Integrating multi-level interactive network and in vivo/vitro studies to explore the protective mechanism of Ampelopsis grossedentata in hyperuricemia.

The strategies or drugs for preventing and treating Hyperuricemia (HUA) are still lacking. As a traditional Chinese medicine (TCM) with a profound history, Ampelopsis grossedentata has been shown to play diverse biological roles. The purpose of the present study was to evaluate hypouricemic effect of A. grossedentata, and investigate its involved material basis and mechanism. A HUA mice model was established to evaluate the therapeutic effects of A. grossedentata. And then some extracts from A. grossedentata were prepared, isolated and analyzed. Furthermore, network pharmacology, based on the above results, was used to discover potential active ingredients and therapeutic targets, and they were further verified and explored by molecular docking and in vitro experiments. In vivo experiments showed that A. grossedentata exerted hypouricemic effect on mice of HUA. The core active ingredients (quercetin, myricetin and dihydromyricetin etc.) and core targets (PTGS2, XOD and ABCG2 etc.) for A. grossedentata to treat HUA were predicted by network pharmacology. And molecular docking showed that the spontaneous binding activities of above components and targets were marvelous. In vitro experiments further demonstrated that A. grossedentata exerted hypouricemic effect by decreasing the levels of UA, XOD, antioxidant factors, inflammatory factors, GLUT9 and URAT1 in HK-2 cells of HUA. Taken together, this study integrates multi-level interaction network with in vivo/vitro experiments to systematically reveal the material basis and mechanism of A. grossedentata in treating HUA, which provides a scientific basis for further study of A. grossedentata and HUA.

Dieting alleviates hyperuricemia and organ injuries in uricase-deficient rats via down-regulating cell cycle pathway.

Dieting is a basic treatment for lowering hyperuricemia. Here, we aimed to determine the optimal amount of dietary food that lowers serum uric acid (SUA) without modifying the dietary ingredients in rats. Increased SUA was found in food-deprived 45-day-old uricase-deficient rats (Kunming-DY rats), and the optimal amount of dietary food (75% dietary intake) to lower SUA was established by controlling the amount of food given daily from 25% to 100% for 2 weeks. In addition to lowering SUA by approximately 22.5 ± 20.5%, the optimal amount of dietary food given for 2 weeks inhibited urine uric acid excretion, lowered the uric acid content in multiple organs, improved renal function, lowered serum triglyceride, alleviated organ injuries (e.g., liver, kidney and intestinal tract) at the histological level, and down-regulated the Kyoto Encyclopedia of Genes and Genome (KEGG) pathway of the cell cycle (ko04110). Taken together, these results demonstrate that 75% dietary food effectively lowers the SUA level without modifying dietary ingredients and alleviates the injuries resulting from uricase deficiency or hyperuricemia, the mechanism of which is associated with the down-regulation of the cell cycle pathway.

Jian Pi Shen Shi formula alleviates hyperuricemia and related renal fibrosis in uricase-deficient rats via suppression of the collagen-binding pathway.

AIM: Jian Pi Shen Shi Formula (JPSSF) is a beneficial treatment for hyperuricemia and related tissue damage in the clinical setting. This study was designed to investigate its therapeutic potential and underlying mechanisms in uricase-deficient rats (Uox-/- rats). METHODS: Uox-/- rats were used to assess the therapeutic potential of JPSSF on hyperuricemia. Protein extracts from renal tissues of a Uox-/- group and a JPSSF group were analyzed using tandem mass tag labeling quantitative proteomic workflow. Collagen deposition in Uox-/- rat kidneys was analyzed by Masson trichromatic staining. The gene expression associated with collagen-binding-related signaling pathways in the kidneys was further explored using quantitative polymerase chain reaction assay. The protein expressions of collagen 1a1 (col1a1), col6a1, and α-smooth muscle actin were measured by Western blotting and immunohistochemistry. RESULTS: JPSSF significantly decreased renal function indices and alleviated renal injuries. The action of JPSSF was manifested by down-regulation of col6a1 and interleukin-1 receptor-associated kinase-like 2, which blocked the binding sites on collagen and further prevented kidney injury. The anti-renal fibrosis effect of JPSSF was confirmed by reducing the collagen deposition and hydroxyproline concentrations. JPSSF treatment also intensely down-regulated the mRNA and protein expressions of col6a1, col1a1, and α-smooth muscle actin, which inhibited the function of the collagen-binding-related signaling pathway. CONCLUSION: Our results indicated that JPSSF notably ameliorated hyperuricemia and related renal fibrosis in Uox-/- rats through lowering uric acid and down-regulating the function of the collagen-binding pathway. This suggested that JPSSF is a potential empirical formula for treating hyperuricemia and accompanying renal fibrosis.

