Wenxiao Powder Alleviates Depression by Promoting Neurogenesis via BDNF/TrkB/ERK/CREB Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo decipher the mechanism of Wenxiao powder in alleviating corticosterone-induced depression-like behaviors in mice. MethodMale ICR mice were randomized into normal， model， paroxetine （20 mg·kg-1）， and low- and high-dose （3.27， 6.54 g·kg-1， respectively） Wenxiao powder groups. The mice in normal and model groups received equal volume of saline. Other groups except the normal group were injected with corticosterone subcutaneously 0.5 h after gavage to induce depression. Mice were tested for depression-like behaviors after drug administration. Enzyme-linked immunosorbent assay （ELISA） was performed to measure the corticosterone content in the serum. Nissl staining was performed to observe the damage of hippocampal neurons. Immunofluorescence staining was employed to observe the expression of double cortin （DCX） in the dentate gyrus （DG） of the hippocampus. Western blot was employed to determine the expression of proteins in the brain-derived neurotrophic factor （BDNF）/tyrosine kinase receptor B （TrkB）/extracellular signal-regulated kinase （ERK）/cAMP-response element-binding protein （CREB） pathway in the hippocampus. ResultCompared with the normal group， the model group showed decreased sucrose preference rate， increased immobility time in the tail suspension test （P<0.01）， and reduced residence time in the central area of the open field and the total movement distance （P<0.05， P<0.01）. In addition， the modeling elevated the corticosterone level in the serum （P<0.01）， decreased the volume and intensified the nuclear staining of hippocampal neurons in the DG area， reduced the expression of DCX in the DG area， and down-regulated the protein levels of BDNF， phosphorylated （p）-TrkB， p-ERK， and p-CREB in the hippocampus （P<0.05， P<0.01）. Compared with the model group， low-dose Wenxiao powder improved the mouse behavivors in the sucrose preference， open field， and tail suspension tests （P<0.05， P<0.01）， and high-dose Wenxiao powder improved the behaviors in the sucrose preference and open field tests （P<0.05， P<0.01）. In addition， Wenxiao powder lowered the serum corticosterone level （P<0.01） and recovered the structure and morphology of neurons with obvious nuclei and presence of Nissl bodies in the DG area of the hippocampus. Moreover， Wenxiao powder at both doses promoted the expression of DCX in the DG area， and high-dose Wenxiao powder up-regulated the protein levels of BDNF， p-TrkB， p-ERK， and p-CREB in the hippocampus （P<0.05， P<0.01）. ConclusionWenxiao powder can alleviate corticosterone-induced depression-like behaviors and promote neurogenesis in mice possibly by activating the BDNF/TrkB/ERK/CREB signaling pathway.

The nuclear factor E2-related factor 2 and age-related macular degeneration / O fator 2 relacionado a NF-E2 e a degeneração macular relacionada à idade

ABSTRACT After the discovery of anti-vascular endothelial growth factor agents as treatment of wet age-related macular degeneration, the number of studies with the objective to understand the molecular mechanisms involved in the age-re lated macular degeneration genesis has increased. The importance of the nuclear factor e2-related factor 2 lies in its activation-derived proteins being involved in the maintenance of the redox balance and consequent prevention of degenerative macular disease. This article aims to present the characteristics of nuclear factor e2-related factor 2 and describe the main nuclear factor e2-related factor 2-activated antioxidant enzymes that contribute to the preservation of vision.

RESUMO Após a descoberta do anti fator de crescimento en dotelial vascular no tratamento da degeneração macular relacionada à idade úmida, muitas pesquisas têm sido realizadas com o intuito de elucidar os mecanismos moleculares envolvidos na gênese da degeneração macular relacionada à idade. O fator nuclear eritroide 2 relacionado ao fator 2 destaca-se pelo fato de diversas proteínas, oriundas de sua ativação, estarem envolvidas na manutenção do equilíbrio do estado redox e consequente prevenção da doença macular degenerativa. Este artigo mostra as características do fator nuclear eritroide 2 relacionado ao fator 2 e descreve as principais enzimas antioxidantes originadas da ativação que contribuem para a preservação da visão.

Effect of edaravone on ERK-CREB signaling pathway in hippocampus of aged rats with postoperative cognitive dysfunction / 中华麻醉学杂志

Objective:To evaluate the effect of edaravone on the extracellular signal-regulated kinase (ERK)-cAMP responsive element binding protein (CREB) signaling pathway in the hippocampus of aged rats with postoperative cognitive dysfunction (POCD).Methods:Sixty SPF healthy male Sprague-Dawley rats, aged 20 months, weighing 650-700 g, were divided into 4 groups ( n=15 each) using a random number table method: control group (group C), POCD group (group P), edaravone group (group E) and ERK inhibitor group (group I). The rats received laparotomy under 3% sevoflurane anesthesia to prepare POCD model in P, E and I groups. Edaravone 3 mg/kg was intraperitoneally injected at 30 min before operation in E and I groups, ERK inhibitor PD98059 0.3 mg/kg was injected via the tail vein in group I. The open field test was performed at 3 days after operation to evaluate the spontaneous activity of rats, then Morris water maze test was performed to evaluate the cognitive function of rats on 3-7 days after operation. The rats were sacrificed after the end of Morris water maze test, and hippocampal tissues were obtained for determination of the expression of phosphorylated ERK (p-ERK), phosphorylated CREB (p-CREB), synaptophysin and postsynaptic density protein 95 (PSD-95) (by Western blot) and dendrite length and density of dendrites in hippocampal CA1 area (using Golgi staining). Results:Compared with group C, the escape latency was significantly prolonged after operation, the number of crossing the original platform was reduced, the expression of p-ERK, p-CREB, synaptophysin and PSD-95 was down-regulated, and the dendritic length and density of hippocampal neurons were reduced in group P ( P<0.05). Compared with group P, the escape latency was significantly shortened, the number of crossing the original platform was increased, the expression of p-ERK, p-CREB, synaptophysin and PSD-95 was up-regulated, and the dendritic length and density of hippocampal neurons were increased in group E ( P<0.05). Compared with group E, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the expression of p-ERK, p-CREB, synaptophysin and PSD-95 was down-regulated, the dendritic length of hippocampal neurons was shortened, and the density of hippocampal neurons was decreased in group I( P<0.05). Conclusions:The mechanism by which edaravone improves POCD may be related to activating ERK/CREB signaling pathway and changing synaptic plasticity in hippocampal CA1 region in aged rats.

