Moniezia benedeni drives the SNAP-25 expression of the enteric nerves in sheep's small intestine.

BACKGROUND: The neuroimmune network plays a crucial role in regulating mucosal immune homeostasis within the digestive tract. Synaptosome-associated protein 25 (SNAP-25) is a presynaptic membrane-binding protein that activates ILC2s, initiating the host's anti-parasitic immune response. METHODS: To investigate the effect of Moniezia benedeni (M. benedeni) infection on the distribution of SNAP-25 in the sheep's small intestine, the recombinant plasmid pET-28a-SNAP-25 was constructed and expressed in BL21, yielding the recombinant protein. Then, the rabbit anti-sheep SNAP-25 polyclonal antibody was prepared and immunofluorescence staining was performed with it. The expression levels of SNAP-25 in the intestines of normal and M. benedeni-infected sheep were detected by ELISA. RESULTS: The results showed that the SNAP-25 recombinant protein was 29.3 KDa, the titer of the prepared immune serum reached 1:128,000. It was demonstrated that the rabbit anti-sheep SNAP-25 polyclonal antibody could bind to the natural protein of sheep SNAP-25 specifically. The expression levels of SNAP-25 in the sheep's small intestine revealed its primary presence in the muscular layer and lamina propria, particularly around nerve fibers surrounding the intestinal glands. Average expression levels in the duodenum, jejunum, and ileum were 130.32 pg/mg, 185.71 pg/mg, and 172.68 pg/mg, respectively. Under conditions of M. benedeni infection, the spatial distribution of SNAP-25-expressing nerve fibers remained consistent, but its expression level in each intestine segment was increased significantly (P < 0.05), up to 262.02 pg/mg, 276.84 pg/mg, and 326.65 pg/mg in the duodenum, jejunum, and ileum, and it was increased by 101.06%, 49.07%, and 89.16% respectively. CONCLUSIONS: These findings suggest that M. benedeni could induce the SNAP-25 expression levels in sheep's intestinal nerves significantly. The results lay a foundation for further exploration of the molecular mechanism by which the gastrointestinal nerve-mucosal immune network perceives parasites in sheep.

Screening of polysaccharides from the differently processed products of Angelica sinensis with the best liver protection effect on chicken and the intervention mechanism study based on tandem mass tag proteomics and multiple reaction monitoring approach.

The incidence of colibacillosis in poultry is on the rise, significantly affecting the chicken industry. Ceftiofur sodium (CS) is frequently employed to treat this disease, resulting in lipopolysaccharide (LPS) buildup. Processing plays a vital role in traditional Chinese veterinary medicine. The potential intervention in liver injury by polysaccharides from the differently processed products of Angelica sinensis (PDPPAS) induced by combined CS and LPS remains unclear. This study aims to investigate the protective effect of PDPPAS on chicken liver injury caused by CS combined with LPS buildup and further identify the polysaccharides with the highest hepatoprotective activity in chickens. Furthermore, the study elucidates polysaccharides' intervention mechanism using tandem mass tag (TMT) proteomics and multiple reaction monitoring (MRM) methods. A total of 190 1-day-old layer chickens were randomly assigned into 12 groups, of which 14 chickens were in the control group and 16 in other groups, for a 10-day trial. The screening results showed that charred A. sinensis polysaccharide (CASP) had the most effective and the best hepatoprotective effect at 48 h. TMT proteomics and MRM validation results demonstrated that the intervention mechanism of the CASP high-dose (CASPH) intervention group was closely related to the protein expressions of FCER2, TBXAS1, CD34, AGXT, GCAT, COX7A2L, and CYP2AC1. Conclusively, the intervention mechanism of CASPH had multitarget, multicenter regulatory features.

Yujin powder improves large intestine dampness-heat syndrome by regulating gut microbiota and serum metabolism.

