, bovine serum albumin （BSA）, a protein that is considered to have a lesser allergenic potential, and potato acid phosphatase （PAP）, a non-allergenic protein when administered to BN rats via different routes of exposure （intraperitoneally or by gavage）. IgG and IgE antibody responses were determined by ELISA and PEA, respectively. An immunoassay kit was used to determine the plasma histamine level. In addition, possible systemic effect of allergens was investigated by monitoring blood pressure. RESULTS： OVA provoked very vigorous protein-specific IgG and IgE responses, low grade protein-specific IgG and IgE responses were elicited by BSA, while by neither route did PAP elicit anything. In either routes of exposure, plasma histamine level in BN rats sensitized with OVA was higher than that of BSA or PAP. In addition, an oral challenge with BSA and PAP did not induce any effect on blood pressure, while a temporary drop in systolic blood pressure in few animals of each routes of exposure was found by an oral challenge with OVA. CONCLUSION： BN rat model might be a useful and predictive animal model to study the potential allergenicity of novel food proteins.

Quantitative Traits of Ion Beam Induced Mutagenesis in Triticum aestivum

[Objective] The aim of this study was to elucidate the quantitative traits of plants mutagenized by ion beam. [Method] The particular variation phenotypes, a- gronomic traits, and protein and wet gluten contents of progenies derived from the same ion beam induced mutant were investigated. [Result] Morphological polymor- phism existed in some individuals. Plant height, spike length and protein content were significantly influenced by ion beam, and effective tiller number and wet gluten content were moderately influenced. Multiple comparisons of all the indices within groups indicated genomic instability among these groups. Coefficient of variation im- plied the differences within group were very low. [Conclusion] Ion beam irradiation displayed characteristics of multi-directivity and non-directiveness. It aroused multiple variations in the same mutant. Instability among progeny indicates cells had different fate even in the same irradiated tissue. It may take several generations for mutants to stabilize particular phenotypes. The effects of ion beam irradiation may be the in- terrelated direct irradiation damage, indirect irradiation damage and late effect, such as bystander effect and adaptive response.

Proteomic analysis of glutathione S-transferase isoforms in mouse liver mitochondria

AIM:To survey glutathione(GSH) S-transferase(GST) isoforms in mitochondria and to reveal the isoforms' biological significance in diabetic mice.METHODS:The presence of GSTs in mouse liver mitochondria was systematically screened by two proteomic approaches,namely,GSH affinity chromatography/two dimensional electrophoresis(2DE/MALDI TOF/TOF MS) and SDS-PAGE/LC ESI MS/MS.The proteomic results were further confirmed by Western blotting using monoclonal antibodies against GSTs.To evaluate the liver mitochondrial GSTs quantitatively,calibration curves were generated by the loading amounts of individual recombinant GST protein vs the relative intensities elicited from the Western blotting.An extensive comparison of the liver mitochondrial GSTs was conducted between normal and db/db diabetic mice.Student's t test was adopted for the estimation of regression and significant difference.RESULTS:Using GSH affinity/2DE/MALDI TOF/TOF MS,three GSTs,namely,alpha3,mu1 and pi1,were identified;whereas five GSTs,alpha3,mu1,pi1,kappa1 and zeta1,were detected in mouse liver mitochondria using SDS-PAGE/LC ESI MS/MS,of these GSTs,GST kappa1 was reported as a specific mitochondrial GST.The R 2 values of regression ranged between values of about 0.86 and 0.98,which were acceptable for the quantification.Based on the measurement of the GST abundances in liver mitochondria of normal and diabetic mice,the four GSTs,alpha3,kappa1,mu1 and zeta1,were found to be almost comparable between the two sets of animals,whereas,lower GST pi1 was detected in the diabetic mice compared with normal ones,the signal of Western blotting in control and db/db diabetic mice liver mitochondria is 134.61 ± 53.84 vs 99.74 ± 46.2,with P < 0.05.CONCLUSION:Our results indicate that GSTs exist widely in mitochondria and its abundances of mitochondrial GSTs might be tissue-dependent and disease-related.

Effects of Waterlogging in Different Growth Stages on the Photosynthesis,Growth,Yield,and Protein Content of Three Wheat Cultivars in Jianghan Plain