Effect of surgery under propofol anesthesia during mid-pregnancy on cognitive function and hippocampal HDAC2-CREB-NR2B signaling pathway in offspring rats / 中华麻醉学杂志

Objective:To evaluate the effect of surgery under propofol anesthesia during mid-pregnancy on the cognitive function and hippocampal histone deacetylase 2 (HDAC2)-cAMP response element-binding protein (CREB)-N-methyl-D-aspartate (NMDA) receptor 2B subunit (NR2B)-containing NMDA receptor (NR2B) signaling pathway in the offspring rats.Methods:Thirty healthy Sprague-Dawley rats at 14 days of gestation were divided into 3 groups ( n=10 each) using a random number table method: propofol anesthesia group (P group), surgery under propofol anesthesia group (S group) and control group (C group). In S group, propofol 20 mg/kg was injected via the caudal vein, and then propofol was continuously infused at a rate of 20 mg·kg -1·h -1 to maintain anesthesia for 4 h, and exploratory laparotomy was performed. Group P received no exploratory laparotomy and the other treatments were similar to those previously described in group S. The equal volume of normal saline was given instead in group C. The learning and memory of the offspring rats was assessed using Morris water maze test on postnatal day 30. The expression of HDAC2, phosphorylated CREB (p-CREB), NR2B, brain-derived neurotriphic factor (BDNF) and phosphorylated tyrosine kinase B (p-TrkB) in offspring′s hippocampi was evaluated by Western blot. Apoptosis in hippocampal neurons was detected by TUNEL staining. Results:Compared with group C, the escape latency was significantly prolonged, the frequency of crossing the original platform was decreased, the time spent in the second quadrant was shortened, the expression of HDAC2 was up-regulated, the expression of p-CREB, NR2B, BDNF and p-TrkB was down-regulated, and the apoptosis rate of the hippocampal neurons was increased in P and S groups ( P<0.05). Compared with P group, the escape latency was significantly prolonged, the frequency of crossing the original platform was decreased, the time spent in the second quadrant was shortened, the expression of HDAC2 was up-regulated, the expression of p-CREB, NR2B, BDNF and p-TrkB was down-regulated, and the apoptosis rate of the hippocampal neurons was increased in S group ( P<0.05). Conclusions:Surgery under propofol anesthesia during mid-pregnancy can decrease the cognitive function of offspring rats, and the mechanism is related to the regulation of HDAC2-CREB-NR2B signaling pathway and the promotion of apoptosis in hippocampal neurons.

Effects of treadmill exercise on learning and memory of the vascular dementia rats and its mechanism / 解剖学报

Objective To observe the effects of 4-week low intensity treadmill exercise on the learning and memory, amino acid levels and the protein expression of protein kinase A ( PKA) , cyclic adenosine monophosphate response element binding protein( CREB) and brain-derived neurotrophic factor(BDNF) in the prefrontal cortex (PFC) of the vascular dementia (VD) rats. Methods Thirty-nine SD rats were randomly allocated to 3 groups, sham group (sham, n= 13) , vascular dementia group (VD, n= 13) and vascular dementia treaded with exercise group (VD + EX, n= 13). Chronic cerebral ischemia model in VD group and VD+EX group rats were established by permanent ligation of bilateral, then VD+EX group rats were submitted to 4-week low intensity treadmill exercise. After exercise, spatial learning and memory ability were evaluated by Moms water maze test ( MWM ) , glutamic ( Glu ) and gamma-aminobutyric acid (GABA) levels in the PFC were measured by high performance liquid chromatography( HPLC) ; the protein expression of PKA, CREB and BDNF in the PFC of rats were detected by Western blotting. Results The result of the MWM showed the average escape latency of rats in the VD group on the 1 -5 days was significantly higer than sham group, the time to first find the original platform was significantly prolonged and the platform crossings decreased significantly ( P 0. 05 ) between the two groups. Conclusion Four-week low-intensity running exercise improves the learning and memory ability of VD rats through enhancing the Glu level and activating PKA-CREB-BDNF signaling in the PFC of rats.

Nerve repair effect of olanzapine-mediated cyclic AMP response element binding protein/brain-derived neurotrophic factor/receptor tyrosine kinase receptors B pathway on schizophrenic rats / 解剖学报

