The mechanism of reactive oxygen species generation, DNA damage and apoptosis in hemocytes of Litopenaeus vannamei under ammonia nitrogen exposure.

Ammonia-N poses a significant threat to aquatic animals. However, the mechanism of ROS production leading to DNA damage in hemocytes of crustaceans is still unclear. Additionally, the mechanism that cells respond to DNA damage by activating complex signaling networks has not been well studied. Therefore, we exposed shrimp to 0, 2, 10, and 20 mg/L NH4Cl for 0, 3, 6, 12, 24, 48, and 72 h, and explored the alterations in endoplasmic reticulum stress and mitochondrial fission, DNA damage, repair, autophagy and apoptosis. The findings revealed that ammonia exposure led to an increase in plasma ammonia content and neurotransmitter content (DA, 5-HT, ACh), and significant changes in gene expression of PLC and Ca2+ levels. The expression of disulfide bond formation-related genes (PDI, ERO1) and mitochondrial fission-related genes (Drp1, FIS1) were significantly increased, and the unfolded protein response was initiated. Simultaneously, ammonia-N exposure leads to an increase in ROS levels in hemocytes, resulting in DNA damage. DNA repair and autophagy were considerably influenced by ammonia-N exposure, as evidenced by changes in DNA repair and autophagy-related genes in hemocytes. Subsequently, apoptosis was induced by ammonia-N exposure, and this activation was associated with a caspase-dependent pathway and caspase-independent pathway, ultimately leading to a decrease in total hemocytes count. Overall, we hypothesized that neurotransmitters in the plasma of shrimp after ammonia-N exposure bind to receptors on hemocytes membrane, causing endoplasmic reticulum stress through the PLC-IP3R-Ca2+ signaling pathway and leading to mitochondrial fission. Consequently, this process resulted in increased ROS levels, hindered DNA repair, suppressed autophagy, and activated apoptosis. These cascading effects ultimately led to a reduction in total hemocytes count. The present study provides a molecular support for the understanding of the detrimental toxicity of ammonia-N exposure to crustaceans.

Mechanisms of neurocentral-eyestalk-intestinal immunotoxicity in whiteleg shrimp Litopenaeus vannamei under ammonia nitrogen exposure.

Ammonia-N, as the most toxic nitrogenous waste, has high toxicity to marine animals. However, the interplay between ammonia-induced neuroendocrine toxicity and intestinal immune homeostasis has been largely overlooked. Here, a significant concordance of metabolome and transcriptome-based "cholinergic synapse" supports that plasma metabolites acetylcholine (ACh) plays an important role during NH4Cl exposure. After blocking the ACh signal transduction, the release of dopamine (DA) and 5-hydroxytryptamine (5-HT) in the cerebral ganglia increased, while the release of NPF in the thoracic ganglia and NE in the abdominal ganglia, and crustacean hyperglycemic hormone (CHH) and neuropeptide F (NPF) in the eyestalk decreased, finally the intestinal immunity was enhanced. After bilateral eyestalk ablation, the neuroendocrine system of shrimp was disturbed, more neuroendocrine factors, such as corticotropin releasing hormone (CRH), adrenocorticotropic-hormone (ACTH), ACh, DA, 5-HT, and norepinephrine (NE) were released into the plasma, and further decreased intestinal immunity. Subsequently, these neuroendocrine factors reach the intestine through endocrine or neural pathways and bind to their receptors to affect downstream signaling pathway factors to regulate intestinal immune homeostasis. Combined with different doses of ammonia-N exposure experiment, these findings suggest that NH4Cl may exert intestinal toxicity on shrimp by disrupting the cerebral ganglion-eyestalk axis and the cerebral ganglion-thoracic ganglion-abdominal ganglion axis, thereby damaging intestinal barrier function and inducing inflammatory response.

Mechanisms of ammonotelism, epithelium damage, cellular apoptosis, and proliferation in gill of Litopenaeus vannamei under NH4Cl exposure.

