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Animal medicine is a large category of Chinese medicinecommonly used in clinical practice and has important scientific and therapeutic value. Animal medicine isscarcer than herbal medicine. In recent years, with the vigorous development of traditional Chinese medicine(TCM),the contradiction between the increasing industrial demand andsupply of scarce and even endangered medicinal animals has become increasingly prominent. The continuous lack of medicinal animal resources affects the clinical demandandalso causes serious damage to the ecological environment. Only relying on artificial breeding is not enough to alleviate the current condition of depletion. In the face of this dilemma, it is a major challenge for the current industrial development to protect animal resources and meet clinical and industrial needs withquot;available medicinesquot;. The application of substitutes for animal medicines isthe key focus to alleviate this problem, and it is also the key scientific issue to be solved urgently in the modernization of TCM. This paper summarizedand reviewedthe history, current situation, strategies, and methods of animal medicinesubstitution and put forward the point of view ofquot;similar chemical characteristics, similar efficacy, and higher safetyquot; to provide references for scientific substitution and resource protection of rare animals.

Natural product bufotenine (5) which could be isolated from Venenum Bufonis, has been widely used as a tool in central nervous system (CNS) studies. We present here its quaternary ammonium salt (6) which was synthesized with high yields using 5-benzyloxyindole as raw materials, and we firstly discover its analgesic effects in vivo. The analgesic evaluation showed that compounds 5 and 6 had stronger effects on the behavior of formalin induced pain in mice. Moreover, the combination of compound 6 and morphine has a synergistic effect. We intended to explain the molecular mechanism of this effect. Therefore, 36 analgesic-related targets (including 15 G protein-coupled receptors, 6 enzymes, 13 ion channels, and 2 others) were systemically evaluated using reverse docking. The results indicate that bufotenine and its derivatives are closely related to acetyl cholinesterase (AChE) or α4β2 nicotinic acetylcholine receptor (nAChR). This study provides practitioners a new insight of analgesic effects.

The inhibiting effect of Norcantharidin (NCTD) on IL-6 (interleukin-6) and STAT3 and the involvement of the IL-6/STAT3 pathway in hepcidin expression prompted us to speculate that NCTD could affect iron metabolism.We examined the effects of NCTD on serum iron (SI) and transferrin (Tf) saturation, iron and ferritin light chain (FTL), transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), ferroportin 1 (Fpn1), iron regulatory protein 1 (IRP1) and hepcidin, as well as IL-6 and STAT3 in the liver, spleen and duodenum of mice treated with lipopolysaccharide (LPS) in vivo, using RT-PCR, Western blotting and immunofluorescence analysis.NCTD could increase SI and Tf saturation and reduce tissue iron and FTL content by affecting expression of cell-iron transport proteins TfR1, DMT1 and Fpn1. The impact of NCTD on TfR1, DMT1 and Fpn1 expression is mediated by up-regulating IRP1 and down-regulating hepcidin expression, while NCTD-induced down-regulation of hepcidin is mediated by the IL-6/STAT3 signalling pathway in LPS-treated mice.NCTD affects iron metabolism by modifying the expression of IL-6/JAK2/STAT3/hepcidin and IRP1 and suggest that the ability of NCTD to reduce tissue iron contents may be a novel mechanism associated with the anti-cancer effects of NCTD.

To observe the effect of moxibustion on the regulation of nuclear factor-kappa B (NF-κB) and inflammatory factors by multiple microRNAs (miRNAs) in rats with diarrhea-predominant irritable bowel syndrome (IBS-D), and to explore the anti-inflammatory mechanism of moxibustion on IBS-D.Twelve of 52 newborn rats were randomly selected into a normal group. The remaining rats were made into IBS-D model. A total of 36 rats with successful model were randomly divided into a model group, a medication group and a moxibustion group, 12 rats in each group. The rats in the medication group were intraperitoneally injected with pyrrolidine dithiocarbamate (PDTC). The rats in the moxibustion group were treated with moxibustion at "Tianshu" (ST 25) and "Shangjuxu" (ST 37) for 20 min each time. All the intervention was given once a day for 7 days. Before and after modeling as well as after intervention, the body mass, loose stool rate and the minimum volume threshold of abdominal withdrawal reflex (AWR) were measured. After intervention, the contents of serum tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-8 were detected by ELISA method; the morphology of colon tissues was observed by HE staining, and the expressions of miR-155, miR-125b, miR-29b, miR-31, miR-18a and NF-κB p65 mRNA in colon tissues were detected by real-time PCR. The expressions of NF-κB p65, TNF-α, IL-1β and IL-8 protein in colon tissues were detected by immunofluorescence.After modeling, the body mass and the minimum volume threshold of AWR in the model group were lower than those in the normal group (The anti-inflammatory mechanism of moxibustion at "Tianshu" (ST 25) and "Shangjuxu" (ST 37) for IBS-D rats may be related to regulating multiple miRNAs to inhibit NF-κB signal pathway and reduce the expression of inflammatory factors.