To maintain high wheat grain yield in areas where frequent and periodic waterlogging occurs,the effects of waterlogging on the photosynthesis,growth,yield,and protein content of three wheat cultivars,namely Xiangmai55(X55),Jingmai102(J102),and Zhengmai9023(Z9023),in four different growth stages were investigated.Experiments were conducted in specially designed experimental tanks in the middle and lower reaches of the Yangtze River during the 2012-2013 and 2013-2014 wheat growing seasons in China.Results showed that X55 was the most susceptible to waterlogging,followed by J102 and Z9023.Chlorophyll content reduction and leaf senescence,which resulted in decreased green-to-total leaf number ratio,were induced to the greatest extents by waterlogging in booting and flowering stages,followed by milky stage.Meanwhile,chlorophyll content in flag leaf,plant height were significantly decreased by waterlogging in jointing stage but effectively recovered after waterlogging withdrawal,and recovery ability varied among the cultivars.Plant biomass and grain yield were most significantly decreased by waterlogging in booting and flowering stages,followed by milky and jointing stages.Grain protein content was also considerably affected by waterlogging depending on growth stage and cultivars.The decreased grain yield caused by waterlogging was mostly due to the sharp decline in 1 000-grain mass.Waterlogging led to reduced protein yield in all growth stages in three wheat cultivars.Above all,in this experiment,waterlogging decreased grain yield significantly,and waterlogging at booting stage and flowering stage was most serious.Comparing the three cultivars,X55 was most sensitive to waterlogging.

Designing the lipid raft marker protein for synaptic vesicles

Lipid rafts are cholesterol-enriched microdomains and implicated in many essential physiological ac-tivities such as the neurotransmitter release.Many studies have been carried out on the function of rafts inthe plasma membranes,whereas little is known about the information of such microdomains in subcellularcompartments especially synaptic vesicles(SVs).In the well-studied plasma membranes,several proteinshave been recognized as raft markers,which are used to label or trace rafts.But the raft marker proteinon SVs has not been identified yet.Although some SV proteins,including VAMP and CPE,have beenfound in raft fractions,they cannot be used as markers due to their low abundance in rafts.In this work,we designed several chimera proteins and tested their characteristics for using as SV raft makers.First,we detected whether they located in SVs,and then the chimeras exhibiting the better localization in SVswere further examined for their enrichment in raft using detergent treatment and gradient density floatationanalysis.Our results indicate that one of the chimeric proteins is primarily located in SVs and distributedin raft microdomains,which strongly suggests that it could be served as a raft marker for SVs.

Expression of BMP-7 and its receptors in renal tubolo-interstitial fibrosis induced with unilateral ureteral obstruction in rats

Objective:To study the changes of the expression of bone morphogenetic protein-7(BMP-7) and its receptors(BMPR-Ⅱ,ALK2,ALK3 and ALK6) in the renal tubulointerstitial fibrosis induced with unilateral ureteral obstruction in rats. Methods: Sixty Wistar male rats were divided randomly into the normal control,sham-operation and unilateral ureteral obstruction(UUO) groups and the rats were killed on the 1st,3rd,7th and 14th postoperative days respectively.The mRNA level of BMP-7,BMPR-Ⅱ,ALK2,ALK3 and ALK6 was determined with RT-PCR.The site and level of protein expression of BMP-7 were observed with immunohistochemical staining. Results: The mRNA level of BMP-7,BMPR-Ⅱ,ALK2 and ALK3 was significantly decreased in the rats of UUO group than in those of the sham-operation group but the mRNA level of ALK6 showed no obvious changes in all the rats.Immunohistochemical staining revealed that the protein of BMP-7 was mainly expressed in the renal tubules and interstitial tissue of the kidneys in normal rats but it was decreased gradually along with the unilateral ureteral obstruction. Conclusion: It is found that the loss of BMP-7 and its receptors including BMPR-Ⅱ,ALK2 and ALK3 occurs in the early phase of renal tubulointerstitial fibrosis before the appearance of other pathological changes in the kidney and may play an important role in the occurrence and progress of renal tubulointerstitial fibrosis.

D-Tyr-tRNA^(Tyr) Deacylase,a New Role in Alzheimer's-associated Disease in SAMP8 Mice

Objective To assess the expression level of D-Tyr-tRNATyr deacylase(DTD) in SAMP8 mice and speculate the function of DTD in disorders associated with Alzheimer's disease(AD).Methods Altogether 12 SAMP8 mice and 12 SAMR1 mice were used in this study.Semi-quantita-tive reverse transcription-polymerase chain reaction(RT-PCR) and Western blot were performed to detect the mRNA and protein levels of DTD in the mice.Purified DTD protein was injected into lateral ventricle to investigate the function of DTD in SAMP mice.The behavior of the mice was tested by using a Step-through Test System.Results Both mRNA and protein levels of DTD were found to be significantly lower in SAMP8 mice compared with those in SAMR1 mice(P<0.05).In vivo injection of DTD protein did not lead to an obvious change in behavior of SAM mice.Conclusions DTD might function in the process of AD-associated pathology and could possibly participate in physiology process in a long-term manner to orchestrate with other regulators in order to maintain the balance of organism.