Excessive ammonia-N in coastal environment and aquaculture threatens the health of marine organisms. To explore the mechanism of gill damage induced by ammonia-N, transcriptome of Litopenaeus vannamei 's gill was carried out under 20 mg/L NH4Cl for 0, 6, and 48 h. K-means clustering analysis suggested that ammonia excretion and metabolism-related genes were elevated. GO and KEGG enrichment analysis suggested that glycosyltransferase activity and amino acid metabolism were affected by ammonia. Moreover, histological observation via three staining methods gave clues on the changes of gill after ammonia-N exposure. Increased mucus, hemocyte infiltration, and lifting of the lamellar epithelium suggested that gill epithelium was suffering damage under ammonia-N stress. Meanwhile, the composition of extracellular matrix (ECM) in connective tissue changed. Based on the findings of transcriptomic and histological analysis, we further investigated the molecular mechanism of gill damage under multiple concentrations of NH4Cl (0, 2, 10, 20 mg/L) for multiple timepoints (0, 3, 6, 12, 24, 48, 72 h). First, ammonia excretion was elevated via ion channel, transporter, and exocytosis pathways, but hemolymph ammonia still kept at a high level under 20 mg/L NH4Cl exposure. Second, we focused on glycosaminoglycan metabolism which was related to the dynamics of ECM. It turned out that the degradation and biosynthesis of chondroitin sulfate (CS) were elevated, suggesting that the structure of CS might be destructed under ammonia-N stress and CS played an important role in maintaining gill structure. It was enlightening that the destructions occurred in extracellular regions were vital to gill damage. Third, ammonia-N stress induced a series of cellular responses including enhanced apoptosis, active inflammation, and inhibited proliferation which were closely linked and jointly led to the impairment of gill. Our results provided some insights into the physiological changes induced by ammonia-N and enriched the understandings of gill damage under environmental stress.

The immunotoxicity mechanism of hemocytes in Chlamys farreri incubated with noradrenaline and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide alone or in combination.

Benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) is the active intermediate metabolite of benzo[a]pyrene (B[a]P) and is considered the ultimate immunotoxicant. The neuroendocrine immunoregulatory network of bivalves is affected under pollutant stress. Besides, bivalves are frequently affected by pollutants in marine environments, yet the combined effects of neuroendocrine factors and detoxification metabolites on bivalves under pollutant stress and the signal pathways that mediate this immunoregulation are not well understood. Therefore, we incubated the hemocytes of Chlamys farreri with the neuroendocrine factor noradrenaline (NA) and the B[a]P detoxification metabolite BPDE, alone or in combination, to examine the immunotoxic effects of NA and BPDE on the hemocytes in C. farreri. Furthermore, the effects of NA and BPDE on the hemocyte signal transduction pathway were investigated by assessing potential downstream targets. The results revealed that NA and BPDE, alone or in combination, resulted in a significant decrease in phagocytic activity, bacteriolytic activity and the total hemocyte count. In addition, the immunotoxicity induced by BPDE was further exacerbated by co-treatment with NA, and the two showed synergistic effects. Analysis of signaling pathway factors showed that NA activated G proteins by binding to α-AR, which transmitted information to the Ca2+-NF-κB signaling pathway to regulate the expression of phagocytosis-associated proteins and regulated cytokinesis through the cAMP signaling pathway. BPDE could activate PTK and affect phagocytosis and cytotoxicity proteins through Ca2+-NF-κB signal pathway, also affect the regulation of phagocytosis and cytotoxicity by inhibiting the AC-cAMP-PKA pathway to down-regulate the expression of NF-κB and CREB. In addition, BPDE and NA may affect the immunity of hemocytes by down-regulating phagocytosis-related proteins through inhibition of the lectin pathway, while regulating the expression of cytotoxicity-related proteins through the C-type lectin. In summary, immune parameters were suppressed through Ca2+ and cAMP dependent pathways exposed to BPDE and the immunosuppressive effects were enhanced by the neuroendocrine factor NA.

The assessment of bioavailability and environmental risk of dissolved and particulate polycyclic aromatic hydrocarbons in the seawater of typical bays.