Large intestine dampness-heat syndrome (LIDHS) is a common syndrome type in animal diarrheal diseases. Yujin powder (YJP) is one of the classic prescriptions for treating damp-heat diarrhea. The aim of this study was to investigate the regulatory effects of YJP on gut microbiota and serum metabolism in LIDHS rats using 16S rRNA sequencing and nontargeted metabolomics. The LIDHS rat model was induced through a high-sugar and high-fat diet, exposure to a high-temperature and high-humidity environment, and infection with Escherichia coli. The results demonstrated that the administration of YJP resulted in a decrease in the abundance of Desulfovibrio, Parabacteroides, Bacteroides, Allobaculum, Escherichia, Butyricimonas, Parasutterella, and Blautia and an increase in Ruminococcus, Akkermansia, Roseburia, and Lachnoclostridium. A total of 25 potential biomarkers were identified in three groups of rats. These metabolites were primarily involved in glycerophospholipid metabolism, taurine and hypotaurine metabolism, glycerol ester metabolism, arachidonic acid metabolism, primary bile acid synthesis, and tryptophan metabolism. Our study demonstrated that YJP has the potential to alleviate LIDHS by modulating gut microbial and serum metabolic homeostasis. These results establish a foundation and offer valuable guidance for the utilization of YJP in the treatment of LIDHS.

Optimization of Ethanol Extraction Technology for Yujin Powder Using Response Surface Methodology with a Box-Behnken Design Based on Analytic Hierarchy Process-Criteria Importance through Intercriteria Correlation Weight Analysis and Its Safety Evaluation.

Here, we aimed to optimize the ethanol extraction technology for Yujin powder (YJP) and evaluate its safety. The ultrasonic-assisted ethanol reflux extraction method refluxing was used to extract YJP. The parameters were optimized through a combination of single-factor and response surface methodology (RSM). The comprehensive Y value score calculated using the content of 13 active ingredients in YJP ethanolic extracts (YEEs) and the yield of the dry extract were used as measuring criteria. RSM with a Box-Behnken design using three factors and three levels was adopted to optimize the ethanol extraction technology for YJP. Finally, acute and subchronic toxicity tests were performed to evaluate its safety. The results revealed the best technological parameters: a liquid-material ratio of 24:1, an ethanol concentration of 69%, assistance of ultrasound (40 °C, 50 kHZ, 30 min), reflux time of 53 min, and reflux temperature of 50 °C. In acute toxicity tests, the maximum administration dosage in mice was 28.21 g/kg, which is higher than 10 times the clinical dosage. Adverse effects in the acute and subchronic toxicity tests were not observed. All clinical indexes were normal. In conclusion, the RSM based on AHP-CRITIC weight analysis could be used to optimize the ethanol extraction technology for YJP and YEEs prepared under the above conditions and ensure high safety.

Moniezia benedeni infection enhances neuromedin U (NMU) expression in sheep (Ovis aries) small intestine.

BACKGROUND: Neuromedin U (NMU) plays an important role in activating the group 2 innate lymphoid cells (ILC2s) and initiating the host's anti-parasitic immune responses. It is aimed to explore the distribution characteristics of NMU in the sheep small intestine and the influence of Moniezia benedeni infection on them. In the present study, the pET-28a-NMU recombinant plasmids were constructed, and Escherichia coli. BL21 (DE3) were induced to express the recombinant protein. And then, the rabbit anti-sheep NMU polyclonal antibody was prepared and immunofluorescence staining was performed with it. The expression levels of NMU in the intestine of normal and Moniezia benedeni-infected sheep were detected by ELISA. RESULTS: The results showed that the molecular weight of the obtained NMU recombinant protein was consistent with the expected molecular (13 kDa) and it was expressed in the form of inclusion body. The titer and specificity of obtained rabbit anti-sheep NMU polyclonal antibody were good. The results of immunofluorescence analysis showed that the nerve fibers which specifically expressed NMU mainly extended from the ganglion in the submucosal to lamina propria (LP) in the sheep small intestine, and the expression level was relatively high; especially on the nerve fibers of LP around the intestinal glands. The expression levels were gradually increased from the duodenum to the ileum, and the levels in the jejunum and ileum were significantly higher than that in the duodenum (P < 0.05). In addition, scattered NMU positive cells were distributed in the epithelium of the jejunal crypts. Moniezia benedeni infection increased the expression of NMU in each intestinal segment, especially in the jejunum and ileum there were significant increase (P < 0.05). CONCLUSIONS: It was suggested that Moniezia benedeni infection could be detected by the high expression of NMU in sheep enteric nervous, and which laid the foundation for further studies on whether NMU exerts anti-parasitic immunity by activating ILC2s. In addition, NMU was expressed in some intestinal gland epitheliums, which also provided a basis for studying its roles in regulation of the immune homeostasis. The present study laid the foundation for further revealing the molecular mechanism of sheep's neural-immune interaction network perceiving the colacobiosis of parasites.