Objective To stud)' the nerve repair effect of olanzapine on schizophrenia model rats through its effect on cyclic AMP response element binding protein (CREB)/brain-derived neurotrophic factor (BDNF)/receptor tyrosine kinase receptors B (TrkB) pathway. Methods Total 60 rats were divided into control group, model group, olanzapine low, middle and high dose group. The rats in the model group, olanzapine low, middle and high dose groups were injected intraperitoneally with MK-801[0. 2 mg/(kg-d) ], while the control injected with the same amount of normal saline. The low, middle and high dose olanzapine groups were perfused with olanzapine solution of 0. 5 mg/(kg-d),1. 0 mg/(kg-d) and 1. 5 mg/(kg-d) respectively. The behavior of rats was scored according to ataxia and stereotyped behavior standards, cognitive function and learning ability were evaluated by Moms water maze test, serum tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6) levels were detected by ELISA method, hippocampal histopathology was observed under microscope, and apoptosis and expression of CREB/BDNF/TrkB pathway related proteins in hippocampus were detected. Results Compared with the control group, the ataxia, the score of stereotyped behavior, the expression of TNF-a, IL-6 and the rate of apoptosis in the model group increased significantly (P < 0 . 01). Compared with the control group, the number of crossing the platform, the time of staying in the target quadrant and the relative expression of CREB, p-CREB, p-TrkB, TrkB and BDNF protein in the model group decreased significantly (P<0. 01), and those in the low and middle dose olanzapine groups decreased significantly (P < 0 . 05). Compared with the model group, the times of crossing the platform and the stay time in the target quadrant increased significantly in the low and middle dose olanzapine groups (P< 0. 05). In the model group and the low dose olanzapine group, the hippocampal cells were swollen obviously, the nucleus was broken and divided, pyknosis, and the tissue aiTangement was disorderly, while the phenomenon of fragmentation and nuclear pyknosis was rarely seen in the middle and high dose olanzapine groups. Conclusion The nerve repair mechanism of olanzapine on schizophrenic model rats is related to improving cognitive impainnent, protecting hippocampal neurons and activating the expression of CREB/BDNF/TrkB signal pathway in rats.

A cell model for high-throughput screening GLP-1 receptor agonists / 中国药理学通报

Aim To establish a high-throughput screening cell model for GLP-1 receptor agonists. Methods A pEGFP-GLP-1R-3 C recombinant plasmid was constructed and transfected into HEK293T cells. The cells were screened with G418 and flow cytometry. The established stable cell line was named HEK293TGLP-lR-3C-eGFP cell line. The expression level of GLP-1 R-3C-eGFP protein was confirmed by Western blotting and laser confocal microscopy. Then cyclic adenosine monophosphate (cAMP) response element reporter gene was transfected into the HEK293T-GLP-lR-3C-eGFP cells. The luminescence values were detected by One-Step Luciferase Reporter Gene Assay Kit after stimulation with different concentrations of GLP-1 peptide. The luminescence values reflected the cellular cAMP level, which was verified using the cAMP kit (E L I S A). Results HEK293T-GLP-lR-3C-eGFP cell line was successfully constructed. The relative light unit change trend after stimulation with different concentrations of GLP-1 was similar to that of the cellular cAMP level change trend. The value of Z' in this experiment was 0.52. Conclusions A recombinant HEK293T cell line is established, which can be used for high-throughput screening of GLP-1 receptor agonists.

Mechanism of Modified Shenqiwan in Relieving Renal Interstitial Fibrosis in Diabetic Mice Based on GSK-3β/CREB Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effects of modified Shenqiwan on renal function and fibrosis in diabetic nephropathy mice and explore the underlying mechanism based on the glycogen synthase kinase-3β （GSK-3β）/cyclic adenosine monophosphate （cAMP） response element-binding protein （CREB） signaling pathway. MethodFifty male db/db mice and 10 db/m mice were used in this study. The fifty db/db mice were randomly divided into model group， irbesartan group， and low-， medium-， and high-dose modified Shenqiwan groups. The 10 db/m mice were assigned to the normal group. The mice in the low-， medium-， and high-dose modified Shenqiwan groups were administered with modified Shenqiwan in the dosage form of suspension of Chinese medicinal granules by gavage， those in the irbesartan group were given irbesartan suspension by gavage， and those in the normal and model groups were given distilled water of equal volume by gavage. The intervention lasted for 12 weeks. The blood glucose levels， urine albumin-to-creatinine ratio （UACR）， and the protein expression levels of GSK-3β， CREB， transforming growth factor-β1 （TGF-β1）， E-cadherin， Vimentin， fibronectin （FN）， plasminogen activator inhibitor-1 （PAI-1）， and Collagen type Ⅳ （Coll Ⅳ） in the mouse kidneys were recorded before and after treatment. The extent of renal pathological damage was also observed. ResultCompared with the normal group， the model group showed significant increases in blood glucose levels， UACR levels， and the protein expression levels of GSK-3β， TGF-β1， E-cadherin， Vimentin， FN， PAI-1， and Coll Ⅳ in the kidneys （P<0.05）， decreased protein expression level of CREB （P<0.05）， and severe renal pathological damage. Compared with the model group， the low-， medium-， and high-dose modified Shenqiwan groups and the irbesartan group showed varying degrees of decreases in blood glucose levels， UACR levels， and the protein expression levels of GSK-3β， TGF-β1， E-cadherin， Vimentin， FN， PAI-1， and Coll Ⅳ in the kidneys （P<0.05）， increased expression level of CREB protein （P<0.05）， and improved renal pathological damage. ConclusionModified Shenqiwan can effectively reduce blood glucose levels， improve renal function， and alleviate fibrosis， and the mechanism of action is related to the inhibition of the GSK-3β/CREB signaling pathway.