The pollution of dissolved and particulate polycyclic aromatic hydrocarbons (PAHs) in coastal waters has been increasing in recent decades. However, limited research has been conducted on the characteristics of dissolved and particulate PAHs in seawater and their associated risk assessment. Here, we focused on the bioavailability and environmental risk of PAHs in four typical bays of Shandong Province, China, and used scallop Chlamys farreri and clam Mactra veneriformis as sentinel species. The results revealed that dissolved PAHs tended to bioaccumulate in scallop C. farreri, and their ecological risk exhibited a significant correlation with the health risk of bioaccumulated PAHs and the bioeffect of screened biomarkers in scallop. Conversely, particulate PAHs demonstrated a higher bioaccumulation potential in the clam M. veneriformis, showing a stronger correlation between their ecological risk, health risk, and bioeffect in clams. This study provides the first elucidation of the connection between the ecological risk, health risk, and bioeffect of PAHs. Furthermore, based on the better correlation of health risk and bioeffect caused by PAHs with total PAHs in seawater, we propose that the clam M. veneriformis is a more suitable sentinel species for assessing environmental risk in typical bays of Shandong Province.

Source-specific ecological and health risks of polycyclic aromatic hydrocarbons in the adjacent coastal area of the Yellow River Estuary, China.

Industrialization and urbanization have led to increasing levels of PAH pollution in highly urbanized estuaries and their adjacent coastal areas globally. This study focused on the adjacent coastal area of the Yellow River Estuary (YRE) and collected surface seawater, surface sediment, and clams Ruditapes philippinarum and Mactra veneriformis at four sites (S1 to S4) in May, August, and October 2021 to analyze the source-specific ecological and health risks and bioeffects. The findings revealed that the main sources of PAHs were traffic emission (25.2% to 28.5%), petroleum sources (23.3% to 29.5%), coal combustion (24.7% to 27.5%), and biomass combustion (19.8% to 20.7%). Further, the PMF-RQ and PMF-ILCR analyses indicated that traffic emission was the primary contributor to ecological risks in seawater and health risks in both clam species, while coal combustion was the major contributor in sediment. Taken together, it is recommended to implement control strategies for PAH pollution following the priority order: traffic > coal > petroleum > biomass, to reduce the content and risk of PAHs in the YRE.

Lead exposure induces neuronal apoptosis via NFκB p65/RBBP4/Survivin signaling pathway.

Lead (Pb), as a heavy metal that is easily exposed in daily life, can cause damage to various systems of body. Apoptosis is an autonomous cell death process regulated by genes in order to maintain the stability of internal environment, which plays an important role in the development of nervous system. RB binding protein 4 (RBBP4) is one of the core histone binding subunits and is closely related to the apoptosis process of nervous system cells. However, it is not known whether RBBP4 can regulate neuronal apoptosis in lead-exposed environments. We exposed PC12 cells to 0 µM (control group), 1 µM, and 100 µM PbAc for 24 h to obtain cell samples. The female rats ingested drinking water containing 0, 0.5 g/L, and 2.0 g/L PbAc from the first day of pregnancy to three weeks after delivery to obtain hippocampal tissue samples from mammary rats. The results of TUNEL showed that lead exposure promoted the onset of apoptosis in cells and hippocampus. The mRNA and protein levels of the apoptosis-related protein Survivin were significantly reduced in the lead-exposed group compared to the control group. In addition, we found that lead exposure reduces the mRNA and protein levels of RBBP4 in PC12 cells and hippocampus, and increases the mRNA and protein levels of NFκB p65. Moreover, inhibiting NFκB p65 can reverse the decrease in RBBP4 expression in the lead exposure model. Overexpression of RBBP4 increased Survivin expression and reduced apoptosis induced by lead exposure. This suggests that lead exposure induces apoptosis through the NFκB p65/RBBP4/Survivin signaling pathway.

The ban on the sale of new petrol and diesel cars: Can it help control prospective marine pollution of polycyclic aromatic hydrocarbons (PAHs) in Shandong Province, China?

