The detoxification activities and mechanisms of microcystinase towards MC-LR.

Microcystins (MCs) are the most common and toxic cyanotoxins that are hazardous to human health and ecosystems. Microcystinase is the enzyme in charge of the initial step in the biodegradation of MCs. The characterization, application conditions, and detoxification mechanisms of microcystinase from an indigenous bacterium Sphingopyxis sp. YF1 towards MC-LR were investigated in the current study. The microcystinase gene of strain YF1 was most similar to Sphingomonas sp. USTB-05 and contained a CAAX-family conversed abortive Infection (ABI) domain. The microcystinase was successful obtained and purified by overexpression in Escherichia coli. The highest degradation rate of MC-LR was 1.0 µg/mL/min under the optimal condition of 30 â„ƒ, pH 7, 20 µg/mL MC-LR, and 400 µg/mL microcystinase. The MC-degrading product was identified as linearized MC-LR, which possessed a much lower inhibitory activity against protein phosphatase 2A than MC-LR. Microcystinase interacted with MC-LR via amino acid residues involved in through the formation of conventional Hydrogen Bond, Pi-Pi T-shapes, Van der Waals force, and so on. The optimal MC-degrading condition of pure microcystinase and its detoxification mechanisms against MC-LR were revealed. The toxicity of purified linearized MC-LR was explored for the first time. These findings suggest that pure microcystinase may efficiently detoxify MCs and it is promising in the bioremediation of MC-polluted environments.

Glucocorticoid-induced loss of beneficial gut bacterial extracellular vesicles is associated with the pathogenesis of osteonecrosis.

Osteonecrosis of the femoral head (ONFH) commonly occurs after glucocorticoid (GC) therapy. The gut microbiota (GM) participates in regulating host health, and its composition can be altered by GC. Here, this study demonstrates that cohousing with healthy mice or colonization with GM from normal mice attenuates GC-induced ONFH. 16S rRNA gene sequencing shows that cohousing with healthy mice rescues the GC-induced reduction of gut Lactobacillus animalis. Oral supplementation of L. animalis mitigates GC-induced ONFH by increasing angiogenesis, augmenting osteogenesis, and reducing cell apoptosis. Extracellular vesicles from L. animalis (L. animalis-EVs) contain abundant functional proteins and can enter the femoral head to exert proangiogenic, pro-osteogenic, and antiapoptotic effects, while its abundance is reduced after exposure to GC. Our study suggests that the GM is involved in protecting the femoral head by transferring bacterial EVs, and that loss of L. animalis and its EVs is associated with the development of GC-induced ONFH.

Ultrasensitive label-free electrochemical biosensor for detecting linear microcystin-LR using degrading enzyme MlrB as recognition element.

A label-free electrochemical biosensor was firstly constructed to detect linear microcystin-LR (L-MC-LR) with high sensitivity. Degradation enzyme MlrB was used as recognition element for specific recognition of L-MC-LR. The electrode was modified with -COOH functionalized multi-walled carbon nanotube to increase the specific surface area and improve the conductivity, which was then applied to immobilize MlrB. The electrochemical signal was changed with the reaction between MlrB and L-MC-LR, which was recorded by using square wave voltammetry. The electrochemical biosensor showed superior sensitivity, with a dynamic range of 1 pg/mL to 100 ng/mL and a detection limit of 0.127 pg/mL. Moreover, the fabricated electrochemical biosensor exhibited excellent specificity toward L-MC-LR in real water samples. The concentrations of spiked L-MC-LR were 0.100, 5.00, 50.0 ng/mL, and the recovery rates were 95.0-104% with relative standard deviation (RSD) of 0.900-2.30% and 74.0-93.0% with RSD of 2.30-3.50% in lake water and tap water, respectively. Furthermore, the selectivity, reproducibility, and stability demonstrated the potential of degradation enzymes as recognition element in detection of cyanotoxins.