Macromolecules in polysorbate 80 for injection: an important cause of anaphylactoid reactions.

Polysorbate 80 for injection (TW80) is a common excipient used for injection whose macromolecular impurities, including those that cause anaphylactoid reactions, are frequently ignored. The main aim of this study was to prove that the macromolecular impurities in the excipient are an important cause of anaphylactoid reactions. Component A (containing macromolecules > 100 kDa), Component B (containing macromolecules from 10 to 100 kDa), and Component C (containing substances < 10 kDa) were prepaired from the original TW80 using ultrafilters. The original TW80 contained numerous substances with molecular weights > 10kD. The original TW80 and Components A and B caused strong anaphylactoid reactions in both guinea pigs and rabbits by intravenous administration. Moreover, the original TW80 and Components A and B even caused strong passive cutaneous anaphylactoid (PCA) reactions and pulmonary capillary permeability. The PCA reaction and increased permeability were partly prevented by cromolyn sodium. Additionally, the original TW80 and Components A and B caused vasodilation and severe hemolysis in vitro. The anaphylactoid reactions were associated with histamine release but not with mast cell degranulation. Nevertheless, Component C almost caused no anaphylactoid reactions or hemolysis and was weaker in the few reactions that ocurred. Taken together, these results suggest that macromolecular substances are one of the main risk factors responsible for anaphylactoid reactions and hemolysis caused by TW80.

Uricase-deficient rats with similarly stable serum uric acid to human's are sensitive model animals for studying hyperuricemia.

The aim of this study was to provide a sensitive model animal for studying hyperuricemia. Male uricase-deficient rats, named Kunming-DY rats, were raised for 130 days, or orally administered with purines and other chemicals. Serum uric acid (SUA) in the animals was assayed, and the UA level in their organs and their 24-h excretion was determined. Genes in the jejunum, ileum, kidney and liver related to UA synthesis and transportation were detected by quantitative RNA sequencing. Uricase-deficient rats have a high level of SUA and are sensitive to xanthine, adenosine, inosine, allopurinol, and alcohol. Besides, the high level of SUA in male uricase-deficient rats was stable, much higher than that in wild-type rats but similar to that in men. The distribution pattern of UA in uricase-deficient rats' organs was different from that in wild-type rats. The kidney, liver, and small intestine were the top three organs where UA distributed, but the UA in the small intestine, colon, lung, thymus, and brain was less affected by uricase deficiency, indicating that these organs are constitutive distribution organs in UA. The 24-h UA excreted by a uricase-deficient rat was about five times higher than that excreted by a wild-type rat. However, the 24-h UA excreted through feces was not significantly changed. Both the urine volume and UA in uricase-deficient rats significantly increased, and more than 90% of UA was excreted via urine. The expression of xanthine dehydrogenase was not upregulated. Some genes of transporter associated with uric acid excretion in the kidney were significantly regulated, though not sufficient to explain the increase in SUA. In conclusion, male uricase-deficient rats' UA metabolism is similar to that of men. The elevation of SUA in uricase-deficient rats is caused by uricase deficiency, and uricase-deficient rats are a sensitive model for studying hyperuricemia.

Uricase deficiency causes mild and multiple organ injuries in rats.

Uricase-deficient rats could be one of the optimal model animals to study hyperuricemia. The present study aimed to find the biological differences between uricase-deficient (Kunming-DY rats) and wild-type male rats. Uricase-deficient rats and wild-type rats were commonly bred. Their body weight, water and food consumption, 24-h urine and feces, uric acid in serum and organs, and serum indexes were recorded or assayed. Organs, including the heart, liver, spleen, lung, kidney, thymus, stomach, duodenum, and ileum, were examined using a routine hematoxylin-eosin staining assay. We found that the growth of male uricase-deficient rats was retarded. These rats excreted more urine than the wild-type rats. Their organ indexes (organ weight body weight ratio), of the heart, liver, kidney, and thymus significantly increased, while those of the stomach and small intestine significantly decreased. The uricase-deficient rats had a significantly higher level of serum uric acid and excreted more uric acid via urine at a higher concentration. Except for the liver, uric acid increased in organs and intestinal juice of uricase-deficient rats. Histological examination of the uricase-deficient rats showed mild injuries to the heart, liver, spleen, lung, kidney, thymus, stomach, duodenum, and ileum. Our results suggest that uricase-deficient rats have a different biological pattern from the wild-type rats. Uricase deficiency causes growth retardation of young male rats and the subsequent increase in serum uric acid results in mild organs injuries, especially in the kidney and liver.