Effect of Guazuma Leaves （Guazuma ulmifolia） Inclusion as Protein Source on Growing Calves

The present work was undertaken in a cattle ranch of the north of Puebla state, Mexico. The aim was to incorporate Guazuma leaves （Guazuma ulmifolia） in the diet of growing calves and evaluate its effect on weight gain. Twenty-four Cebu × Swiss calves （approximately live weight 250 kg） were used during all the experimental period （64 d）. They were assigned to a completely randomized design under two treatments （n = 12 for each treatment）： control （T1） consisted of commercial feed based on 15% crude protein （CP） ＋ cut Taiwan grass and the treatment （T2） consisted of a formulated diet containing 14.7% CP including Guazuma. The composition of the formulated diet consisted of： Guazuma （60%）, rolled corn （12%）, ground sorghum （9%）, oats in grain （7%）, wheat bran （9%） and vitamins and minerals. The feed was offered twice a day at a rate of 3% of live weight for 64 d. The mean daily gain for T1 and T2 was 1.15 g/animal and 1.52 g/animal, respectively （p 〈 0.05）. Results indicate that Guazuma-based supplements resulted in greater productive yield in growing calves.

Banana-dairy Small Scale Farming System for the North Rift Region of Kenya

Feeding costs could be reduced by use of alternative energy and protein sources. Leguminous trees remain green through the dry season, serving as a crude protein bank besides improving soil fertility through nitrogen fixation, nutrients recycling and stabilising soil on terraces. Some Kenyan farmers intercrop bananas with forages to cope with the perennial feed shortages. A simulation model assumed a family of six （husband, wife and four children） owning one cow producing 15 kg milk per day, living on 0.4 ha of land. Bananas grown on 0.36 ha in alleys of alternating rows ofLeucaena diversifolia and Calliandra calothyrses, intercropped with Guinea grass （Panicum maximum） and Desmodium uncinatum. Napier （Pennisetum purpureum） was planted in the alleys as a pure stand on 0.08 ha. Model analysis showed the household had adequate energy and protein all the year and was financially secured, with income exceeding routine expenditure. There were positive balances for nitrogen and phosphorus, but potassium application was required. A replica of the model was then tested.

Moxibustion eases chronic inflammatory visceral pain through regulating MEK, ERK and CREB in rats

AIM To investigate the effects of herb-partitioned moxibustion(HPM) on phosphorylation of mitogen-activated extracellular signal-regulated kinase(MEK)1, extracellular signal-regulated kinase(ERK)1/2 and c AMP response element binding protein(CREB) in spinal cord of rats with chronic inflammatory visceral pain(CIVP), and to explore the central mechanism of HPM in treating CIVP.METHODS Male Sprague-Dawley rats were randomized into normal, model, HPM, sham-HPM, MEK-inhibitor and dimethyl sulfoxide(DMSO) groups. The CIVP model was established using an enema mixture of trinitrobenzene sulfonic acid and ethanol. HPM was applied at bilateral Tianshu(ST25) and Qihai(CV6) acupoints in the HPM group, while in the sham-HPM group, moxa cones and herb cakes were only placed on the same points but not ignited. The MEK-inhibitor and DMSO groups received L5-L6 intrathecal injection of U0126 and 30% DMSO, respectively. Abdominal withdrawal reflex(AWR), mechanical withdrawal threshold(MWT) and thermal withdrawal latency(TWL) were applied for the assessment of pain behavior. The colonic tissue was observed under an optical microscope after hematoxylin-eosin staining. Expression of phosphor(p)MEK1, p ERK1/2 and p CREB in rat spinal cord was detected using Western blotting. The levels of MEK, ERK and CREB m RNA in rat spinal cord were detected using real-time polymerase chain reaction. RESULTS Compared with the normal group, the AWR scores were increased significantly(P < 0.01) and the MWT and TWL scores were decreased significantly(P < 0.05) in the model, sham-HPM and DMSO groups. Compared with the model group, the AWR scores were decreased significantly(P < 0.01) and the MWT and TWL scores were increased significantly in the HPM and MEK-inhibitor groups(P < 0.05). Compared with the sham-HPM and DMSO groups, the AWR scores were decreased significantly(P < 0.01) and the MWT and TWL scores were increased significantly(P < 0.05) in the HPM and MEK-inhibitor groups. Compared with the normal group, the expression of p MEK1, p ERK1/2 and p CREB proteins and the levels of MEK, ERK and CREB m RNA in rat spinal cord were increased significantly in the model, sham-HPM and DMSO groups(P < 0.01 or < 0.05). Compared with the model group, the expression of p MEK1, p ERK1/2 and p CREB proteins and the levels of MEK, ERK and CREB m RNA in rat spinal cord were reduced significantly in the HPM and MEK-inhibitor groups(P < 0.01 or < 0.05). Compared with the sham-HPM and DMSO groups, expression of p MEK1, p ERK1/2 and p CREB proteins and the levels of MEK, ERK and CREB m RNA in rat spinal cord were reduced significantly in the HPM and MEK-inhibitor groups(P < 0.01 or < 0.05). CONCLUSION HPM down-regulates protein phosphorylation of MEK1, ERK1/2 and CREB, and m RNA expression of MEK, ERK and CREB, inhibiting activation of the MEK/ERK/CREB signaling pathway in the spinal cord of CIVP rats, which is possibly a critical central mechanism of the analgesic effect of HPM.