Hepatic fibrosis is characterized by hepatic stellate cell (HSC) activation and transdifferentiation-mediated extracellular matrix (ECM) deposition, which both contribute to cirrhosis. However, no antifibrotic regimen is available in the clinic. microRNA-23b/27b/24-1 cluster inhibition of transforming growth factor-β (TGF-β) signaling during hepatic development prompted us to explore whether this cluster inhibits HSC activation and hepatic fibrosis.Experimental fibrosis was studied in carbon tetrachloride (CClIn this study, we showed that increasing the miR-23b/27b/24-1 level through intravenous delivery of miR-23b/27b/24-1 lentivirus ameliorated mouse hepatic fibrosis. Mechanistically, the miR-23b/27b/24-1 cluster directly targeted messenger RNAs, which reduced the protein expression of 5 secretory profibrotic genes (TGF-β2, Gremlin1, LOX, Itgα2, and Itgα5) in HSCs. Suppression of the TGF-β signaling pathway by down-regulation of TGF-β2, Itgα2, and Itgα5, and activation of the bone morphogenetic protein signaling pathway by inhibition of Gremlin1, decreased extracellular matrix secretion of HSCs. Furthermore, down-regulation of LOX expression softened the ECM. Moreover, a reduction in tissue inhibitors of metalloproteinase 1 expression owing to weakened TGF-β signaling increased ECM degradation.Hepatic overexpression of the miR-23b/27b/24-1 cluster blocked hepatic fibrosis and may be a novel therapeutic regimen for patients with hepatic fibrosis.

Background: High mobility group box 1 (HMGB1), an important “late” inflammatory mediator, is a therapeutic target for bacterial sepsis. Ethyl pyruvate (EP) has anti-inflammatory effects, and prevents bacterial sepsis by decreasing HMGB1 levels. However, the role of HMGB1 in fungal sepsis is still unclear. Therefore, we investigated HMGB1 mRNA and protein levels in serum and tissue from patients and mice with invasive C. albicans infection. We explored the anti-HMGB1 effects of EP, which may be of benefit in the diagnosis and treatment of invasive C. albicans infections. Methods: We measured serum HMGB1 mRNA and protein levels in four different patient groups: 23 control subjects (group 1), 35 patients with sepsis without fungal infection (group 2), 35 patients with severe sepsis without fungal infection (group 3), and 23 patients with severe sepsis with C. albicans infection (group 4). We collected clinical indices to estimate correlations between HMGB1 levels and disease severity. Furthermore, we experimentally stimulated mice with C. albicans and C. albicans + EP, and examined HMGB1 mRNA and protein levels from serum and tissue. We determined liver and kidney functions, investigated pathological changes in tissues and assessed mortality. We also investigated serum levels of the proinflammatory cytokines, tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) in these animals. Results: Serum HMGB1 mRNA and protein levels in patients with severe sepsis with C. albicans infection were elevated. Increased HMGB1 levels were correlated with procalcitonin (PCT), C-reactive protein (CRP), 1,3-β-D-Glucan (BDG) and C. albicans sepsis severity. HMGB1 mRNA and protein levels in serum and tissues were significantly increased within seven days after mice were infected with C. albicans. The administration of EP inhibited HMGB1 levels, decreased tissue damage, increased survival rates and inhibited the release of TNF-α and IL-6.Conclusions: Increased HMGB1 levels in patients with severe sepsis with C. albicans infection were positively correlated with PCT, CRP, BDG, and disease severity. EP prevented C. albicans lethality by decreasing HMGB1 expression and release. We suggest that HMGB1 plays a vital role in invasive C. albicans infections and may provide an effective diagnostic and therapeutic target for such infections in clinical settings.