A new method providing complementary explanation of the blood-enriching function and mechanism of unprocessed Angelica sinensis and its four kinds of processed products based on tissue-integrated metabolomics and confirmatory analysis.

Angelica sinensis (AS) is a common Traditional Chinese Medicine used for tonifying blood in China. Unprocessed AS and its four kinds of processed products (ASs) are used to treat blood deficiency syndrome in the country. The different blood-tonifying mechanisms of ASs remain unclear. In this work, a novel method integrating metabolomics and hematological and biochemical parameters was established to provide a complementary explanation of blood supplementation mechanism of ASs. Our results revealed that different ASs exhibited various blood supplementation effect, and that AS parched with alcohol demonstrated the best blood supplementation effect. Eight metabolites from liver tissue and 12 metabolites from spleen tissue were considered to be potential biomarkers. These biomarkers were involved in four metabolic pathways. Correlation analysis results showed that l-aspartic acid and l-alanine (spleen tissue), linoleic acid, and l-cystathionine (liver tissue) exhibited a high positive or negative correlation with the aforesaid biochemical indicators. The blood-supplementation effect mechanism of ASs were related to four metabolic pathways. l-Aspartic acid and l-alanine (spleen tissue), linoleic acid, and l-cystathionine (liver tissue) were the four key metabolites associated with the blood supplementation effect of ASs. This study gives a complementary explanation of the blood supplementation effect and mechanism of action of ASs.

Magnesium Modified ß-Tricalcium Phosphate Induces Cell Osteogenic Differentiation In Vitro and Bone Regeneration In Vivo.

In vitro, in vivo, and clinical studies have shown how the physicochemical and biological properties of ß-tricalcium phosphate (ß-TCP) work in bone regeneration. This study aimed to improve the properties of ß-TCP by achieving optimum surface and bulk ß-TCP chemical/physical properties through the hydrothermal addition of magnesium (Mg) and to later establish the biocompatibility of ß-TCP/Mg for bone grafting and tissue engineering treatments. Multiple in vitro and in vivo analyses were used to complete ß-TCP/Mg physicochemical and biological characterization. The addition of MgO brought about a modest rise in the number of ß-TCP surface particles, indicating improvements in alkaline phosphatase (ALP) activity on day 21 (p < 0.05) and in the WST-1assay on all days (p < 0.05), with a corresponding increase in the upregulation of ALP and bone sialoprotein. SEM analyses stated that the surfaces of the ß-TCP particles were not altered after the addition of Mg. Micro-CT and histomorphometric analysis from rabbit calvaria critical defects resulted in ß-TCP/Mg managing to reform more new bone than the control defects and ß-TCP control at 2, 6, and 8 weeks (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001). The hydrothermal addition of MgO to the ß-TCP surfaces ameliorated its biocompatibility without altering its surface roughness resulting from the elemental composition while enhancing cell viability and proliferation, inducing more bone regeneration by osteoconduction in vivo and osteoblastic differentiation in vitro.

Screening study of blood-supplementing active components in water decoction of Angelica sinensis processed with yellow rice wine based on response surface methodology.