The constantly increasing amount of road vehicles causes massive exhaust emissions of pollutants, including polycyclic aromatic hydrocarbons (PAHs), necessitating a global responsibility to implement the policy of the ban on the sale of new petrol and diesel cars. Here, we assessed the policy control efficiency on marine pollution of PAHs in China through scenario modeling and prediction models, based on pollution monitoring, risk assessment, and source apportionment of PAHs in typical bays of Shandong Province. The results showed that in 2021, the pollution risk levels were relatively low (HI: 0.008-0.068, M-ERM-Q: 0.001-0.016, IBR: 1.23-2.69, ILCR: 8.11 ×10-6-1.99 ×10-5), and PAHs were mainly derived from traffic emissions (24.9%-35.2%), coal combustion (25.2%-32.9%), petroleum (17.2%-28.9%), and biomass combustion (17.6%-22.8%). In 2050, the predicted decrease of pollution risk values after the implementation of the policy was significant (12%-26%), and the gap between 2021 and 2050 was also significantly huge (18%-85%) without considering possible substitution of conventional energy. Collectively, this study built systematic approaches for assessing prospective marine pollution of PAHs. However, due to the particularity of Shandong Province, i.e., its national predominance of conventional energy consumption, the policy may be more effective when it comes to other coastal areas worldwide, calling for a larger scale research.

The immunotoxicity mechanism of NH4Cl exposure to Litopenaeus vannamei based on the cerebral ganglion-eyestalk-haemocytes axis.

Ammonia nitrogen, as a water environmental toxin, poses a potential threat to aquatic animals. Although NH4Cl stress is known to cause immunotoxicity, mechanistic pathways linking stress networks in the neuroendocrine system to immunotoxicity remain poorly understood. In this study, firstly, using transcriptome analysis of cerebral ganglion and eyestalk in shrimp, we identified significant changes in genes related to biogenic amines, acetylcholine, crustacean hyperglycemic hormones, and neuropeptide F. Additionally, expression patterns of neuroendocrine factors in different tissues of shrimp were evaluated to explore the sources of these factors. Here, we showed that NH4Cl exposure activates acetylcholine (ACh) neurons in cerebral ganglion of shrimp and dramatically upregulates high affinity choline transporter 1 (ChT1) gene expression. The knockdown of ChT1 gene enhanced the immunity of haemocytes in shrimp compared with saline and GFP dsRNA groups. And after eyestalk ablation, the levels of neuroendocrine factors in the cerebral ganglion and thoracic ganglion were disturbed, and haemocytes parameters induced by NH4Cl were significantly decreased. Combined with different doses of NH4Cl exposure experiments, we demonstrated that: (1) In a short period of NH4Cl exposure, the neuroendocrine factors CRH-ACTH-cortisol and 5-HT-DA in the cerebral ganglion-eyestalk axis of shrimp play a major role in regulating haemocytes immunity; (2) With the prolongation of exposure, the immunotoxicity induced by NH4Cl was mainly due to the release of more ACh in the cerebral ganglion, which promoted the release of NPF in the thoracic ganglion, and CHH and NPF in the eyestalk, as well as weakened the effect of biogenic amines. Subsequently, these neuroendocrine factors regulate immunity through intracellular signaling pathways. Collectively, these results established a new mechanism that NH4Cl might directly regulate haemocytes immunotoxicity through the cerebral ganglion and thoracic ganglion; or through the cerebral ganglion-eyestalk axis or cerebral ganglion-thoracic ganglion axis cause haemocytes immunotoxicity.

Potential molecular mechanism underlying the harmed haemopoiesis upon Benzo[a]pyrene exposure in Chlamys farreri.

Benzo[a]pyrene (B[a]P), a ubiquitous contamination in the marine environments, has the potential to impact the immune response of bivalves by affecting the hemocyte parameters, especially total hemocyte count (THC). THC is mainly determined by haematopoietic mechanisms and apoptosis of hemocytes. Many studies have found that B[a]P can influence the proliferation and differentiation of hemocytes. However, the link between the toxic mechanisms of haematopoietic and environmental pollutants is not explicitly stated. This study is to investigate the toxic effects of B[a]P on haematopoietic mechanisms in C. farreri. Through the tissue expression distribution experiment and EDU assay, gill is identified as a potential haematopoietic tissue in C. farreri. Subsequently, the scallops were exposed to B[a]P (0.05, 0.5, 5 µg/L) for 1d, 3d, 6d, 10d and 15d. Then BPDE content, DNA damage, gene expression of haematopoietic factors and haematopoietic related pathways were determined in gill and hemocytes. The results showed that the expression of CDK2 was significantly decreased under B[a]P exposure through three pathways: RYR/IP3-calcium, BPDE-CHK1 and Notch pathway, resulting in cell cycle arrest. In addition, B[a]P also significantly reduced the number of proliferating hemocytes by affecting the Wnt pathway. Meanwhile, B[a]P can significantly increase the content of ROS, causing a downregulation of FOXO gene expression. The gene expression of Notch pathway and ERK pathway was also detected. The present study suggested that B[a]P disturbed differentiation by multiple pathways. Furthermore, the expression of SOX11 and CD9 were significantly decreased, which directly indicated that differentiation of hemocytes was disturbed. In addition, phagocytosis, phenoloxidase activity and THC were also significant decreased. In summary, the impairment of haematopoietic activity in C. farreri further causes immunotoxicity under B[a]P exposure. This study will improve our understanding of the immunotoxicity mechanism of bivalve under B[a]P exposure.