The aim of this study was to clarify the microRNA (miRNA) expression profiles of RAW264.7 macrophages infected by Candida albicans to elucidate the roles of differentially expressed miRNAs and to further explore the mechanisms underlying the immune response to C. albicans infection.High-throughput miRNA microarray analysis was performed to detect differentially expressed miRNAs in control and C. albicans-infected RAW264.7 cells. Quantitative real-time PCR analysis was used to verify the microarray results. Target genes of differentially expressed miRNAs were predicted with bioinformatics software. The cell biological processes and signaling pathways of these miRNA-targeted genes involved in C. albicans infection were predicted by gene ontology (GO) enrichment and pathway analyses.Significant upregulation of eight miRNAs (mmu-miR-140-5p, mmu-miR-96-5p, mmu-miR-8109, mmu-miR-466i-3p, mmu-miR-222-5p, mmu-miR-301b-3p, mmu-miR-466g, and mmu-miR-7235-5p) and downregulation of eight miRNAs (mmu-miR-3154, mmu-miR-223-3p, mmu-miR-494-3p, mmu-miR-6908-5p, mmu-miR-188-5p, mmu-miR-6769b-5p, mmu-miR-7002-5p, and mmu-miR-1224-5p) were observed, as compared with the control (fold change ≥2.0 and P 0.05). GO analysis revealed that both mmu-miR-140-5p and mmu-miR-223-3p participated in immune responses, inflammatory reactions, and cell apoptosis in C. albicans infection. Also, the MAPK signaling pathway was found to play an important role in the immune response against C. albicans infection.This study revealed comprehensive expression and functional profiles of differentially expressed miRNAs in macrophage RAW264.7 cells infected by C. albicans. These findings should help to further elucidate the mechanisms underlying the immune response to C. albicans infection.

The aim of this study was to observe the effect of baicalin on the growth state of attention deficit hyperactivity disorder animal model and its regulation on Ca MKⅡand ERK1/2.In the present study,a total of 40 SHR rats were randomly divided into model group,methylphenidate hydrochloride group,and low,medium,and high dose baicalin groups,with 8 rats in each group.Eight WKYrats were selected as a normal control group.The methylphenidate hydrochloride group(0.07 g·L~(-1))and the low(3.33 g·L~(-1)),medium(6.67 g·L~(-1)),and high dose(10 g·L~(-1))baicalin groups received corresponding drugs by gavage administration according to the body weight(0.015 m L·g~(-1)),while the normal group and the model group received the same volume of normal saline by gavage.Thegavage administration lasted for 4 weeks,twice a day.The body weight of the rats and the amount of remaining feed were weighed daily,and the growth state of the rats was statistically evaluated weekly.Percoll density gradient centrifugation was used to prepare brain synaptosomes and an electron microscope was used to observe their structures.The Ca MKⅡand ERK1/2 protein and mRNA expression levels were detected with Western blot and Real-time PCR methods,respectively.RESULTS: showed that baicalin did not affect the normal eating and weight gain of rats,and the weight gain of rats was even more significant than that in the normal group(P0.05).In the study of its effects on Ca MKⅡand ERK1/2 protein expression in rat synaptosomes,the expression of both proteins in each drug-administered group was higher than that in the model group(P0.05);besides,the expression levels of Ca MKⅡand ERK1/2 protein were significantly increased in both baicalin high dose group and the methylphenidate hydrochloride group(P0.05).The relative expression of Ca MKⅡand ERK1/2 mRNA in synaptosome was detected by PCR.The results showed that medium and high doses of baicalin and methylphenidate hydrochloride significantly increased the relative expression of Ca MKⅡand ERK1/2 mRNA in synaptosomes of SHR rats(P0.05).In conclusion,baicalin does not affect the normal growth and development of SHR rats,so it is safe for administration.Both baicalin and methylphenidate hydrochloride could up-regulate the relative expression of Ca MKⅡand ERK1/2 in mRNA and protein,and the pharmacodynamic stability of baicalin is in a dose-dependent manner to certain extent.

Association of both iron/hepcidin and apolipoprotein E (ApoE) with development of Alzheimer disease (AD) and atherosclerosis led us to hypothesize that ApoE might be required for body iron homeostasis. Here, we demonstrated that ApoE knock-out (KO) induced a progressive accumulation of iron with age in the liver and spleen of mice. Subsequent investigations showed that the increased iron in the liver and spleen was due to phosphorylated extracellular regulated protein kinases (pERK) mediated up-regulation of transferrin receptor 1 (TfR1), and nuclear factor erythroid 2-related factor-2 (Nrf2)-dependent down-regulation of ferroportin 1. Furthermore, replenishment of ApoE could partially reverse the iron-related phenotype in ApoE KO mice. The findings imply that ApoE may be essential for body iron homeostasis and also suggest that clinical late-onset diseases with unexplained iron abnormality may partly be related to deficiency or reduced expression of ApoE., Graphical abstract Image 1, Highlights • Apolipoprotein E deficiency induces a progressive increase in tissue iron contents with age in mice. • ApoE−/− induced a progressive accumulation of iron with age in the liver and spleen of mice. • The increased iron was due to upregulation of TfR1 and downregulation of Fpn1. • Replenishment of ApoE could partially reverse the iron-related phenotype in ApoE KO mice. • ApoE may be essential for body iron homeostasis.