CONTEXT: Angelica sinensis (Oliv.) Diels (Apiaceae) (syn. Angelica polymorpha Maxim var. sinensis Oliver) processed with yellow rice wine (WAS) has a blood-supplementing effect. OBJECTIVE: To establish an optimal technology for preparing water decoction of WAS (WASD), and screen blood-supplementing fractions. MATERIALS AND METHODS: Ferulic acid and crude polysaccharide were used in optimizing the preparation technology for WASD through response surface methodology. The independent variables were liquid-solid ratio, soaking time, and extraction time. Eighty Kunming mice were randomly divided into normal control, model, and six intervention groups (n = 10). The intervention groups were given different WASD fractions by gavage (5 or 10 g/kg). The model intervention groups received acetylphenyl hydrazine (subcutaneous injection) and cyclophosphamide (intraperitoneal injection). Duration of study, 9 days. The components of blood-supplementing fractions were analyzed. RESULTS: The optimum extraction parameters were liquid-solid ratio, 7.69:1 mL/g; soaking time, 119.78 min; and extraction time, 143.35 min. The optimal OD value was 0.8437. RBC, WBC, and Hb in the water fraction (5, 10 g/kg) and n-butanol fraction (10 g/kg) intervention groups increased significantly compared with the model group (p < 0.05). Polysaccharide and caffeic acid contents of water fraction were 252.565 and 0.346 µg/mg, respectively; ferulic acid was not detected. Caffeic acid and ferulic acid contents of n-butanol fraction were 1.187 and 0.806 µg/mg, respectively, polysaccharide was not detected. CONCLUSIONS: The optimum preparation technology of WASD was obtained, and the water, n-butanol fractions were blood-supplementing fractions. This study provides a theoretical foundation for further application of WAS in the pharmaceutical industry.

Urinary metabolomics analysis reveals the effect of volatile oil from Angelica sinensis on LPS-induced inflammation rats.

Lipopolysaccharide (LPS)-induced inflammation occurs commonly and volatile oil from Angelica sinensis (VOAS) can be used as an anti-inflammatory agent. The molecular mechanisms that allow the anti-inflammatory factors to be expressed are still unknown. In this paper, we applied gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography-time-of-flight mass spectrometry (LC-Q/TOF-MS) based on a metabolomics platform coupled with a network approach to analyze urine samples in three groups of rats: one with LPS-induced inflammation (MI); one with intervention with VOAS; and normal controls (NC). Our study found definite metabolic footprints of inflammation and showed that all three groups of rats, MI, intervention with VOAS and NC have distinct metabolic profiles in urine. The concentrations of 48 metabolites differed significantly among the three groups. The metabolites in urine were screened by the GC-MS and LC-Q/TOF-MS methods. The significantly changed metabolites (p < 0.05, variable importance in projection > 1.5) between MI, NC and VOAS were included in the metabolic networks. Finally, hub metabolites were screened, including glycine, arachidonic acid, l-glutamate, pyruvate and succinate, which have high values of degree (k). the Results suggest that disorders of glycine, arachidonic acid, l-glutamate, pyruvate and succinate metabolism might play an important part in the predisposition and development of LPS-induced inflammation. By applying metabolomics with network methods, the mechanisms of diseases are clearly elucidated.

Metabolomics study on promoting blood circulation and ameliorating blood stasis: Investigating the mechanism of Angelica sinensis and its processed products.

Angelica sinensis (Danggui, DG) parched with alcohol (Jiu Danggui, JDG) and charred DG are the main processed products of DG, which are used to treat blood stasis syndrome (BSS). However, their therapeutic effect and mechanisms are still unclear. Based on an acute rat BSS model, the intervention effects of DG and its processed products (DGPPs) were evaluated by the hemorheology and coagulation function parameters. Meanwhile, plasma and urine metabolites were detected and analyzed by liquid chromatography coupled to quadrupole time-of-flight mass spectrometry and multivariate statistical analysis method. The results of hemorheology, coagulation function parameters and metabolomics all showed that the BSS model was successfully established, DGPPs intervention could significantly relieve rats BSS and the therapeutic effect of JDG was best. Moreover, 23 differential metabolites (14 in plasma and nine in urine) were identified that were closely related to the BSS, involving seven potential target metabolic pathways. DGPP intervention showed different degrees of reverse effect on these metabolites. JDG was the most effective owing to extensive regulation effect on differential metabolites. This study provides a reference for understanding the pathological mechanism of BSS and the mechanism of DGPPs, which lays a theoretical foundation for the rational use of DGPPs in clinical practice.

Metabolomics analysis of Pulsatilla decoction on treatment of wetness-heat-induced diarrhea in rats based on UPLC-Q/TOF-MS/MS.