Potential pathway and mechanisms underlining the immunotoxicity of benzo[a]pyrene to Chlamys farreri.

The long-distance migration of polycyclic aromatic hydrocarbons (PAHs) promotes their release into the marine environment, posing a serious threat to marine life. Studies have shown that PAHs have significant immunotoxicity effects on bivalves, but the exact mechanism of immunotoxicity remains unclear. This paper aims to investigate the effects of exposure to 0.4, 2, and 10 µg/L of benzo(a)pyrene (B[a]P) on the immunity of Chlamys farreri under environmental conditions, as well as the potential molecular mechanism. Multiple biomarkers, including phagocytosis rate, metabolites, neurotoxicity, oxidative stress, DNA damage, and apoptosis, were adopted to assess these effects. After exposure to 0.4, 2, and 10 µg/L B[a]P, obvious concentration-dependent immunotoxicity was observed, indicated by a decrease in the hemocyte index (total hemocyte count, phagocytosis rate, antibacterial and bacteriolytic activity). Analysis of the detoxification metabolic system in C. farreri revealed that B[a]P produced B[a]P-7,8-diol-9,10-epoxide (BPDE) through metabolism, which led to an increase in the expression of protein tyrosine kinase (PTK). In addition, the increased content of neurotransmitters (including acetylcholine, γ -aminobutyric acid, enkephalin, norepinephrine, dopamine, and serotonin) and related receptors implied that B[a]P might affect immunity through neuroendocrine system. The changes in signal pathway factors involved in immune regulation indicated that B[a]P interfered with Ca2+ and cAMP signal transduction via the BPDE-PTK pathway or neuroendocrine pathway, resulting in immunosuppression. Additionally, B[a]P induced the increase in reactive oxygen species (ROS) content and DNA damage, as well as an upregulation of key genes in the mitochondrial pathway and death receptor pathway, leading to the increase of apoptosis rate. Taken together, this study comprehensively investigated the detoxification metabolic system, neuroendocrine system, and cell apoptosis to explore the toxic mechanism of bivalves under B[a]P stress.

Toxicological mechanism of ammonia-N on haematopoiesis and apoptosis of haemocytes in Litopenaeus vannamei.

Ammonia, as an important pollutant, contributed to the reduction of immunity, disruption of physiology in animals. RNA interference (RNAi) was performed to understand the function of astakine (AST) in haematopoiesis and apoptosis in Litopenaeus vannamei under ammonia-N exposure. Shrimps were exposed to 20 mg/L ammonia-N from 0 to 48 h with injection of 20 µg AST dsRNA. Further, shrimps were exposed to 0, 2, 10 and 20 mg/L ammonia-N also from 0 to 48 h. The results showed that the total haemocytes count (THC) decreased under ammonia-N stress and the knockdown of AST resulted in a further decrease of THC, suggesting that 1) the proliferation was decreased through the reduction of AST and Hedgehog, the differentiation was interfered by Wnt4, Wnt5 and Notch, and the migration was inhibited by the decrease of VEGF; 2) oxidative stress was induced under ammonia-N stress, leading to the increase of DNA damage with the up-regulated gene expression of death receptor, mitochondrial and endoplasmic reticulum stress pathways; 3) the changes of THC resulted from the decrease of proliferation, differentiation and migration of haematopoiesis cells and the increase of apoptosis of haemocytes. This study helps to deepen our understanding of risk management in shrimp aquaculture.