The gene therapy of cancer, due to the limit of its efficiency and safety, has not been widely used in clinical. Recently, bacterial magnetic particles (BMPs), which are membrane-bound nanocrystals found in magnetotactic bacteria, have been exploited as a new gene delivery system. However, its application on gene therapy remains to be explored. In our previous study, we found that a combination of cecropin B (ABPs) and apoptin (VP3) could serve as an effective gene therapeutic agent. Thus, in this study, we used BMPs to deliver the co-expression plasmid of these two gene, namely pVAX1-VA, and evaluated its therapeutic effect on human hepatocellular carcinoma (HepG2). Our results showed that BMPs significantly improved the efficiency of gene transfection (almost 3-fold than Lipofectamine 2000 at 48 h, P .001), which led to stronger apoptosis (in a peak almost 2-fold than Lipofectamine 2000-pVAX1-VA, P .01) and growth inhibition of HepG2 cells. More importantly, compared with Lipofectamine 2000-pVAX1-VA group, BMP-pVAX1-VA strikingly inhibited tumor growth (0.60 ± 0.09 g vs. 0.88 ± 0.11 g, P .05) in nude mouse tumor models and increased the tumor-infiltrating lymphocytes considerably without apparent cytotoxicity. These findings suggest that BMPs could be an attractive gene delivery system for gene therapy and provide a potential available treatment for human hepatocellular carcinoma and maybe some other kinds of tumors.

OBJECTIVE: To investigate the effect of baicalin on the behavioral characteristics of rats with attention deficit hyperactivity disorder (ADHD), and to provide a basis for further research on baicalin in the treatment of ADHD. METHODS: A total of 40 SHR rats were randomly divided into model group, methylphenidate hydrochloride (MPH) group, and low-, medium-, and high-dose baicalin groups, with 8 rats in each group. Eight WKY rats were selected as normal control group. The rats in the MPH group (0.07 mg/mL) and the low-(3.33 mg/mL), medium-(6.67 mg/mL), and high-dose (10 mg/mL) baicalin groups were given the corresponding drugs (1.5 mL/100 g) by gavage twice a day, and those in the normal control group and the model group were given an equal volume of normal saline by gavage twice a day. The course of treatment was 4 weeks for all groups. The open field test was performed to observe total moving distance and average moving speed on day 0 of experiment and at 7, 14, 21, and 28 days after gavage and to evaluate the control effects of drugs on hyperactivity and impulsive behavior. The Morris water maze test was used to observe the latency, time spent in the target quadrant, and number of platform crossings and to evaluate the effects of drugs on attention. RESULTS: The open field test showed that the model group and the drug treatment groups had a significantly longer total moving distance and a significantly higher average moving speed than the normal control group on day 0 (P < 0.05). On day 7, the MPH group had significant reductions in total moving distance and average moving speed compared with the model group (P < 0.05). On day 14, the MPH group and the high-dose baicalin group had significant reductions in total moving distance and average moving speed compared with the model group (P < 0.05). The data on days 21 and 28 showed that compared with the model group, the low-, medium-, and high-dose baicalin groups had gradual reductions in total moving distance and average moving speed (P < 0.05). The water maze test showed that compared with the model group, the MPH group and the medium-and high-dose baicalin groups had a significantly longer time spent in the target quadrant (P < 0.05), and the MPH group and the high-dose baicalin group had a significantly higher proportion of the moving distance in the target quadrant in total moving distance (P < 0.05). The high-dose baicalin group had the highest number of platform crossings among all groups (P < 0.05). CONCLUSIONS: Both baicalin and MPH can regulate the motor ability and learning and memory abilities of SHR rats with ADHD and thus control the core symptoms of ADHD, i.e., hyperactivity, impulsive behavior, and inattention. Baicalin exerts its effect in a dose-dependent manner, and high-dose baicalin has the most significant effect, but compared with MPH, it needs a longer time to play its therapeutic effect.