Shenling Baizhusan Improves Spermatogenesis in Hyperuricemia Oligoasthenospermia Mice by Regulating Nrf2/ARE Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effect and mechanism of Shenling Baizhusan on the treatment of oligoasthenospermia with hyperuricemia （HUA）. MethodThirty-two male Kunming （KM） mice were randomly divided into blank group （n=6）， model group （n=6）， high-dose Shenling Baizhusan group （n=7）， low-dose Shenling Baizhusan group （n=7）， and febuxostat group （n=6）. Except for the blank group， all other groups received intraperitoneal injection of potassium oxazinate suspension （600 mg·kg-1） for 7 days. After modeling， the high-dose Shenling Baizhusan group and the low-dose Shenling Baizhusan group were orally administered with 20.14 g·kg-1 and 10.07 g·kg-1 of Shenling Baizhusan， respectively. The Febuxostat group was orally administered with 0.25 g·kg-1 of Febuxostat， while the blank group and model group were orally administered with the same volume of physiological saline. Oral administration was performed once a day for 14 consecutive days， after which samples were collected. Biochemical methods were used to measure serum uric acid （UA）， superoxide dismutase （SOD） and malondialdehyde （MDA） in testicular tissue. Hematoxylin-eosin （HE） staining was used to observe the histopathological changes in testicular tissue and evaluate the spermatogenesis function. Automated sperm analyzer was used to measure sperm density and motility. Single-cell gel electrophoresis （SCGE） was used to assess sperm DNA integrity. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） was used to detect testicular cell apoptosis rate. Western blot analysis was performed to measure the protein expression levels of Kelch-like ECH-associated protein 1 （Keap1）， nuclear factor E2-related factor 2 （Nrf2）， heme oxygenase-1 （HO-1）， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， and Caspase-3 in testicular tissue. Real-time polymerase chain reaction （PCR） was conducted to evaluate the mRNA expression levels of Keap1， Nrf2， and HO-1 in testicular tissue. ResultCompared with the blank group， the model group showed elevated serum UA level （P<0.01）， decreased testicular spermatogenesis function， sperm density， and motility （P<0.01）， and increased sperm trailing rate and testicular cell apoptosis rate （P<0.01）. Compared with the model group， the high-dose Shenling Baizhusan group showed significant improvements in the above-mentioned indicators （P<0.05， P<0.01）. Additionally， the expression levels of Keap1， Bax， and Caspase-3 in testicular tissue were reduced， while the expression levels of Nrf2， HO-1， and Bcl-2 increased （P<0.05， P<0.01）. The mRNA level of Keap1 decreased （P<0.05， P<0.01）， while the mRNA levels of Nrf2 and HO-1 increased （P<0.05， P<0.01）. ConclusionShenling Baizhusan can significantly improve HUA oligoasthenospermia， and its mechanism may be related to the Nrf2/antioxidant response element （ARE） signaling pathway.

Development and optimization of a cell screening system for farnesoid X receptor agonist / 生物工程学报

This study aims to develop an improved cell screening system for farnesoid X receptor (FXR) agonists based on a dual luciferase reporter gene system. FXR response element (FXRE) fragments from FXR target genes were cloned and inserted into upstream of firefly luciferase (Luc) gene in the plasmid pGL4-luc2P-Hygro. In combination with the internal reference plasmid containing renilla luciferase, a dual luciferase reporter gene system was developed and used for high throughput screening of FXR agonists. After studying the effects of over-expression of RXR, mouse or human FXR, various FXRE fragments, and different ratio of FXR plasmid amount to reporter gene plasmid, induction efficiency of the screening system was optimized by the known FXR agonist GW4064, and Z factor for the system reached 0.83 under optimized conditions. In summary, an improved cell screening system based on double luciferase reporter gene detection system was developed to facilitate the discovery of FXR agonists, where a new enhanced FXRE element was formed by a superposition of multiple FXRE fragments from FXR target genes, instead of a superposition of traditional IR-1 (inverted repeats-1) fragments.

Protective Effect of Alcohol Extract of Phyllanthi Fructus on Silicosis Mice and Its Correlation with Nrf2/ARE Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the effect and underlying mechanism of alcohol extract of Phyllanthi Fructus on silicosis mice induced by silicon dioxide （SiO2）. MethodThirty-six male Kunming mice of SPF grade were randomly divided into a blank group，a model group，high-， medium， and low-dose Phyllanthi Fructus groups （800， 400， 200 mg·kg-1），and a tetrandrine group （0.039 mg·kg-1），with six mice in each group. The silicosis model was induced by static SiO2 exposure in mice except for those in the blank group. After 28 days of administration by gavage，the lung tissues were collected and the organ coefficient was calculated. Hematoxylin-eosin（HE）staining and Masson staining were used to detect the morphology of lung tissues. The content of hydroxyproline （HYP），superoxide dismutase （SOD），malondialdehyde （MDA）， and catalase （CAT） in serum was detected by enzyme-linked immunosorbent assay （ELISA）. Western blot and Real-time polymerase chain reaction（Real-time PCR） were used to detect the protein and mRNA expression of nuclear factor E2-related factor 2 （Nrf2），heme oxygenase-1 （HO-1），NAD（P）H：quinone oxidoreductase 1 （NQO1），and Kelch-like ECH-associated protein 1 （Keap1）， respectively. ResultCompared with the blank group，the model group showed seriously damaged morphological structure of lung tissues with inflammatory cell infiltration and fibrous tissue proliferation， reduced serum content of SOD and CAT（P<0.01），increased content of HYP and MDA（P<0.01）， down-regulated protein and mRNA expression of Nrf2，HO-1， and NQO1（P<0.01），and up-regulated protein and mRNA expression of Keap1 （P<0.05，P<0.01）. Compared with the model group，the high- and medium-dose Phyllanthi Fructus groups showed significantly restored morphological structure of lung tissues with reduced collagen deposition， increased serum content of SOD and CAT（P<0.05，P<0.01），decreased content of HYP and MDA（P<0.01）， up-regulated protein and mRNA expression of Nrf2，HO-1， and NQO1 （P<0.05，P<0.01），and down-regulated protein and mRNA expression of Keap1（P<0.05，P<0.01）. ConclusionThe alcohol extract of Phyllanthi Fructus can inhibit pulmonary fibrosis in silicosis mice，and the underlying mechanism may be related to the regulation of the Nrf2/antioxidant response element （ARE） signaling pathway.