Extracellular Vesicles from Child Gut Microbiota Enter into Bone to Preserve Bone Mass and Strength.

Recently, the gut microbiota (GM) has been shown to be a regulator of bone homeostasis and the mechanisms by which GM modulates bone mass are still being investigated. Here, it is found that colonization with GM from children (CGM) but not from the elderly (EGM) prevents decreases in bone mass and bone strength in conventionally raised, ovariectomy (OVX)-induced osteoporotic mice. 16S rRNA gene sequencing reveals that CGM reverses the OVX-induced reduction of Akkermansia muciniphila (Akk). Direct replenishment of Akk is sufficient to correct the OVX-induced imbalanced bone metabolism and protect against osteoporosis. Mechanistic studies show that the secretion of extracellular vesicles (EVs) is required for the CGM- and Akk-induced bone protective effects and these nanovesicles can enter and accumulate into bone tissues to attenuate the OVX-induced osteoporotic phenotypes by augmenting osteogenic activity and inhibiting osteoclast formation. The study identifies that gut bacterium Akk mediates the CGM-induced anti-osteoporotic effects and presents a novel mechanism underlying the exchange of signals between GM and host bone.

Optimization of microcystin biodegradation by bacterial community YFMCD4 using response surface method.

The increasing production of microcystin-LR (MC-LR) causing animal and human health issues is found in eutrophic water bodies, marine habitats and desert environments. The health threat posed by MC-LR has led to the establishment of World Health Organization's water guideline value of 1 µg/mL. Combating this has increased the search for cost-effective approach to degrade MC-LR. The study aimed to optimize the MC-degrading environmental factors of bacterial community YFMCD4. Response surface methodology (RSM) was employed to evaluate the influence of varying temperatures, pH and initial MC-LR concentration on the biodegradation efficiency of MC-LR by bacterial community YFMCD4. The optimal MC-LR biodegradation environmental factors were found to be 30 °C, pH 7 and 2 µg/mL initial MC-LR. The biodegradation rate reached 100% after 10 h. YFMCD4 mainly consisted of genera Alacligenes, Sphingobacterium and Pseudomonas using High-throughput pyrosequencing technology. The mlrA gene encoding MlrA enzyme considered most important for MC-LR biodegradation was obtained from YFMCD4. Data demonstrated that the bacterial structure and biodegradation efficiency of YFMCD4 varied with the change of environmental factors including temperature, pH and MC-LR concentrations. RSM is considered a good method to examine the optimal biodegradation environmental conditions for MC-LR. To date, RSM and High-throughput pyrosequencing technology are employed to optimize the biodegradation conditions (30 °C, pH 7 and 2 µg/mL initial MC-LR) and analyze the structure of bacterial community for the first time.

Characterization and Mechanism of Linearized-Microcystinase Involved in Bacterial Degradation of Microcystins.

Microcystins (MCs) are extremely hazardous to the ecological environment and public health. How to control and remove MCs is an unsolved problem all over the world. Some microbes and their enzymes are thought to be effective in degrading MCs. Microcystinase can linearize microcystin-leucine-arginine (MC-LR) via a specific locus. However, linearized MC-LR is also very toxic and needs to be removed. How linearized MC-LR was metabolized by linearized-microcystinase, especially how linearized-microcystinase binds to linearized MC-LR, has not been defined. A combination of in vitro experiments and computer simulation was applied to explore the characterization and molecular mechanisms for linearized MC-LR degraded by linearized-microcystinase. The purified linearized-microcystinase was obtained by recombinant Escherichia coli overexpressing. The concentration of linearized MC-LR was detected by high-performance liquid chromatography, and linearized MC-LR degradation products were analyzed by the mass spectrometer. Homology modeling was used to predict the structure of the linearized-microcystinase. Molecular docking techniques on the computer were used to simulate the binding sites of linearized-microcystinase and linearized MC-LR. The purified linearized-microcystinase was obtained successfully. The linearized-microcystinase degraded linearized MC-LR to tetrapeptide efficiently. The second structure of linearized-microcystinase consisted of many alpha-helices, beta-strands, and colis. Linearized-microcystinase interacted the linearized MC-LR with hydrogen bond, hydrophobic interaction, electrostatic forces, and the Van der Waals force. This study firstly reveals the characterization and specific enzymatic mechanism of linearized-microcystinase for catalyzing linearized MC-LR. These findings encourage the application of MC-degrading engineering bacteria and build a great technique for MC-LR biodegradation in environmental engineering.