Effects of ammonia-N stress on molecular mechanisms associated with immune behavior changes in the haemocytes of Litopenaeus vannamei.

High concentration of ammonia-N will inhibit the immune defense of aquatic animals. The neuroendocrine-immune (NEI) regulatory mechanism under ammonia-N stress has been systematically studied, but the final response mechanism of ammonia-N affecting the immune system remains unclear. To investigate the relationship among immune factors of Litopenaeus vannamei (L. vannamei) exposed to 0, 2, 10 and 20 mg/L ammonia-N, the determination of complement components, C-type lectins, proPO system, signal transduction pathway and phagocytosis as well as exocytosis were performed. The results showed that the expressions of complement components including C1q, MBL, ficolin and alpha-2 macroglobulin (A2M) and the complement receptor integrin were decreased significantly in ammonia-N treatment groups at 6,12 and 24 h. C-type lectins and signal transduction factors changed significantly. The decrease of phagocytosis-related genes and phagocytic activity were similar to the changes of complement components, C-type lectins and the signal pathway. The mRNA abundance of exocytosis-related genes was significantly down-regulated under ammonia-N exposure. Correspondingly, significantly changes occurred in the expressions of PPAE and PPO3, immune factors-related genes (Pen3, crustin, stylicins, ALFs and LYC) and inflammatory factors (HSP90, TNFα, IL-16) in haemocytes. Eventually, the serine proteinase activity, PO activity, antibacterial activity and bacteriolytic activity in plasma were decreased significantly. In addition, we speculated that under ammonia-N stress, phagocytosis and exocytosis were affected by complement components, and C-type lectins through intracellular signal transduction pathway. Complement components may involve in the regulation of proPO-activating system to response to ammonia-N stress. This study helped to further understanding the relationship among immune factors of crustacean in response to environmental stress, which implied that when it comes to the decrease of immunity affected by environmental stress, we should not only focus on the mechanism of upstream neuroendocrine response, but also pay attention to the role of immune factors.

The injection of one recombinant C-type lectin (LvLec) induced the immune response of hemocytes in Litopenaeus vannamei.

To explore the immune function of C-type lectin in shrimp, one recombinant C-type lectin (LvLec) was injected into Litopenaeus vannamei. There were four treatments in the experiment: saline group (as control group), recombinant C-type lectin group (LvLec, 1 mg mL-1), Vibrio harveyi group (V. harveyi, 106 cfu mL-1) and recombinant C-type lectin combined with Vibrio harveyi group (LvLec + V. harveyi, 1 mg mL-1 + 106 cfu mL-1). The sampling time was set at 0, 3, 6, 9, 12, 24 h after the injection. The results showed that the total hemocyte count decreased significantly and the phagocytic activity improved notably after the injection of LvLec, V. harveyi or LvLec + V. harveyi. Prophenoloxidase (proPO) activity decreased, while phenoloxidase (PO) activity increased and the changing degree of each group exhibited a significant difference. The hemagglutinating activity and bacteriolytic activity improved significantly, while the antimicrobial activity did not show a remarkable change in all of the groups. There were also changes that occurred in the levels of second messengers (cAMP, cGMP) and protein kinase (PKA, PKG). After the injection of LvLec, V. harveyi or LvLec + V. harveyi, the concentration of cGMP and PKA increased significantly, while the concentration of cAMP and PKG did not change remarkably. The results above suggested that rLvLec could induce nonspecific immune response, including phagocytosis, release of PO, hemagglutination and bacteriolysis through cGMP-PKA pathway in vivo.

Mitochondrial uncoupling protein 2 is regulated through heterogeneous nuclear ribonucleoprotein K in lead exposure models.