Mechanism of Shaoyaotang in Regulating Water Metabolism and Intestinal Epithelial Permeability in Ulcerative Colitis Through cAMP/PKA/CREB Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the role of cyclic adenosine monophosphate （cAMP）/protein kinase A （PKA）/cAMP-response element binding protein （CREB） signaling pathway in water metabolism and intestinal epithelial permeability in ulcerative colitis （UC） and the intervention mechanism of Shaoyaotang based on the theory of large intestine governing fluids. MethodSixty male SD rats were divided into blank group， model group， mesalazine group （0.42 g·kg-1）， Shaoyaotang low-dose group （11.1 g·kg-1）， Shaoyaotang medium-dose group （22.2 g·kg-1） and Shaoyaotang high-dose group （44.4 g·kg-1）， with 10 in each group. The UC rat model of internal retention of dampness-heat was established by compound factors. The blank group and the model group were given normal saline （ig）. The mesalazine group was given mesalazine （ig）， and Shaoyaotang low-， medium- and high-dose groups were administrated with corresponding doses of Shaoyaotang （ig）. The treatment lasted for 14 days. The diarrhea score and fecal moisture content of rats in each group were observed. The contents of diamine oxidase （DAO） and D-lactic acid in plasma were detected by enzyme-linked immunosorbent assay （ELISA）. The protein expressions of aquaporin （AQP）8， AQP4， ZO-1 and Occludin in colon tissues were detected by immunohistochemistry， while those of cAMP， PKA and CREB in colon tissues were determined by Western blot. ResultCompared with the normal group， the model group had elevated diarrhea score and fecal moisten content （P<0.01）， increased contents of DAO and D-lactic acid in plasma （P<0.01） and decreased protein expressions of ZO-1， Occludin， AQP8， AQP4， cAMP， PKA and CREB in colon （P<0.01）. Compared with the conditions in the model group， the contents of DAO and D-lactic acid in plasma in each administration groups were lower （P<0.01）， while the protein expressions of ZO-1， Occludin， AQP8， AQP4， cAMP， PKA and CREB in colon were higher （P<0.01）. ConclusionShaoyaotang alleviates the diarrhea in UC， probably through activating cAMP/PKA/CREB signaling pathway， up-regulating expressions of AQPs， enhancing tight junctions in intestinal epithelium and thus improving the water metabolism in colon and the intestinal mucosal permeability.

Neuroprotection of Natural Products Based on Keap1/Nrf2/ARE Signaling Pathway： A Review / 中国实验方剂学杂志

The pathological manifestations of neurodegenerative diseases， such as Alzheimer's disease， Parkinson's disease， and multiple sclerosis， are abnormal protein aggregation and accumulation， microglia activation， and mitochondrial dysfunction， which eventually lead to the gradual loss of neuronal structure or function and deteriorate over time. These pathological processes are related to the production of reactive oxygen species （ROS）， which can cause oxidative stress and damage proteins， lipids， and DNA， leading to cell and tissue injuries. The Kelch-like ECH-associated protein 1 （Keap1）/nuclear factor E2-related factor 2 （Nrf2）/antioxidant response element （ARE） signaling pathway is the main mechanism to maintain the redox balance of the body and defend against oxidative stress injury. Nrf2 activates the expression of a series of antioxidant genes related to ARE through the dissociation of Keap1 and nuclear transfer in the cytoplasm to protect the body from oxidative damage. Therefore， the discovery and study of the Keap1/Nrf2/ARE signaling pathway activator is of great significance for the prevention and treatment of neurodegenerative diseases. Because of the remarkable biological activity and slight side effects， natural products are a treasure trove for new drug research and development. Studies have shown that a variety of natural products can activate the Keap1/Nrf2/ARE signaling pathway and play a neuroprotective role. According to the structural characteristics， natural products can be divided into flavonoids， terpenoids， volatile oils， polyphenols， and phenylpropanoids. This study summarized the underlying mechanism of the Keap1/Nrf2/ARE signaling pathway in regulating diseases and reviewed the research progress on natural products based on this signaling pathway in neuroprotection to provide references for the development of clinical drugs for the prevention and treatment of neurodegenerative diseases.

Buyang Huanwutang Treats Diabetic Peripheral Neuropathy via Mitochondrial Transport in Rats / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Buyang Huanwutang in treating diabetic peripheral neuropathy （DPN） via mitochondrial transport. MethodDiabetes in SD rats was induced by a high-carbohydrate/high-fat diet and intraperitoneal injection of streptozotocin （STZ）. The 45 diabetic rats were randomly assigned into a DPN group， an alpha-lipoic acid （60 mg·kg-1·d-1） group， and a Buyang Huanwutang （15 g·kg-1·d-1） group， with 15 rats in each group. Fifteen normal SD rats were fed with the standard diet and set as the control group. The rats were administrated with corresponding drugs by gavage for 12 weeks. The paw withdraw threshold （PWT） and motor nerve conduction velocity （MNCV） were measured at the end of medication， and the sciatic nerve and the bilateral dorsal root ganglia of L4-5 were collected. The injury model of NSC34 cells was established by treating with 50 mmol·L-1 glucose and 250 μmol·L-1 sodium palmitate. The NSC34 cells were then randomly assigned into a blank （10% blank serum） group， a DPN （10% blank serum） group， an apha-lipoic acid （10% apha-lipoic acid-containing serum） group， a Buyang Huanwutang （10% Buyang Huanwutang-containing serum） group， and a Buyang Huanwutang + Compound C （CC） （10% Buyang Huanwutang-containing serum + 10 μmol·L-1 CC） group. The cell intervention lasted for 24 h. The immunofluorescence method， immunohistochemistry， and Western blot were employed to determine the expression levels of phosphorylated adenosine monophosphate-activated protein kinase （p-AMPK）， phosphorylated cAMP-response element binding protein （p-CREB）， kinesin family member 5A （KIF5A）， and dynein cytoplasmic 1 intermediate chain 2 （DYNC1I2）. ResultCompared with the control group， the DPN group of rats showed increased fasting blood glucose （P<0.01）， decreased MNCV and PWT （P<0.01）， down-regulated expression of KIF5A， p-AMPK/AMPK， and p-CREB/CREB （P<0.01）， and up-regulated expression of DYNC1I2 （P<0.01）. Compared with the DPN group， drug intervention groups showed increased MNCV and PWT （P<0.01）， up-regulated expression of KIF5A， p-AMPK/AMPK， and p-CREB/CREB （P<0.05， P<0.01）， and down-regulated expression of DYNC1I2 （P<0.05， P<0.01）. The Buyang Huanwutang group had higher levels of MNCV and KIF5A （P<0.05） and lower level of DYNC1I2 （P<0.01） than the apha-lipoic acid group. Compared with the blank group， the DPN group of NSC34 cells showed decreased levels of KIF5A， p-AMPK/AMPK， and p-CREB/CREB （P<0.01） and increased level of DYNC1I2 （P<0.01）. The apha-lipoic acid group and Buyang Huanwutang group had higher levels of KIF5A， p-AMPK/AMPK， and p-CREB/CREB （P<0.05， P<0.01） and lower level of DYNC1I2 （P<0.01） in NSC34 cells than the DPN group. Buyang Huanwutang group had higher KIF5A level （P<0.05） in NSC34 cells than the apha-lipoic acid group. Moreover， the Buyang Huanwutang + CC group had lower levels of KIF5A， DYNC1I2， p-AMPK/AMPK， and p-CREB/CREB （P<0.01） in NSC34 cells than the Buyang Huanwutang group. ConclusionBuyang Huanwutang may regulate mitochondrial anterograde transport via the AMPK/CREB pathway to prevent and treat DPN.