Aboriginal Bacterial Flora in the Uricase-Deficient Rat Gut is Not the Main Factor Affecting Serum Uric Acid.

The relationship between intestinal bacteria and hyperuricemia is a hot research topic. To better understand this relationship, uricase-deficient Sprague-Dawley rats (Kunming-DY rats) were used. The wild-type rats and Kunming-DY rats were used as controls. Kunming-DY rats were treated with ampicillin (90 mg/kg) and ciprofloxacin (150 mg/kg) for 5 days. Bacterial 16S rDNA in the fresh stool was sequenced, and the abundance was calculated. The rats' serum uric acid (SUA) level was assayed, and the rats' intake and output in 24 h were recorded. The bacterial diversity in three groups' fresh stool was analyzed. The gut bacterial diversity and abundance changed in the Kunming-DY rats. More than 99% of bacteria were inhibited or killed by the combination of antibiotics. In contrast to each of the antibiotics alone, the combination of antibiotics lowered the Kunming-DY rats' SUA level; it also caused mild diarrhea, which increased uric acid excretion through stool. These results suggested that the aboriginal gut bacteria in uricase-deficient rats play a minor role in determining the SUA levels. It is too early to conclude that aboriginal gut bacteria are a tempting target for lowering SUA levels.

A Network Pharmacology Approach to Explore the Potential Mechanisms of Yifei Sanjie Formula in Treating Pulmonary Fibrosis.

OBJECTIVE: Yifei Sanjie Formula (YFSJF) is an effective formula on pulmonary fibrosis (PF), which has been used in clinic for more than 30 years. In order to investigate the molecular mechanism of YFSJF in treating PF, network pharmacology was used to predict the cooperative ingredients and associated pathways. METHODS: Firstly, we collected potential active ingredients of YFSJF by TCMSP databases. Secondly, we obtained PF-associated targets through OMIM and Genecards database. Finally, metascape was applied for the analysis of GO terms and KEGG pathways. RESULTS: We screened out 76 potential active ingredients and 98 associated proteins. A total of 5715 items were obtained by GO enrichment analysis (P < 0.05), including 4632 biological processes, 444 cellular components, and 639 molecular functions. A total of 143 related KEGG pathways were enriched (P < 0.05), including IL-17 signaling pathway, T cell receptor signaling pathway, TNF signaling pathway, calcium signaling pathway, TH17 cell differentiation, HIF-1 signaling pathway, and PI3K-Akt signaling pathway. CONCLUSION: YFSJF can interfere with immune and inflammatory response through multiple targets and pathways, which has a certain role in the treatment of PF. This study lays a foundation for future experimental research.

Corrigendum to "Excretory Function of Intestinal Tract Enhanced in Kidney Impaired Rats Caused by Adenine".

[This corrects the article DOI: 10.1155/2016/2695718.].

Macromolecular substances as a dangerous factor in traditional Chinese medicine injections were determined by size-exclusive chromatography.

Macromolecular substances in traditional Chinese medicine injections (TCMIs) are expected to be a main dangerous factor causing anaphylactic or anaphylactoid reaction. The main aim of the study was to verify the macromolecular substances' anaphylactic or anaphylactoid reaction in guinea pigs and establish a size-exclusive chromatographic method to detect them. The macromolecular substances from six TCMIs (Danshen injection, Dengzhanxixin injection, Honghua injection, Qingkailing injection, Shuanghuanglian injection and Shuxuening injection) were prepared by removing substances with molecular weight less than 10 kDa with an ultra-filter. The anaphylactic and anaphylactoid reactions caused by original TCMIs, injections rich in or free of macromolecules were assayed in guinea pigs. The relationship between the amount of the macromolecular substances and peak area of chromatogram was established by size-exclusive chromatography. Injections free of macromolecules were not likely to cause anaphylactic and anaphylactoid reactions, but injections rich in macromolecular substances were more likely to do so. If the macromolecular substances with molecular weight bigger than 10 kDa were removed, the signal of macromolecular substances in TCMIs was quantitatively reduced. All the results suggested that macromolecular substances in TCMIs are a dangerous factor causing safety problems, and the macromolecular substances can be quantitatively detected with size-exclusive chromatography.