Genome-Wide Analysis Reveals Genetic Potential for Aromatic Compounds Biodegradation of Sphingopyxis.

Members of genus Sphingopyxis are frequently found in diverse eco-environments worldwide and have been traditionally considered to play vital roles in the degradation of aromatic compounds. Over recent decades, many aromatic-degrading Sphingopyxis strains have been isolated and recorded, but little is known about their genetic nature related to aromatic compounds biodegradation. In this study, bacterial genomes of 19 Sphingopyxis strains were used for comparative analyses. Phylogeny showed an ambiguous relatedness between bacterial strains and their habitat specificity, while clustering based on Cluster of Orthologous Groups suggested the potential link of functional profile with substrate-specific traits. Pan-genome analysis revealed that 19 individuals were predicted to share 1,066 orthologous genes, indicating a high genetic homogeneity among Sphingopyxis strains. Notably, KEGG Automatic Annotation Server results suggested that most genes pertaining aromatic compounds biodegradation were predicted to be involved in benzoate, phenylalanine, and aminobenzoate metabolism. Among them, ß-ketoadipate biodegradation might be the main pathway in Sphingopyxis strains. Further inspection showed that a number of mobile genetic elements varied in Sphingopyxis genomes, and plasmid-mediated gene transfer coupled with prophage- and transposon-mediated rearrangements might play prominent roles in the evolution of bacterial genomes. Collectively, our findings presented that Sphingopyxis isolates might be the promising candidates for biodegradation of aromatic compounds in pollution sites.

A complete route for biodegradation of potentially carcinogenic cyanotoxin microcystin-LR in a novel indigenous bacterium.

Microcystin-leucine-arginine (MC-LR), a cyclic potentially carcinogenic hepatotoxin, occurs frequently in aquatic habitats worldwide and seriously threatens ecosystem and public health. Limited effectiveness of physicochemical treatments to remove MC-LR from drinking water has led to a search for alternative cost-effective and environment friendly biodegradation strategies. Obtaining MC-degrading bacteria and understanding their MC-degrading mechanisms are outstanding challenges. Here, a novel indigenous bacterium named Sphingopyxis sp. YF1 with a high efficient capacity for MC-degradation was successfully isolated from eutrophic Lake Taihu. Through integrating mass spectrometer and multi-omics analyses accompanied by functional verification of certain genes and proteins, a complete MC-degradation pathway was firstly identified, in which MC-LR was sequentially degraded into linearized MC-LR, tetrapeptide, Adda, phenylacetic acid, and finally potential product CO2. Some specific proteins such as microcystinase, linearized-microcystinase, tetrapeptidease and PAAase responsible for this pathway were identified. This study pioneeringly demonstrated that MC-LR can be completely degraded through natural remediation processes and revealed a significant potential for MC-LR biodegradation in both natural environment and engineered systems.

Simultaneous Microcystis algicidal and microcystin synthesis inhibition by a red pigment prodigiosin.