Pulsatilla decoction (PD) is a classical prescription in traditional Chinese medicine that has therapeutic effects on wetness-heat-induced diarrhea (WHD). To investigate the therapeutic effects of PD in the treatment of WHD and elucidate the potential mechanism, we used a metabolomics strategy on the base of ultraperformance liquid chromatography coupled with quadrupole time-of-flight/mass spectrometry (UPLC-Q/TOF-MS/MS) and analyzed the serum samples of 32 rats to identify differential metabolites and pathways associated with the PD treatment of WHD. With variable importance for projection >1.0 in the Orthogonal partial least-squares discriminant analysis (OPLS-DA ) models and FC ≥1.2 or ≤0.8, 67 differential metabolites in the model and control groups and 33 differential metabolites in the model and PD groups were screened. A total of 23 differential metabolites were selected based on Venny analysis. Functional analysis showed that the differential metabolites identified were primarily involved in pentose and glucuronate interconversions, glycerophospholipid metabolism, tryptophan metabolism, starch and sucrose metabolism, and glycerolipid metabolism. This study suggested that PD exerts inhibitory effects on WHD. In particular, the significant roles of PD for treating WHD lie in regulating perturbed energy metabolism, glycerophospholipid metabolism and glycerolipid metabolism, and promoting lysoPC production restoring the function of intestinal tract.

Establishment of a human hepatocellular cell line capable of maintaining long-term replication of hepatitis B virus.

Hepatitis B virus (HBV) is a virus whose replication cycle cannot be completely reproduced using cultured cell lines. Here, we report an engineered cell line capable of supporting the complete HBV life cycle. We generated HepG2 cells over-expressing the HBV entry receptor human NTCP (sodium taurocholate cotransporting polypeptide), and defective in RIG-I (retinoic acid-inducible gene-I)-like receptor signaling, by knocking down the IPS-1 (IFNß-promoter stimulator-1) adaptor molecule. The resultant NtG20.i7 cells were susceptible to HBV, and its replication was detectable at 14 days post-infection and persisted for at least 35 days with a gradual increase of HBV core expression. The cells produced infectious HBV in the culture supernatant, and the addition of preS1 peptide myr47-WT, which blocks HBV entry, impaired the persistence of the infection. These findings suggest that the persistence of the infection was maintained by continuous release of infectious HBV virions and their re-infection. This system is useful for expanding our basic understanding of the HBV replication cycle and for screening of anti-HBV chemicals.

Effects of Tao-Hong-Si-Wu decoction on acute blood stasis in rats based on a LC-Q/TOF-MS metabolomics and network approach.

A novel approach using metabolomics coupled with a metabolic network was used to investigate the effects of Tao-Hong-Si-Wu decoction (THSWD) on the rat model of acute blood stasis syndrome. Acute blood stasis syndrome was induced by placing the rats in ice-cold water following two injections with epinephrine. The hemorheological indicators [whole blood viscosity (WBV) and plasma viscosity (PV)] and the blood coagulation indicators [thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen (FIB)] were detected. The nonparametric univariate method and multivariate statistical analysis were performed for determining the potential biomarkers. A correlation map was structured between biochemical indicators and hub metabolites to explain the effects mechanism of THSWD. After the administration of THSWD, the levels of WBV, PV, TT, APTT and FIB returned to levels observed in the control group. According to metabolomics coupled with metabolic network analysis, the intervention of THSWD in rats with acute blood stasis syndrome induced substantial and characteristic changes in their metabolic profiles. Fifteen metabolites were screened, which mainly involved 10 pathways and five hub metabolites, namely, l-glutamate, l-phenylalanine, N-acylsphingosine, arachidonic acid and phosphatidate. The biochemical indicators and hub metabolites could be adjusted to close to normal levels by THSWD. Therefore, combining metabolomics and metabolic network helped to evaluate the effects of THSWD on acute blood stasis.

Effects of volatile oils of Angelica sinensis on an acute inflammation rat model.