Mechanism of miR-26a-5p/cAMP response element binding protein 1 molecular axis regulating osteogenic differentiation of adipose-derived mesenchymal stem cells / 中国修复重建外科杂志

OBJECTIVE@#To investigate the regulatory effects of miR-26a-5p on the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) by regulating cAMP response element binding protein 1 (CREB1).@*METHODS@#The adipose tissues of four 3-4 weeks old female C57BL/6 mice were collected and the cells were isolated and cultured by digestion separation method. After morphological observation and identification by flow cytometry, the 3rd-generation cells were subjected to osteogenic differentiation induction. At 0, 3, 7, and 14 days after osteogenic differentiation induction, the calcium deposition was observed by alizarin red staining, ALP activity was detected, miR- 26a-5p and CREB1 mRNA expressions were examined by real-time fluorescence quantitative PCR, and CREB1 protein and its phosphorylation (phospho-CREB1, p-CREB1) level were measured by Western blot. After the binding sites between miR-26a-5p and CREB1 was predicted by the starBase database, HEK-293T cells were used to conduct a dual-luciferase reporter gene experiment to verify the targeting relationship (represented as luciferase activity after 48 hours of culture). Finally, miR-26a-p inhibitor (experimental group) and the corresponding negative control (control group) were transfected into ADSCs. Alizarin red staining, ALP activity, real-time fluorescent quantitative PCR (miR-26a-5p) and Western blot [CREB1, p-CREB1, Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN)] were performed at 7 and 14 days after osteogenic induction culture.@*RESULTS@#The cultured cells were identified as ADSCs. With the prolongation of osteogenic induction culture, the number of calcified nodules and ALP activity significantly increased ( P<0.05). The relative expression of miR-26a-5p in the cells gradually decreased, while the relative expressions of CREB1 mRNA and protein, as well as the relative expression of p-CREB1 protein were increased. The differences were significant between 7, 14 days and 0 day ( P<0.05). There was no significant difference in p-CREB1/CREB1 between different time points ( P>0.05). The starBase database predicted that miR-26a-5p and CREB1 had targeted binding sequences, and the dual-luciferase reporter gene experiment revealed that overexpression of miR-26a-5p significantly suppressed CREB1 wild-type luciferase activity ( P<0.05). After 7 and 14 days of osteogenic induction, compared with the control group, the number of calcified nodules, ALP activity, and relative expressions of CREB1, p-CREB1, OCN, and RUNX2 proteins in the experimental group significantly increased ( P<0.05). There was no significant difference in p-CREB1/CREB1 between the two groups ( P>0.05).@*CONCLUSION@#Knocking down miR-26a-5p promoted the osteogenic differentiation of ADSCs by up-regulating CREB1 and its phosphorylation.

Effect of the aqueous extracts of ganoderma leucocontextum on cognitive function of aging rats / 解剖学报

Objective To investigate the effects of the aqueous extracts of ganoderma leucocontextum (GLAE) on cognitive decline of aging rats and possible regulation mechanism. Methods Fifty rats were divided into five groups, control group, model group, GLAE low-dose group, GLAE middle-dose group and GLAE high-dose group. Aging SD rat models were made by D-galactose, and then treated continuously with different doses (0, 50, 100, 200 mg/ kg) of GLAE. The novel object recognition and step down test were performed to detect the changes of rats cognitive function. The brain tissue was stained with toluidine blue, Giemsa and HE staining and observed. The cerebral cell DNA damage was detected by comet assay. Expressions of protein kinase A (PKA) / cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB) signaling pathway related factors in brain were respectively detected by ELISA, Western blotting and Real-time PCR. Results Compared with the model group, administration of GLAE could obviously alleviate rats cognitive decline and pathological change. The levels of cell DNA damage reduced markedly (P<0. 05). The contents of cAMP, brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF), and also expression levels of mRNA and protein of PKA, BDNF, NGF, CREB in the brain increased significantly in each medicated group (P<0. 01, P< 0. 05). Conclusion GLAE can improve cognitive function, and its mechanism may be related to activation of brain PKA/ CREB signaling pathway, increase in neurotrophic factor content and inhibition of cell DNA damage.