Microcystis blooms and their secondary metabolites microcystins (MCs) occurred all over the world, which have damaged aquatic ecosystems and threatened public health. Techniques to reduce the Microcystis blooms and MCs are urgently needed. This study aimed to investigate the algicidal and inhibitory mechanisms of a red pigment prodigiosin (PG) against the growth and MC-producing abilities of Microcystis aeruginosa (M. aeruginosa). The numbers of Microcystis cells were counted under microscope. The expression of microcystin synthase B gene (mcyB) and concentrations of MCs were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme linked immunosorbent assay (ELISA) methods, respectively. The inhibitory effects of PG against M. aeruginosa strain FACHB 905 with 50% algicidal concentration (LC50) at 120 h was 0.12 µg/mL. When M. aeruginosa cells exposed to 0.08 µg/mL, 0.16 µg/mL, 0.32 µg/mL PG, the expression of mcyB of M. aeruginosa was down-regulated 4.36, 8.16 and 18.51 times lower than that of the control at 120 h. The concentrations of total MC (TMC) also were 1.66, 1.72 and 5.75 times lower than that of the control at 120 h. PG had high algicidal effects against M. aeruginosa, with the activities of superoxide dismutase (SOD) initially increased and then decreased after 72 h, the contents of malondialdehyde (MDA) increase, the expression of mcyB gene down-regulation, and MCs synthesis inhibition. This study was first to report the PG can simultaneously lyse Microcystis cells, down-regulate of mcyB expression and inhibit MCs production effectively probably due to oxidative stress, which indicated PG poses a great potential for regulating Microcystis blooms and MCs pollution in the environment.

Identification and characterization of the dominant Microcystis sp. cyanobacteria detected in Lake Dong Ting, China.

The presence of cyanobacteria in drinking water, aquatic foods and bathing water has created a significant major problem to global public health as these toxins induce damage in various organ including liver, cardiovascular, intestinal and central nervous systems. Although the morphologic, phylogenetic and toxicogenetic characteristics of cyanobacteria were identified in several lakes in China, many freshwater sources such as Dong Ting Lake, Hunan Province, China remain to be determined. Since the presence of these cyanobacteria may potentially affect human health, the aim of this study was to isolate, identify and characterize the most frequent occurring bloom-forming cyanobacteria in Dong Ting Lake, Hunan Province, China, which can provide information on the safety of utilization of this water source for drinking water, agriculture and recreation. Samples collected from the surface water of Dong Ting Lake were subjected to serial dilution in the lab for morphological analysis. Data demonstrated the morphological features were 2-5 µm diameters with rounded shapes and green color resembling Microcystis sp. The isolated cyanobacterial strain obtained from surface water samples in Dong Ting Lake was termed Microcystis sp. YFM2. The MC concentration was detected by enzyme-linked immunosorbent assay (ELISA) and found to be 92.88 µg/107 cells in Microcystis sp. YFM2. By polymerase chain reaction (PCR) results indicated that Microcystis sp. YFM2 isolated from Dong Ting Lake contained synthetase genes (mcyA-C). Our findings indicated that the dominant cyanobacteria Microcystis sp. YFM2 isolated from the freshwater Dong Ting Lake demonstrated morphologic, phylogenetic and toxicogenetic properties resembling a toxin generating cyanobacterium. Based upon this knowledge, it is essential to monitor the use of this Lake for future domestic, agricultural and recreational purposes.

Aromatic hydrocarbon compound degradation of phenylacetic acid by indigenous bacterial Sphingopyxis isolated from Lake Taihu.