OBJECTIVE: To study the effect of baicalin on synaptosomal adenosine triphosphatase (ATPase) and lactate dehydrogenase (LDH) and its regulatory effect on the adenylate cyclase (AC) /cyclic adenosine monophosphate (cAMP) /protein kinase A (PKA) signaling pathway in rats with attention deficit hyperactivity disorder (ADHD). METHODS: A total of 40 SHR rats were randomly divided into five groups:ADHD model, methylphenidate hydrochloride treatment (0.07 mg/mL), and low-dose (3.33 mg/mL), medium-dose (6.67 mg/mL), and high-dose (10 mg/mL) baicalin treatment (n=8 each). Eight WKY rats were selected as normal control group. Percoll density gradient centrifugation was used to prepare brain synaptosomes and an electron microscope was used to observe their structure. Colorimetry was used to measure the activities of ATPase and LDH in synaptosomes. ELISA was used to measure the content of AC, cAMP, and PKA. RESULTS: Compared with the normal control group, the ADHD model group had a significant reduction in the ATPase activity, a significant increase in the LDH activity, and significant reductions in the content of AC, cAMP, and PKA (P < 0.05). Compared with the ADHD model group, the methylphenidate hydrochloride group and the medium-and high-dose baicalin groups had a significant increase in the ATPase activity (P < 0.05), a significant reduction in the LDH activity (P < 0.05), and significant increases in the content of AC, cAMP, and PKA (P < 0.05). Compared with the methylphenidate hydrochloride group, the high-dose baicalin group had significantly greater changes in these indices (P < 0.05). Compared with the low-dose baicalin group, the high-dose baicalin group had a significant increase in the ATPase activity (P < 0.05); the medium-and high-dose baicalin groups had a significant reduction in the LDH activity (P < 0.05) and significant increases in the content of AC, cAMP, and PKA (P < 0.05). Compared with the medium-dose baicalin group, the high-dose baicalin group had a significant increase in the ATPase activity (P < 0.05). CONCLUSIONS: Both methylphenidate hydrochloride and baicalin can improve synaptosomal ATPase and LDH activities in rats with ADHD. The effect of baicalin is dose-dependent, and high-dose baicalin has a significantly greater effect than methylphenidate hydrochloride. Baicalin exerts its therapeutic effect possibly by upregulating the AC/cAMP/PKA signaling pathway.

Attention deficit hyperactivity disorder (ADHD) is a common behavioral disorder. Previous research has indicated that genetic factors, family education, environment and dietary habits are associated with ADHD. It has been determined that in China many children with ADHD also have allergic rhinitis or asthma. These children are more susceptible to the common cold or upper respiratory infections compared with normal healthy children. Additionally, the common cold or an upper respiratory infection may lead to disease recurrence or worsen the symptoms in these children. Previous studies have determined that ADHD may have a close association with allergic disease. Based on the clinically observed phenomenon and previous studies, it was hypothesized that ADHD is a high inflammation and immune‑associated disease. Therefore, the authors designed clinical and animal experiments to test this hypothesis in the future. Immune system disorders may be a novel part of the etiology of ADHD. The current report may have implications for future clinical practice.

The orphan nuclear receptor NOR1 belongs to the NR4A subfamily of the nuclear hormone receptor superfamily, and is involved in glucose and fat metabolism. However, its potential contribution to cardiovascular diseases remains to be assessed. Here, the roles of NOR1 in cardiac hypertrophy induced by isoprenaline and the underlying molecular mechanisms were investigated.NOR1 was expressed in cardiomyocytes treated with isoprenaline. After NOR1 overexpression or knockdown in neonatal rat cardiomyocytes, cellular hypertrophy was monitored by measuring cell surface area and the mRNA of hypertrophic biomarkers. Interactions between NOR1 and PARP-1 were investigated by co-immunoprecipitation. NOR1 expression and PARP-1 activity were measured in rats with cardiac hypertrophy induced by isoprenaline.Treatment with isoprenaline significantly up-regulated NOR1 expression and PARP-1 activity both in vivo and in vitro. Specific gene silencing of NOR1 attenuated isoprenaline-induced cardiomyocyte hypertrophy, whereas NOR1 overexpression exacerbated cardiac hypertrophy. We identified a physical interaction between NOR1 and PARP-1, which was enhanced by NOR1 transfection and thereby led to PARP-1 activation. Overexpression of NOR1, but not C293Y, a NOR1 mutant lacking the PARP-1 binding activity, increased cellular surface area and the mRNA levels of atrial natriuretic factor and brain natriuretic polypeptide, effects blocked by the PARP-1 inhibitor 3-aminobenzamide or siRNA for PARP-1.This is the first evidence that NOR1 was involved in isoprenaline-induced cardiac hypertrophy. The pro-hypertrophic effect of NOR1 can be partly attributed to its regulation of PARP-1 enzymic activity.