Context Despite several pharmacological studies of volatile oils of Angelica sinensis (Oliv.) Diels (Umbelliferae) (VOAS), its anti-inflammatory mechanism remains unknown. Objective The study investigates the effects of VOAS on the lipopolysaccharide (LPS)-induced acute inflammation rat model and analyzes its possible anti-inflammatory mechanisms. Materials and methods Fourty rats were randomly divided into the control, model, VOAS and dexamethasone (Dex) groups. The VOAS and Dex groups were given VOAS (0.176 mL/kg) and Dex (40 µg/kg), respectively. Rats in all groups except the control group were intraperitoneally injected with LPS (100 µg/kg), their exterior behaviour and liver pathological changes were observed, and the level of white blood cell (WBC), the number of neutrophils (NE)%, glutamic oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), alkaline phosphatase (ALP), tumour necrosis factor (TNF-α), interleukin (IL)-1ß, IL-6, IL-10, histamine (HIS), 5-hydroxytryptamine (5-HT), nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) were detected. Results Compared with the model group, VOAS and Dex significantly accelerated the recovery of the exterior behaviour, the liver pathological changes of rats, and increased the level of IL-10, but decreased the level of WBC, NE%, GOT, GPT, ALP, TNF-α, IL-1ß, IL-6, HIS, 5-HT, NO, PGE2, iNOS and COX-2 (p < 0.05). Conclusion VOAS exhibits anti-inflammatory and liver protection effects by inhibiting the secretion of the pro-inflammatory cytokines (TNF-α, IL-1ß and IL-6), the inflammatory mediators (HIS, 5-HT, PGE2 and NO), the inflammation-related enzymes (iNOS and COX-2), as well as promoting the production of the anti-inflammatory cytokines IL-10.

[Effects of Volatile Oils from Different Preparations of Angelica sinensis Root on Acute Inflammation Induced by LPS in Rats].

Objective: To compare the intervention effects of volatile oils from different preparations of Angelica sinensis root on acute inflammation induced by lipopolysaccharide in rats. Methods: Acute inflammation model was induced by intraperitoneal injection of lipopolysaccharide( 100 µg/kg) in rats. Blood and serum inflammatory mediators and cytokines were detected, combining with the pathological histological observation of lung and liver to evaluate the anti-inflammatory activities of volatile oils from parching Angelica sinensis root with wine( J-VOAS),volatile oils from charred Angelica sinensis root( C-VOAS) and Angelica sinensis root( S-VOAS). Results: Compared with control group, the WBC count, the percentage of NE and PLT count in acute inflammation model group significantly increased ( P < 0. 05),and the percentage of LY significantly decreased( P < 0. 05); the content of IL-1ß,IL-6,NO and TNF-α significantly increased( P < 0. 001) and content of IL-10 significantly decreased( P < 0. 05) in model group; after J-VOAS,C-VOAS and S-VOAS intervention, the blood routine index and serum inflammatory mediators and cytokines significantly reversed( P < 0. 05). The pathological histological study showed that expanded alveoli, massive inflammatory cells infiltration in alveoli and pulmonary interstitium, the liver leaflets diffuse necrosis, hepatic cord derangement, and some of the liver cells degeneration and edema in model group; after J-VOAS intervention, their pathological changes significantly reduced. Conclusion: All volatile oils from different preparations of Angelica sinensis root had intervention on acute inflammation induced by LPS. And J-VOAS had the best effect, followed by C-VOAS and S-VOAS.

[Metabolomic evaluation for anti-inflammatory effect of volatile oils from different preparations of Angelicae Sinensis Radix].

To evaluate the anti-acute inflammation effects of volatile oils from different processed products of Angelicae Sinensis Radix(AS) in the rat model of acute inflammation established by the metabolomic method. Volatile oil of charred AS (C-VOAS), wine-processed AS (J-VOAS), locally processed AS (T-VOAS) and oil-process AS (Y-VOAS) were applied to intervene the rat acute paw swelling inflammation model induced by Carrageenan. Changes in serum HIS, 5-HT, PGE2 and TNF-α content in rats were detected. Gas chromatography-mass spectrometry was used to detect the metabolites in plasma. Potential biomarkers were investigated according to principal component analysis method and partial least-squares discriminant analysis. According to the results, C-VOAS and J-VOAS could significantly inhibit inflammatory mediators Histamine, 5-hydroxytryptamine, prostaglandin-E2 and cytokine tumor necrosis factor-alpha (P<0.01), and T-VOAS and Y-VOAS also showed a significantly inhibitory effect (P<0.05). Compared with the normal group, 14 endogenous metabolite biomarkers showed metabolic disturbance in plasma (P<0.05 or P<0.01). Compared with acute inflammation model group, C-VOAS and J-VOAS could better recover the levels of the endogenous metabolites (P<0.05 or P<0.01) than T-VOAS and Y-VOAS (P<0.05 or P<0.01). This study suggests that C-VOAS and J-VOAS show a better anti-inflammatory effect than T-VOAS and Y-VOAS. Therefore, the metabolomic method could be used to expound the anti-inflammatory mechanism of volatile oils from different processed products of AS, and provide a theoretical basis for clinical application of VOAS.