The aromatic compound phenylacetic acid (PAA) is present in the environment, and released in the catabolism of phenylalanine, 2-phenylethylamine, or environmental contaminants such as ethylbenzene and styrene. PAA was also proposed to be involved in human chronic kidney disease development. Several bacteria and fungi utilize these aromatic acids as sole carbon source either during aerobic or anaerobic conditions. The aromatic structure of PAA makes this compound resistant toward oxidation or reduction, because the stabilizing resonance energy of the aromatic ring system is difficult to overcome. In the case of bacteria that utilize aromatic compounds as growth substrates, the aromatic ring system limits survival due to a lack of carbon source. Sphingopyxis sp. YF1 isolated from Lake Taihu was found to be beneficial in bioremediation of aromatic compounds. This study thus aimed to examine the influence of environmental factors such as temperature, PAA concentration, and pH on the effectiveness of Sphingopyxis sp. YF1 to degrade aromatic compounds using PAA as model compound. Data showed the highest PAA-degrading rate of strain Sphingopyxis sp. YF1 was 7.6 mg/L·h under the condition of 20°C, pH 9 with a 1000 µg/ml concentration of PAA. Evidence indicates that PAA-degrading ability of strain Sphingopyxis sp. YF1 appears to be primarily influenced by the concentration of PAA, followed by temperature and pH. PAA-degrading gene PAAase was identified in this strain using polymerase chain reaction (PCR) method. These results illustrate that the bacteria Sphingopyxis sp. YF1 removes PAA effectively at certain environmental conditions and this proves beneficial in bioremediation of aromatic compounds.

Effects of Chronic Exposure to Microcystin-LR on Kidney in Mice.

Microcystin-LR (MC-LR) is a potent hepatotoxin, but a few studies suggested that it might also induce nephrotoxicity. However, nephrotoxicity induced by prolonged oral exposure to MC-LR is unknown. The aim of this study was to evaluate the potential influence of MC-LR on the kidney in mice following chronic exposure to MC-LR. In this study, we evaluated the nephrotoxicity of MC-LR in mice drinking water at different concentrations (1, 30, 60, 90, and 120 µg/L) for 6 months for the first time. The results showed that the kidney weights and the kidney indexes of mice were not altered in the MC-LR treated mice, compared with the control group. In addition, the renal function indicators revealed that the serum creatinine (SCr) levels were not significant changes after exposure to MC-LR. The blood urea nitrogen (BUN) levels were markedly decreased after exposure to 90 and 120µg/L MC-LR for 3 months. The BUN levels were lower than that of the control group after exposure to 120µg/L MC-LR for 6 months. The histopathological investigation revealed enlarged renal corpuscles, widened of kidney tubules, and lymphocyte infiltration in the interstitial tissue and the renal pelvis after exposure to 60, 90, and 120 µg/L MC-LR. Consequently, our results suggested that long-term exposure to MC-LR might be one important risk of kidney injury, which will provide important clues for the prevention of renal impairment.

Anaerobic degradation of microcystin-LR by an indigenous bacterial Enterobacter sp. YF3.

Microcystin-LR (MC-LR), a known hepatotoxin present in drinking water, and contaminated food and algal dietary supplements poses a threat to environmental and public health and thus needs to be removed. Previously microbial aerobic degradation was considered the predominant catabolic process for MC-LR inactivation, but the potential role of anaerobic microbes still needs to be determined. In our study an anaerobic MC-degrading bacterium Enterobacter sp. YF3 was isolated and identified that was capable of degrading MC-LR. Under optimal conditions the anaerobic Enterobacter sp. YF3 displayed a MC-degrading rate of 0.34 µg/ml/day. This process was dependent on temperature, pH and MC-LR concentration. Further the extracellular secretion of metabolites of anaerobic bacterium degraded MC-LR at 0.22 µg/ml/day. The parent MC-LR as well as two MC-degrading products was identified by high performance liquid chromatography (HPLC). The anaerobic MC-degrading Enterobacter sp. bacterium metabolized MC-LR independent of MC-degrading genes mlrABCD. Data indicate that anaerobic Enterobacter sp. YF3 produces MC-degrading products via a pathway that acts independently of mlrABCD genes which may add to the arsenal of bacteria to degrade microcystins.

Identification and characterization of a novel indigenous algicidal bacterium Chryseobacterium species against Microcystis aeruginosa.