Synaptic plasticity plays an important role in learning and memory in the developing hippocampus. However, the precise molecular mechanism in lead exposure models remains to be studied. UCP2, an inner mitochondrial anion carrier, regulates synaptic plasticity through uncoupling neurons. And hnRNP K, an RNA binding protein, plays a role in modulating the expression of transcripts coding synaptic plasticity. We aim to investigate whether lead exposure affects UCP2 and hnRNP K expression levels. The Sprague-Dawley rats were exposed to different lead acetate concentrations (0 g/l, 0.5 g/l, 2.0 g/l) during gestational and lactational periods. PC12 cells were also exposed to different lead acetate concentrations (0 µM, 1 µM and 100 µM). We found that the expression levels of UCP2 and hnRNP K had significant declines in the lead exposure rat hippocampus and PC12 cells. Furthermore, the up-regulation of hnRNP K expression level could reverse the expression level of UCP2 in lead exposure models. In conclusion, these results suggest that lead exposure can reduce the expression level of UCP2 which is mediated by decreasing the expression level of hnRNP K.

Effects of triptolide on degeneration of dendritic spines induced by Aß1-40 injection in rat hippocampus.

Although the exact cause of Alzheimer's disease (AD) remains elusive, mounting evidence continues to support the involvement of neuroinflammation in the development of AD. Triptolide isolated from the herb Tripterygium wilfordii Hook F has anti-inflammatory and immunosuppressive activities. In this study, we observed the effects of triptolide on dendritic spines of hippocampal neurons in model rats with AD. Thirty male SD rats were randomly divided into control group, AD model group and triptolide-treated group. The AD model group was made with bilateral microinjection of aggregated beta-amyloid protein (Aß)1-40 into hippocampus in rats and the control group rats were injected with normal saline in the same way. The triptolide-treated group rats were administered triptolide intraperitoneally for 30 days after microinjection of aggregated Aß1-40 into hippocampus. Dendritic morphology of hippocampal neurons in each group was analyzed using Golgi staining and ImageJ software. Our data showed that the total number of intersection points of dendrites and spine density in hippocampal neurons in the AD model group were decreased as compared with the control group. However, the total number of intersection points of dendrites and spine density in hippocampal neurons in the triptolide-treated group were increased as compared with the AD model group. Our results indicate that triptolide can alleviate the degeneration of dendritic spines in hippocampal neurons in model rats with AD.

Effects of triptolide on the synaptophysin expression of hippocampal neurons in the AD cellular model.

Due to the immunoinflammatory pathology of Alzheimer's disease (AD) brain, recent studies have begun to focus attention on the role of anti-inflammatory drugs or immunomodulators in AD. Triptolide isolated from the herb Tripterygium wilfordii Hook F has anti-inflammatory and immunosuppressive activities. In this study, we observed the effects of triptolide on synaptophysin expression in AD cellular model. AD cellular model was established by action of Aß-stimulated microglial conditioned medium (MCM) on cultured rat hippocampal neurons (HN). Immunocytochemical staining, western blot and RT-PCR were used to observe the effects of triptolide at different dosages on the synaptophysin expression of hippocampal neurons in AD cellular model at different time points during incubation of cultures. After 24 h of cultivation, the expression level of synaptophysin in MCM/HN model group was decreased as compared with normal HN group and MCM/HN control group, and the expression level of synaptophysin in MCM/HN low-dose triptolide group and MCM/HN high-dose triptolide group was increased as compared with MCM/HN model group. It is concluded that triptolide can promote the synaptophysin expression of hippocampal neurons in the AD cellular model.

[Studies on the chemical constituents of Callicarpa kochiana].

OBJECTIVE: To study on the chemical constituents of Callicarpa kochiana. METHODS: The chemical constituents were isolated by chromatographic methods and structurally elucidated by spectral analysis. RESULTS: Twelve compounds were obtained and identified as alpha-amyrin(I), 2beta, 3beta, 19alpha-trihydroxy-12-en-28-ursolic acid (II), oleanolic aicd (III), alpha-amyrin-3-0-beta-D-glucopyranoside (IV), ursolic acid (V), betulinic acid (VI), 2alpha, 3beta,23-trihydroxy-12-en-28-oic-0-beta-D-glucopyranoside (VII), 0-hydroxybenzoic acid (VI), pomolic acid (IX), myrianthic acid (X), beta-sitosterol (XI), dauricine (XII). CONCLUSION: All of these compounds are isolated from Callicarpa kochiana for the first time and compounds II, IV, VII, VIII, IX and X are reported for the first time from Callicarpa genus.