[Effect of different processed volatile oils from Angelica sinensis on urinary metabolomics rats].

Different processed volatile oils from AS on urine metabolites of normal rats were analyzed to reveal the possible metabolic pathways. Totally 50 male Waster rats were randomly divided into normal control group, C-ASVO group, J-ASVO group, T-ASVO group and Y-ASVO group, with 10 rats in each group. The normal group was given isovolumetric 0.5% polyoxyethylene sorbitan fatty acid ester（Tween-80）, while the other groups were given 0.176 mL•kg⁻¹ different processed volatile oils from AS. Drugs were given for 3 successive days. The urine was collected at 48 h with metabolic cages. GC-MS was employed to detect the metabolic fingerprint of rat urine in different times. Principal component analysis(PCA) and orthogonal partial least-squares discriminant analysis(OPLS-DA) were adopted for a multivariate statistical analysis. Metabolites with potential differences were selected based on the results of variable importance in the projection(VIP) and t test. The metabolic pathway analysis(MetPA) database was built for different metabolites' metabolic pathways. The results showed that compared with the normal group, 31 kinds of endogenous metabolites in the different processed volatile oils from AS groups change significantly(P<0.05). And there were differences in normal rat urine metabolites among the different processed volatile oils from AS, of which the influence degree of J-ASVO was slightly stronger than C-ASVO, T-ASVO, and Y-ASVO. Therefore, the metabolism effect may be focused on energy metabolism, amino acid metabolism, fatty acid metabolism and glucose metabolism. This study focused on metabolism and mechanism of different processed volatile oils from AS, and provided new ideas for pharmacological actions of traditional Chinese medicines.

Autotaxin/Lpar3 signaling regulates Kupffer's vesicle formation and left-right asymmetry in zebrafish.

Left-right (L-R) patterning is essential for proper organ morphogenesis and function. Calcium fluxes in dorsal forerunner cells (DFCs) are known to regulate the formation of Kupffer's vesicle (KV), a central organ for establishing L-R asymmetry in zebrafish. Here, we identify the lipid mediator lysophosphatidic acid (LPA) as a regulator of L-R asymmetry in zebrafish embryos. LPA is produced by Autotaxin (Atx), a secreted lysophospholipase D, and triggers various cellular responses through activation of specific G protein-coupled receptors (Lpar1-6). Knockdown of Atx or LPA receptor 3 (Lpar3) by morpholino oligonucleotides perturbed asymmetric gene expression in lateral plate mesoderm and disrupted organ L-R asymmetries, whereas overexpression of lpar3 partially rescued those defects in both atx and lpar3 morphants. Similar defects were observed in embryos treated with the Atx inhibitor HA130 and the Lpar1-3 inhibitor Ki16425. Knockdown of either Atx or Lpar3 impaired calcium fluxes in DFCs during mid-epiboly stage and compromised DFC cohesive migration, KV formation and ciliogenesis. Application of LPA to DFCs rescued the calcium signal and laterality defects in atx morphants. This LPA-dependent L-R asymmetry is mediated via Wnt signaling, as shown by the accumulation of ß-catenin in nuclei at the dorsal side of both atx and lpar3 morphants. Our results suggest a major role for the Atx/Lpar3 signaling axis in regulating KV formation, ciliogenesis and L-R asymmetry via a Wnt-dependent pathway.

Shaoyao decoction improves damp-heat colitis by activating the AHR/IL-22/STAT3 pathway through tryptophan metabolism driven by gut microbiota.