Harmful Microcystis aeruginosa blooms occurred frequently in many eutrophic lakes and rivers with resultant serious global environmental consequences. Algicidal bacteria may play an important role in inhibiting the growth of Microcystis aeruginosa and are considered as an effective method for preventing the appearance of blooms. In order to counteract the harmful effects of Microcystis aeruginosa, a critical step is to identify, isolate and characterize indigenous algicidal bacteria. This study aimed to isolate a novel indigenous algicidal bacterium identified as Chryseobacterium species based upon its 16S rDNA sequence analysis, and determine whether this bacterium was effective in lysing Microcystis aeruginosa FACHB 905. The influence of environmental factors including temperature, pH, quantity of Chryseobacterium species as well as Microcystis aeruginosa concentration were examined with respect to algae-lysing properties of this bacterial strain. Data demonstrated that the highest algae-lysing activity of 80% against Microcystis aeruginosa FACHB 905 occurred within 72 hr. In addition, the algae-lysing activities of Chryseobacterium species cells were significantly higher than those of cell-free supernatant. In conclusion, data showed the algicidal bacterium Chryseobacterium species exhibited potent Microcystis aeruginosa-lysing activities and attacked Microcystis aeruginosa directly suggesting this algicidal bacterium may be potentially useful for reducing the number of harmful Microcystis aeruginosa blooms.

Effects of Microcystin-LR on the Microstructure and Inflammation-Related Factors of Jejunum in Mice.

The increasing cyanobacterial blooms have recently been considered a severe environmental problem. Microcystin-leucine arginine (MC-LR) is one of the secondary products of cyanobacteria metabolism and most harmful cyanotoxins found in water bodies. Studies show MC-LR negatively affects various human organs when exposed to it. The phenotype of the jejunal chronic toxicity induced by MC-LR has not been well described. The aim of this paper was to investigate the effects of MC-LR on the jejunal microstructure and expression level of inflammatory-related factors in jejunum. Mice were treated with different doses (1, 30, 60, 90 and 120 µg/L) of MC-LR for six months. The microstructure and mRNA expression levels of inflammation-related factors in jejunum were analyzed. Results showed that the microstructure of the jejunum was destroyed and expression levels of inflammation-related factors interleukin (IL)-1ß, interleukin (IL)-8, tumor necrosis factor alpha, transforming growth factor-ß1 and interleukin (IL)-10 were altered at different MC-LR concentrations. To the best of our knowledge, this is the first study that mice were exposed to a high dose of MC-LR for six months. Our data demonstrated MC-LR had the potential to cause intestinal toxicity by destroying the microstructure of the jejunum and inducing an inflammatory response in mice, which provided new insight into understanding the prevention and diagnosis of the intestinal diseases caused by MC-LR.

Synechococcus elongatus PCC7942 secretes extracellular vesicles to accelerate cutaneous wound healing by promoting angiogenesis.

Poor wound healing affects millions of people worldwide each year and needs better therapeutic strategies. Synechococcus elongatus PCC 7942 is a naturally occurring photoautotrophic cyanobacterium that can be easily obtained and large-scale expanded. Here, we investigated the therapeutic efficacy of this cyanobacterium in a mouse model of acute burn injury and whether the secretion of extracellular vesicles (EVs), important mediators of cell paracrine activity, is a key mechanism of the cyanobacterium-induced regulation of wound healing. Methods: The effects of Synechococcus elongatus PCC 7942 on burn wound healing in mice under light or dark conditions were evaluated by measuring wound closure rates, histological and immunofluorescence analyses. A series of assays in vivo and in vitro were conducted to assess the impact of the cyanobacterium on angiogenesis. GW4869 was used to interfere with the secretion of EVs by the cyanobacterium and the abilities of the GW4869-pretreated and untreated Synechococcus elongatus PCC 7942 to regulate endothelial angiogenesis were compared. The direct effects of the cyanobacterium-derived EVs (S. elongatus-EVs) on angiogenesis, wound healing and expressions of a class of pro-inflammatory factors that have regulatory roles in wound healing were also examined. Results: Synechococcus elongatus PCC 7942 treatment under light and dark conditions both significantly promoted angiogenesis and burn wound repair in mice. In vitro, the cyanobacterium enhanced angiogenic activities of endothelial cells, but the effects were markedly blocked by GW4869 pretreatment. S. elongatus-EVs were capable of augmenting endothelial angiogenesis in vitro, and stimulating new blood vessel formation and burn wound healing in mice. The expression of interleukin 6 (IL-6), which has an essential role in angiogenesis during skin wound repair, was induced in wound tissues and wound healing-related cells by S. elongatus-EVs and Synechococcus elongatus PCC 7942. Conclusion: Synechococcus elongatus PCC 7942 has the potential as a promising strategy for therapeutic angiogenesis and wound healing primarily by the delivery of functional EVs, not by its photosynthetic activity. The promotion of IL-6 expression may be a mechanism of the cyanobacterium and its EVs-induced pro-angiogenic and -wound healing effects.