Role of microRNA-132 and Mecp2 in methamphetamine dependence / 中国药理学通报

Aim To explore the roles of miRNA-132 and its related proteins(Mecp2, CREB)in the mechanism of methamphetamine(MA)-induced neurotoxicity and dependence.Methods The rats were intraperitioneally injected(ip)with MA(10 mg·kg-1·d-1)to establish methamphetamine dependence model with different dependent time courses of 1 week, 2 weeks, and 4 weeks respectively.The miRNA-132 and Mecp2 mRNA were detected by RT-qPCR, and the Mecp2, p-Mecp2, CREB and p-CREB proteins were detected by Western blot in the tissues of frontal cortex and hippocampus.Results In the frontal cortex, the miRNA-132 and Mecp2 mRNA were up-regulated in MA-dependent groups(P<0.05 and P<0.01), while the Mecp2 protein were down-regulated(P<0.01).MA could promote the phosphorylation of Mecp2 protein in the frontal cortex(P<0.01).In hippocampus, the miRNA-132 was down-regulated in the MA-dependent groups, but Mecp2 mRNA was up-regulated(P<0.05).Mecp2 protein increased in MA-dependent 1 week group(P<0.05), and then recovered with the prolonged time of MA dependence, then decreased in MA-dependent 4 weeks groups(P<0.05)in hippocampus.The phosphorylation level of Mecp2 was significantly decreased in the 1 week group(P<0.01), and then increased in the 2 weeks group(P<0.01)in hippocampus.Conclusions MA could induce an abnormal expression of miRNA-132 in the frontal cortex and hippocampus, and miRNA-132 might inhibit the translation of Mecp2 mRNA and induce the decrease expression of Mecp2 protein in the frontal cortex.But in hippocampus, miRNA-132 does not show the correlation with the Mecp2 expression trend of the frontal cortex.And miRNA-132 regulation does not depend on the expression of Mecp2 in hippocampus.

Role of PKA-CREB signaling pathway in sevoflurane-induced reduction of cognitive impairment caused by cardiopulmonary bypass in rats / 中华麻醉学杂志

Objective:To evaluate the role of protein kinase A (PKA)-cyclic adenosine monophosphate response element binding protein (CREB) signaling pathway in sevoflurane-induced reduction of cardiopulmonary bypass (CPB)-induced cognitive impairment in rats.Methods:Forty healthy male Sprague-Dawley rats, aged 4 months, weighing 300-350 g, were divided into 4 groups ( n=10 each) using a random number table method: control group (group C), group CPB, CPB+ sevoflurane group (group CS) and CPB+ sevoflurane+ PKA inhibitor H89 group (group CSH). After H89 5 μl was injected into the lateral ventricle in group CSH, the rats in group CS and group CSH were exposed to 2.4% sevoflurane for 1 h, and then the CPB model of beating heart without blood priming for 60 min was developed in CPB, CS and CSH groups.The autonomic movement ability was evaluated using the open field test at 2nd day after CPB.Morris water maze test was used to assess the cognitive function at 3rd day after CPB.The rats were sacrificed after the Morris water maze test, the brain was removed and the hippocampal tissues were isolated for determination of the apoptosis rate of hippocampal neurons (by flow cytometry) and expression of PKA, phosphorylated CREB (p-CREB) and brain-derived neurotrophic factor (BDNF) (by Western blot). Results:There was no significant difference in movement speed, distance and time of staying at the central region among the four groups ( P>0.05). Compared with group C, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the time of staying at the original platform quadrant was shortened, the apoptosis rate of hippocampal neurons was increased, and the expression of PKA, p-CREB and BDNF was down-regulated in the other three groups ( P<0.05). Compared with group CPB, the escape latency was significantly shortened, the number of crossing the original platform was increased, the time of staying at the original platform quadrant was prolonged, the apoptosis rate of hippocampal neurons was decreased, and the expression of PKA, p-CREB and BDNF was up-regulated in group CS ( P<0.05), and no significant change was found in the indexes mentioned above in group CSH ( P>0.05). Compared with group CS, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, and the time of staying at the original platform quadrant was shortened, the apoptosis rate of hippocampal neurons was increased, and the expression of PKA, p-CREB and BDNF was down-regulated in group CSH ( P<0.05). Conclusions:Sevoflurane can reduce the apoptosis in hippocampal neurons by activating PKA-CREB signaling pathway, and thus reducing the cognitive impairment induced by CPB in rats.

Effects of enriched environment on anxiety- and depressive-like behavior and synaptic plasticity in neuropathic pain rats / 中华行为医学与脑科学杂志