ETHNOPHARMACOLOGICAL RELEVANCE: The efficacy of Shaoyao Decoction (SYD), a traditional Chinese medicine prescription, in treating damp-heat colitis is established, but its underlying mechanism remains to be elucidated. AIM OF THE STUDY: Our study aims to investigate the effect and mechanism of action of SYD in treating damp-heat colitis. MATERIALS AND METHODS: A mouse model of damp-heat colitis was induced and treated with SYD via gavage for seven days. The therapeutic efficacy of SYD was assessed through clinical indicators and histopathological examinations. The inflammatory factors and oxidative stress parameters were detected by ELISA and biochemical kits. We also analyzed alterations in the gut microbiome via 16 S rRNA gene sequencing and quantified serum indole derivatives using targeted tryptophan metabolomics. Western blotting and immunofluorescence were used to detect the expressions of AHR, CYP1A1, STAT3 and tight junction (TJ) proteins. The ELISA kit was utilized to detect the content of antibacterial peptides (Reg3ß and Reg3γ) in colon. The immunohistochemistry was employed to detect the expressions of proliferating cell nuclear antigen (PCNA) protein. RESULTS: SYD effectively alleviated symptoms in mice with damp-heat colitis, including body weight loss, shortened colon, elevated DAI, enlarged spleen, and damage to the intestinal mucosa. SYD notably reduced IL-6, TNF-α, IL-1ß and MDA levels in colon tissues, while increasing IL-10 and T-AOC levels. Furthermore, SYD mitigated gut microbiota disturbance, restored microbial tryptophan metabolite production (such as IA, IAA, and IAld), notably increased the protein levels of AHR, CYP1A1 and p-STAT3 in colon tissue, and elevated the IL-22 level. Moreover, the expression levels of Reg3ß, Reg3γ, occludin, ZO-1 and PCNA were increased in SYD group. CONCLUSION: Our study showed that SYD ameliorates damp-heat colitis by restructuring gut microbiota structure, enhancing the metabolism of tryptophan associated with gut microbiota to activate the AHR/IL-22/STAT3 pathway, thereby recovering damaged intestinal mucosa. This research offers novel insights into the therapeutic mechanisms of SYD on damp-heat colitis.

Moniezia benedeni drives CD3+ T cells residence in the sheep intestinal mucosal effector sites.

Introduction: T cells are the core of the cellular immunity and play a key role in the regulation of intestinal immune homeostasis. In order to explore the impact Moniezia benedeni (M. benedeni) infection on distributions of CD3+ T cells in the small intestine of the sheep. Methods: In this study, sheep pET-28a-CD3 recombinant plasmid were constructed and expressed in BL21 receptor cells, then the rabbit anti-sheep CD3 polyclonal antibody was prepared through recombinant protein inducing. The M. benedeni-infected sheep (infection group, n = 6) and healthy sheep (control group, n = 6) were selected, and the distributions of CD3+ T cells in intestinal laminae propria (LP) and mucous epitheliums were observed and analyzed systematically. Results: The results showed that the rabbit anti-sheep CD3 polyclonal antibody had good potency and specificity. In the effector area of small intestine, a large number of CD3+ T cells were mainly diffusely distributed in the intestinal LP as well as in the mucous epitheliums, and the densities of intestinal LP from duodenum to jejunum to ileum were 6.01 cells/104 µm2, 7.01 cells/104 µm2 and 6.43 cells/104 µm2, respectively. Their distribution densities in mucous epitheliums were 6.71 cells/104 µm2, 7.93 cells/104 µm2 and 7.21 cells/104 µm2, respectively; in the infected group, the distributions of CD3+ T cells were similar to that of the control group, and the densities in each intestinal segment were all significantly increased (p < 0.05), meanwhile, the total densities of CD3+ T cells in duodenum, jejunum and ileum were increased by 33.43%, 14.50%, and 34.19%. In LP and mucous epitheliums, it was increased by 33.57% and 27.92% in duodenum; by 25.82% and 7.07% in jejunum, and by 27.07% and 19.23% in ileum, respectively. Discussion: It was suggested that M. benedeni infection did not change the spatial distributions of CD3+ T cells in the small intestine of sheep, but significantly increased their densities, which lays a foundation for further research on the regulatory mechanism of sheep intestinal mucosal immune system against M. benedeni infection.