Uricase-deficient rat is generated with CRISPR/Cas9 technique.

Urate oxidase (uricase, Uox) is a big obstacle for scientists to establish stable animal models for studying hyperuricemia and associated disorders. Due to the low survival rate of uricase-deficient mice, we generated a Uox-knockout model animal from Sprague Dawley (SD) rats using the CRISPR/Cas9 technique by deleting exons 2 to 4 of the Uox gene. The uricase-deficient rats were named "Kunming-DY rats", and were apparently healthy with more than a 95% survival up to one year. The male rats' serum uric acid (SUA) increased to 48.3  ± 19.1 µg/ml, significantly higher than those of wild-type rats. Some indexes of the blood fat like total triglyceride, low density lipoprotein, and renal function indexes including blood urea nitrogen and serum creatinine were significantly different from those of wild-type rats, however, all the indexes were close to or in normal ranges. Histological renal changes including mild glomerular/tubular lesions were observed in these uricase-deficient rats. Thus, "Kunming-DY rats" with stable uricase-deficiency were successfully established and are an alternative model animal to study hyperuricemia and associated diseases mimicking human conditions.

A pilot study on searching for peri-nuclear NeuN-positive cells.

The aim of this study was to find out neuron (-like) cells in peripheral organs by cell markers in rats. Adult male Sprague-Dawley rats were anaesthetized. Their organs including brain, heart, lung, liver, kidney, stomach, duodenum, and ileum were harvested. The mRNA and protein in these organs were extracted. RNA sequencing (RNA-Seq) was carried out, and NeuN, a "specific" marker for neuronal soma, was assayed with Western blotting. The sections of the aforementioned organs were obtained after a routine fixation (4% methanal)-dehydration (ethanol)-embedding (paraffin) process. NeuN in the sections and seven non-neuronal cell lines was analyzed by immunofluorescence (IF) or immunohistochemistry (IHC). Neuronal markers, such as Eno2, NeuN (Rbfox3), choline acetyltransferase (Chat), as well as tyrosine hydroxylase (Th), and neuronal-glial markers, e.g., glial fibrillary acidic protein (Gfap), S100b, 2', 3'-cyclic nucleotide 3'-phosphodiesterase (Cnp), and other related markers, were positively expressed in all the organs at mRNA level. NeuN was further analyzed by Western blotting. The IF and IHC assays showed that NeuN-positive cells were distributed in all the peripheral tissues (mainly peri-nuclear NeuN-positive cells) though with different patterns from that in brain (nuclear NeuN-positive cells), and a NeuN-negative tissue could not be found. Especially, NeuN and Myl3 co-expressed in the cytoplasm of myocardial cells, suggesting that NeuN could possess other functions than neuronal differentiation. Also, the protein was positively expressed in seven non-neuronal cell lines. Our findings suggested that NeuN-positive cells exist widely, and without identification of its distribution pattern, the specificity of NeuN for neurons could be limited.

What Is the Biological Function of Uric Acid? An Antioxidant for Neural Protection or a Biomarker for Cell Death.

The main aim of the present study was to investigate the biological function of uric acid. The level of uric acid in different organs in normal male rats was determined with uric acid assay kits, and the expression level of genes in the organs was determined by RNA quantitative sequencing. The correlation analysis between uric acid in the organs and gene expression (measured by FPKM value) was made. Serum uric acid (SUA) in patients with breast cancer or with breast benign tumor was assayed when the diagnosis was made, and SUA in patients with breast cancer was also assayed just after chemotherapy. There were 1937 mRNAs whose expression level significantly correlated with the level of uric acid, and most of which were associated with purine or nucleoside metabolism, cellular metabolism, cell cycles, and cell death pathways. Further analysis showed that the level of uric acid was highly correlated with cell death rather than cell viability. The level of SUA in patients with breast cancer was higher than that in patients with breast benign tumor, and the SUA increased after chemotherapy. All the results suggested that uric acid was mainly synthesized from local nucleosides degraded from dead cells, and uric acid could be an important biomarker for cell death rather than an antioxidant for neural protection.

Intestinal tract is an important organ for lowering serum uric acid in rats.