Removal of Microcystin-LR by a Novel Native Effective Bacterial Community Designated as YFMCD4 Isolated from Lake Taihu.

Microcystin-LR (MC-LR) is the most toxic and frequently detected monocyclic heptapeptide hepatotoxin produced by cyanobacteria, which poses a great threat to the natural ecosystem and public health. It is very important to seek environment-friendly and cost-efficient methods to remove MC-LR in water. In this study, the MC-degrading capacities of a novel indigenous bacterial community designated as YFMCD4 and the influence of environmental factors including various temperatures, MC concentrations and pH on the MC-degrading activities were investigated utilizing high-performance liquid chromatography (HPLC). In addition, the MC-degrading mechanism of YFMCD4 was also studied using HPLC coupled with a mass spectrometry equipped with electrospray ionization interface (HPLC-ESI-MS). The data showed MC-LR was completely removed at the maximum rate of 0.5 µg/(mL·h) under the optimal condition by YFMCD4. Two pure bacterial strains Alcaligenes faecalis and Stenotrophomonas acidaminiohila were isolated from YFMCD4 degraded MC-LR at a slower rate. The MC-degrading rates of YFMCD4 were significantly affected by different temperatures, pH and MC-LR concentrations. Two intermediates of a tetrapeptide and Adda appeared in the degradation process. These results illustrate that the novel YFMCD4 is one of the highest effective MC-degrading bacterial community, which can completely remove MC-LR and possesses a significant potential to treat water bodies contaminated by MC-LR.

Kai-Xin-San, a traditional Chinese medicine formula, induces neuronal differentiation of cultured PC12 cells: Modulating neurotransmitter regulation enzymes and potentiating NGF inducing neurite outgrowth.

ETHNOPHARMACOLOGICAL RELEVANCE: Kai-Xin-San, an ancient formula composed of Ginseng Radix et Rhizoma, Polygalae Radix, Acori Tatarinowii Rhizoma and Poria, was frequently applied for major depression disorders for thousands of years. However, its molecular mechanism has not clearly been investigated. AIM OF THE STUDY: We aimed to reveal the action mechanism of KXS on anti-depression on inducing neuronal differentiation on PC12 cells. MATERIALS AND METHODS: A chemically standardized water extract of KXS was applied onto cultured PC12 cells in determining its effect on neurotransmitter regulation enzymes and neurite outgrowth. RESULTS: Single KXS treatment showed obvious changes in the expression of neurofilament and neurotransmitter regulation enzymes, which in parallel to treatment of nerve growth factor (NGF). Although KXS by itself did not show significant inductive effect on neurite outgrowth of PC12 cells, KXS could potentiate the NGF induced neurite outgrowth. Among the three ratios, K-652 showed the most powerful effect and cAMP-dependent pathway might play the major role. CONCLUSIONS: KXS might exert the anti-depressant-like action of be inducing neuronal differentiation, which supported the clinically usage of this decoction.