Objective:To explore the effect of enriched environment on pain sensitivity, anxiety- and depressive-like behavior in selective nerve injury(SNI) rats model and its potential mechanism.Methods:A total of 36 male clean grade SD rats aged 6-8 weeks were randomly divided into three groups( n=12 in each group): sham operation+ standard environment group (sham group), SNI+ standard environment group (standard environment group), SNI+ enriched environment group (enriched environment group). The rat model of neuropathic pain was established by SNI.The rats in the enriched enviroment group were placed in an enriched enviroment 7 days before operation until 21 days after operation.The paw withdraw threshold(PWT) and paw withdraw latency (PWL) were performed to assess hyperalgesia.The open field test, elevated plus maze test, novelty suppressed feeding test and forced swimming test were used to assess anxiety and depression like behavior.The expressions of cAMP response element binding protein (CREB), p-CREB, brain-derived neurotrophic factor (BDNF), postsynaptic density-95 (PSD-95) and neuroligin 2 (NLGN2) were detected by Western blot.The expression of CREB and BDNF in contralateral ACC were measured by immunofluorescence.GraphPad prism 8.0 and SPSS 23.0 were used for data analysis.One way ANOVA was used for inter group comparison, repeated measurement ANOVA was used to analyze PWT and PWL results, and Tukey test was used for pairwise comparison. Results:(1) In PWT and PWL experiments, the interaction effect between group and time, group main effect and time main effect of PWT were significant ( F=13.4, 39.6, 369.6, all P<0.05), and the interaction effect between group and time, group main effect and time main effect of PWL were significant ( F=3.8, 10.3, 58.8, all P<0.05). Compared with sham group, PWT((8.0±3.5) g, (2.4±1.4) g, (2.3±1.1) g, (2.2±1.6) g, (1.6±0.5) g) and PWL((8.6±1.3) s, (7.3±1.5) s, (7.9±1.0) s, (6.6±1.1) s, (7.7±1.4) s) in standard environment group decreased at each time point (all P<0.05). (2) Compared with sham group, the number of entrying into the central area (1.3±1.7), the time of entrying into the central area((1.6±1.3) s), the proportion of entering open arms ((8.0±7.8) %) and the proportion of time in the open arms ((1.3±1.2) %) all significantly decreased in standard environment group ( t=4.585, 5.423, 4.682, 5.202, all P<0.05). The eating latency ((365.2±94.4) s) and immobility time ((127.6±24.3) s) dramatically increased ( t=6.008, 14.290, both P<0.05). The number and time of entrying into central area of enriched environment group were both higher than those of standard environment group(both P<0.05), while the eating latency and immobility time of enriched environment group were both lower than those of standard environment group(both P<0.05). (3) Compared with sham group(CREB: (1.6±0.2), (0.8±0.5); BDNF: (0.8±0.5), (1.0±0.4)), the expression of CREB ((1.8±0.1), (1.5±0.2)), BDNF ((0.9±0.6), (1.4±0.3)) in spinal cord and ACC of standard environment group increased (spinal: t=3.283, 4.989; ACC: t=5.502, 4.257, all P<0.05). The expression of PSD-95 ((1.6±0.2), (1.0±0.2) and NLGN2 ((1.5±0.5), (1.1±0.2)) also increased in ACC of standard enviroment group ( t=4.257, 2.214, both P<0.05). Compared with standard environment group, the expression of CREB (1.3±0.3), BDNF (0.7±0.4), PSD-95(1.0±0.3) and NLGN2(1.1±0.4) in spinal cord of enriched environment group decreased ( t=5.007, 2.166, 2.358, 2.322, all P<0.05). The expression of PSD-95(1.2±0.3) and NLGN2(1.1±0.2) also decreased in ACC of enriched environment group ( t=2.674, 2.944, both P<0.05). However, the expression of p-CREB (1.7±0.6) and BDNF (2.4±0.2) increased in ACC ( t=4.180, 7.610, P<0.05). Conclusion:Enriched environment can improve neuropathic pain and anxiety- and depressive-like behavior in SNI rats, which may be related to the change of synaptic plasticity in spinal cord and ACC.

Protective Mechanism of Total Flavonoids of Lavender Against Skin Photoaging in Mice / 中国实验方剂学杂志

ObjectiveTo study the protective effect of total flavonoids of lavender on skin photoaging induced by ultraviolet B （UVB） in mice and to explore its mechanism from the perspective of nuclear factor E2-related factor 2 （Nrf2） antioxidant pathway. MethodEighty-four female KM mice were randomly divided into seven groups， namely blank group， model group， solvent group， vitamin E （0.013 g·kg-1） group， as well as low-， middle-， and high-dose （0.25， 1.25， 2.50 g·kg-1） groups of total flavonoids of lavender. The naked skin on the back of mice was irradiated with UVB for inducing optical damage. Thirty minutes before irradiation， the skin was coated with the total flavonoids of lavender. After continuous irradiation for one week， the skin moisture and elasticity on the back of mice were evaluated， and the effects of total flavonoids of lavender on histopathological changes in mouse skin were investigated by hematoxylin-eosin （HE） and Van Gieson （VG） staining. The levels of malondialdehyde （MDA）， superoxide dismutase （SOD）， total antioxidant capacity （T-AOC）， nitric oxide synthase （NOS）， and glutathione peroxidase （GSH-Px） after skin homogenization were detected by colorimetry， the inflammatory factors interleukin-1 （IL-1）， interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） in skin tissue by enzyme-linked immunosorbent assay （ELISA）， and the mRNA expression levels of Nrf2， Kelch-like epichlorohydrin-associated protein 1 （Keap1）， BTB-CNC homology 1 （Bach1）， heme oxygenase-1 （HO-1）， quinone oxidoreductase 1 （NQO1）， and glutamate-cysteine ligase catalytic subunit （GCLC） by real-time polymerase chain reaction （Real-time PCR）. ResultCompared with the blank group， the model group exhibited significantly increased appearance score （P<0.01）， reduced skin moisture and elasticity （P<0.01）， pronounced pathological changes in the skin tissue like epidermal thickening， scabbing， small abscess， and severe injury， elevated MDA， NOS， IL-1， IL-6 and TNF-α （P<0.05， P<0.01）， lowered SOD， T-AOC， Nrf2， Keap1， NQO1 and GCLC mRNA expression （P<0.05，P<0.01）， and up-regulated Bach1 mRNA expression （P<0.01）. Compared with the model group， the total flavonoids of lavender at the low， middle， and high doses all remarkably reduced the appearance score （P<0.01）， enhanced the skin moisture and elasticity （P<0.01）， diminished the MDA， NOS， IL-1， IL-6， and TNF-α （P<0.05， P<0.01）， increased SOD， T-AOC， Nrf2， Keap1， NQO1， HO-1 and GCLC mRNA expression （P<0.05， P<0.01）， and down-regulated the expression of Bach1 mRNA （P<0.01）. ConclusionThe protective effect of the total flavonoids of lavender against skin photoaging in mice is significant， which may be related to its activation of Keap1/Nrf2/ARE signaling pathway， regulation of oxidative stress， and improvement of inflammatory response.