The kidney was recognized as a dominant organ for uric acid excretion. The main aim of the study demonstrated intestinal tract was an even more important organ for serum uric acid (SUA) lowering. Sprague-Dawley rats were treated normally or with antibiotics, uric acid, adenine, or inosine of the same molar dose orally or intraperitoneally for 5 days. Rat's intestinal tract was equally divided into 20 segments except the cecum. Uric acid in serum and intestinal segment juice was assayed. Total RNA in the initial intestinal tract and at the end ileum was extracted and sequenced. Protein expression of xanthine dehydrogenase (XDH) and urate oxidase (UOX) was tested by Western blot analysis. The effect of oral UOX in lowering SUA was investigated in model rats treated with adenine and an inhibitor of uric oxidase for 5 days. SUA in the normal rats was 20.93±6.98 µg/ml, and total uric acid in the intestinal juice was 308.27±16.37 µg, which is two times more than the total SUA. The uric acid was very low in stomach juice, and attained maximum in the juice of the first segment (duodenum) and then declined all the way till the intestinal end. The level of uric acid in the initial intestinal tissue was very high, where XDH and most of the proteins associated with bicarbonate secretion were up-regulated. In addition, SUA was decreased by oral UOX in model rats. The results suggested that intestinal juice was an important pool for uric acid, and intestinal tract was an important organ for SUA lowering. The uric acid distribution was associated with uric acid synthesis and secretion in the upper intestinal tract, and reclamation in the lower.

Excretory Function of Intestinal Tract Enhanced in Kidney Impaired Rats Caused by Adenine.

The main aim of the study was to prove the compensative effect of intestine for renal function. Rat kidney was impaired by intragastrically administrating adenine (400 mg per day for 5 days). Intestinal tract was harvested and equally divided into 20 segments except cecum. Kidneys were harvested and histologically examined with hematoxylin-eosin staining kits. Uric acid, urea (BUN), and creatinine in serum were determined with assay kits, and BUN and creatinine in every intestinal segment were also determined. The results showed that adenine was able to increase uric acid level in serum from 20.98 ± 6.98 µg/mL to 40.77 ± 7.52 µg/mL and cause renal function damage with BUN (from 3.87 ± 0.62 mM to 12.33 ± 3.27 mM) and creatinine (from 51.48 ± 6.98 µM to 118.25 ± 28.63 µM) increasing in serum and with abnormally micromorphological changes in kidney. The amount of BUN and creatinine distributed in intestinal tract was positively correlated with those in blood. In impaired renal function rats, the amount of BUN (from 4.26 ± 0.21 µMole to 10.72 ± 0.55 µMole) and creatinine (from 681.4 ± 23.3 nMole to 928.7 ± 21.3 nMole) distributed in intestinal tract significantly increased. All the results proved that intestinal tract had excretory function compensative for renal function.

Neurite outgrowth resistance to rho kinase inhibitors in PC12 Adh cell.

Rho kinase (ROCK) inhibitor is a promising agent for neural injury disorders, which mechanism is associated with neurite outgrowth. However, neurite outgrowth resistance occurred when PC12 Adh cell was treated with ROCK inhibitors for a longer time. PC12 Adh cells were treated with ROCK inhibitor Y27632 or NGF for different durations. Neurite outgrowth resistance occurred when PC12 Adh cell exposed to Y27632 (33 µM) for 3 or more days, but not happen when exposed to nerve growth factor (NGF, 100 ng/mL). The gene expression in the PC12 Adh cells treated with Y27632 (33 µM) or NGF (100 ng/mL) for 2 or 4 days was assayed by gene microarray, and the reliability of the results were confirmed by real-time RT-PCR. Cluster analysis proved that the gene expression profile of PC12 Adh cell treated with Y27632 for 4 days was different from that treated with Y27632 for 2 days and those treated with NGF for 2 and 4 days, respectively. Pathway analysis hinted that the neurite outgrowth resistance could be associated with up-regulation of inflammatory pathways, especially rno04610 (complement and coagulation cascades), and down-regulation of cell cycle pathways, especially rno04110.

Combination of fasudil and celecoxib promotes the recovery of injured spinal cord in rats better than celecoxib or fasudil alone.

Resistance mechanisms of rho-associated kinase (ROCK) inhibitors are associated with the enhanced expression of cyclooxygenase-2 (COX-2). The therapeutic effects of ROCK on nervous system diseases might be enhanced by COX-2 inhibitors. This study investigated the synergistic effect of the combined use of the ROCK inhibitor fasudil and a COX-2 inhibitor celecoxib on spinal cord injury in a rat model established by transecting the right half of the spinal cord at T11. Rat models were orally administrated with celecoxib (20 mg/kg) and/or intramuscularly with fasudil (10 mg/kg) for 2 weeks. Results demonstrated that the combined use of celecoxib and fasudil significantly decreased COX-2 and Rho kinase II expression surrounding the lesion site in rats with spinal cord injury, improved the pathomorphology of the injured spinal cord, and promoted the recovery of motor function. Moreover, the effects of the drug combination were better than celecoxib or fasudil alone. This study demonstrated that the combined use of fasudil and celecoxib synergistically enhanced the functional recovery of injured spinal cord in rats.

Nitric oxide and interleukins are involved in cell proliferation of RAW264.7 macrophages activated by viili exopolysaccharides.

Viili has been traditionally regarded as healthy food; viili exopolysaccharides (VEPS) function as antioxidants, but the molecular and cellular mechanisms, especially its immune functions, remain largely unclear. To assess VEPS's immunological roles, VEPS were separated by Sevage's method and purified by anion exchange chromatography. Cell proliferation, phagocytosis, releases of nitric oxide (NO), interleukin (IL)-1ß, and IL-6, the inducible nitric oxide synthase (iNOS) gene expression by reverse transcription polymerase chain reaction (RT-PCR) and iNOS protein by Western blotting, and morphology by scanning electron microscopy in lipopolysaccharides (LPS)/VEPS-stimulated and non-stimulated RAW264.7 macrophages were analyzed. VEPS increased cell proliferation at 50-200 µg/mL. The uptake of neutral red for the indication of phagocytosis and releases of NO, IL-6, and IL-1ß were enhanced after exposure to LPS and VEPS. Gene expressions of iNOS, IL-6, and IL-1ß and protein expressions of iNOS were increased with VEPS. The RAW264.7 cell treated with VEPS became flattened, a strong indication of the activation of macrophages. We concluded that VEPS promoted the activation of macrophages in which NO, IL-6, and IL-1ß were involved; the release of NO and other cytokines may eventually activate lymphocytes, increasing nonspecific (innate) and specific immunity in humans.

Tolerance of neurite outgrowth to Rho kinase inhibitors decreased by cyclooxygenase-2 inhibitor.

In this study, PC12 Adh cells and Neuro-2a cells were treated with Rho-associated kinase inhibitors (Y27632 and Fasudil), a cyclooxygenase-1 selective inhibitor (SC560), and a cyclooxygenase-2 inhibitor (NS398). We found that these cells became tolerant to Rho-associated kinase inhibitors, as neurite outgrowth induced by these inhibitors diminished following more than 3 days of exposure in either cell line. The proteins cyclooxygenase-2 and cytosolic prostaglandin E synthetase were upregulated at day 3. NS398 decreased the tolerance to neurite outgrowth induction in both cell lines, whereas SC560 had almost no effect. These findings indicate that cells become tolerant to neurite outgrowth induced by Rho-associated kinase inhibitors, this is at least partly associated with upregulation of proteins involved in the cyclooxygenase-2 pathway, and cyclooxygenases-2 inhibition prevents this tolerance.

Rho kinase inhibitor Y-27632 down-regulates norepinephrine synthesis and release in PC12 cells.

Rho kinase inhibition is beneficial for neurite outgrowth and nerve disorders, and the Rho kinase inhibitors have been regarded as promising agents to treat neural diseases. The main aim of the study was to elucidate how Rho kinase inhibitor Y-27632 regulates neurotransmitter norepinephrine synthesis and release in PC12 cells when neurite outgrowth was induced. PC12 cells were treated with Y-27632 for 6 days. The amount of norepinephrine synthesized in PC12 cells and the amount released evoked by acetylcholine or by KCl were determined by norepinephrine enzyme-linked immunosorbent assay kits. The results showed that the amount of norepinephrine both synthesized and released was down-regulated with a concentration-dependent relationship. Further results of Western blotting found that the protein expression of tyrosine hydroxylase and synapsin I (especially its active form, synapsin I phosphoSer603) was also down-regulated, which were directly related to synthesis and release of norepinephrine, respectively. All the results suggest that Y-27632 is able to down-regulate norepinephrine synthesis and release, the direct mechanism of which may be associated with down-regulation on expression of some proteins, including tyrosine hydroxylase and synapsin I.