Rat Feces Research Articles

The Effect of Gut Microbiota on the Progression of Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IDD) is the main cause of back pain, and its treatment is a serious socio-economic burden. The safety and treatment of fecal microbiota transplantation (FMT) has been established. However, the relationship between FMT and IDD still unclear. We aimed to explore whether FMT plays a role in IDD to provide a reference for the treatment of IDD. An experimental model of IDD was established using 2-month-old male Sprague-Dawley rats. FMT was performed by intragastric gavage of IDD rats with a fecal bacterial solution. Rat serum, feces, and vertebral disc tissue were collected after surgery for 2 months. The levels of TNF-α, IL-1β, IL-6, matrix metalloproteinase (MMP)-3, MMP-13, Collagen II, and aggrecan in the serum or vertebral disc tissue were measured by an enzyme-linked immunosorbent assay, immunohistochemistry, quantitative real-time polymerase chain reaction, or western blotting. We also examined the pathology of the vertebral disc tissue using hematoxylin and eosin (HE) and safranin O-fast green staining. Finally, we examined the gut microbiota in rat feces using 16 S rRNA gene sequencing. We found that the expression of TNF-α, IL-1β, IL-6, MMP-3, MMP-13, NLRP3 and Caspase-1 increased in the IDD group rats. In contrast, Collagen II and aggrecan levels were downregulated. Additionally, vertebral disc tissue was severely damaged in the IDD group, with disordered cell arrangement and uneven safranin coloration. FMT reversed the effects of IDD modeling on these factors and alleviated cartilage tissue damage. In addition, FMT increased the gut microbiota diversity and microbial abundance in rats treated with IDD. Our findings suggest that FMT has a positive effect in maintaining cellular stability in the vertebral disc and alleviating histopathological damage. It affects the diversity and abundance of gut microbiota in rats with IDD. Therefore, FMT may serve as a promising target for amelioration of IDD.

Folic acid attenuates chronic visceral pain by reducing Clostridiales abundance and hydrogen sulfide production.

Irritable bowel syndrome (IBS) related chronic visceral pain affects 20% of people worldwide. The treatment options are very limited. Although the scholarly reviews have appraised the potential effects of the intestinal microbiota on intestinal motility and sensation, the exact mechanism of intestinal microbiota in IBS-like chronic visceral pain remains largely unclear. The purpose of this study is to investigate whether Folic Acid (FA) attenuated visceral pain and its possible mechanisms. Chronic visceral hyperalgesia was induced in rats by neonatal colonic inflammation (NCI). 16S rDNA analysis of fecal samples from human subjects and rats was performed. Patch clamp recording was used to determine synaptic transmission of colonic-related spinal dorsal horn. Alpha diversity of intestinal flora was increased in patients with IBS, as well as the obviously increased abundance of Clostridiales order (a main bacteria producing hydrogen sulfide). The hydrogen sulfide content was positive correlation with visceral pain score in patients with IBS. Consistently, NCI increased Clostridiales frequency and hydrogen sulfide content in feces of adult rats. Notably, the concentration of FA was markedly decreased in peripheral blood of IBS patients compared with non-IBS human subjects. FA supplement alleviated chronic visceral pain and normalized the Clostridiales frequency in NCI rats. In addition, FA supplement significantly reduced the frequency of sEPSCs of neurons in the spinal dorsal horn of NCI rats. Folic Acid treatment attenuated chronic visceral pain of NCI rats through reducing hydrogen sulfide production from Clostridiales in intestine.

Efficient extraction of small microplastic particles from rat feed and feces for quantification

To date, microplastic is ubiquitously encountered in the environment. Studies analyzing microplastic in terrestrial ecosystems, including animal feces and feed, are few. Microplastic quantification method validation and harmonization are not yet far developed. For the analysis of small microplastic, approximately <0.5 mm, extraction from organic and inorganic materials is fundamental prior to quantitative and qualitative analysis. Method validation, including recovery studies, are necessary throughout the analytical chain. In this study, we developed an optimized, efficient protocol with a duration of 72 h for the digestion of laboratory rat feed and feces. A combination of a mild acidic (H2O2 15%; HNO3 5%) and an alkaline treatment (10% KOH) dissolving the previous filter, followed by enzymatic digestion (Viscozyme®L) proved to be efficient for the extraction and identification of spiked polyamide (15–20 μm) and polyethylene (40–48 μm) from feed and feces samples from rats, showing high recovery rates. Extracted rat feces samples from an in vivo study in which Wistar rats were fed with feed containing microplastic were analyzed with pyrolysis-gas chromatography-Orbitrap™ mass spectrometry, quantifying recovered microplastic in rat feces in environmentally relevant concentrations. The presented three-step protocol provides a suitable, time and cost-effective method to extract microplastic from rat feed and feces.

Application of the Midi Parasep® SF technique for the detection of L1 larvae of Angiostrongylus cantonensis in faeces of infected rats.

We investigated parasitic zoonoses caused by protozoans and helminths in urban and peri-urban rat populations (Rattus norvegicus and R. rattus) in Spanish cities. Rats were trapped and then dissected to remove adult helminths, and the contents of the large intestine were retrieved for the study of parasitic forms. The Midi Parasep® solvent free (SF) technique was used to concentrate the parasites in the intestinal contents. Some of the rats studied (n = 8) were infected by the rat lungworm, Angiostongylus cantonensis, whose first stage larvae (L1) are shed in rat faeces. After the concentration technique, L1 larvae were found in the sediment of 6 of the 8 positive rats. The two negative sediment samples were due to the presence of either only adult females or, in addition to males, only young females in the lungs of the rats. In view of our results, Midi Parasep® SF turned out to be a simple, rapid, inexpensive, and sensitive method to detect nematode larvae, such as the L1 larvae of A. cantonensis (or A. costaricensis), in natural and experimentally infected rats.

Sulfasalazine prevents lung injury due to intra-abdominal sepsis in rats: possible role of Nrf2 and angiopoietin-2

This study aimed to investigate the effect of sulfasalazine in preventing and treating intra-abdominal sepsis-induced acute respiratory distress syndrome (ARDS) in a rat model. Forty male Wistar albino rats were used. The rats were randomly divided into four equal groups, and sepsis was induced in 30 rats by intraperitoneal administration of a fecal saline solution prepared from rat feces. Group 1: normal control (n=10) [non-surgical], Group 2: fecal intraperitoneal injection (FIP) (n=10) [untreated septic group], Group 3: FIP+saline (placebo) (n=10) [saline administered intraperitoneally], Group 4 (n=10): FIP+sulfasalazine [250 mg/kg per day administered intraperitoneally]. Computed tomography was performed and blood samples were collected for biochemical and blood gas analysis. The lungs were removed for histopathological studies. Statistically significant reductions in interleukin (IL)-6, IL1-β, tumor necrosis factor (TNF)-α, malondialdehyde (MDA), and angiopoietin-2 (ANG-2) levels were observed in the sulfasalazine group compared to the FIP+saline group (P<0.001). Nrf2 levels were significantly higher in the sulfasalazine-treated group than in the FIP and FIP+saline groups (P<0.01). Lung tissue scores were significantly reduced in the sulfasalazine group compared to the other sepsis groups. The Hounsfield unit (HU) value was significantly lower in the sulfasalazine group than in the FIP+saline group (P<0.001). PaO2 values were significantly higher in the sulfasalazine-treated group than in the FIP+saline-treated group (P<0.05). Sulfasalazine was shown to be effective in preventing and treating ARDS.

Integration of semi-empirical MS/MS library with characteristic features for the annotation of novel amino acid-conjugated bile acids.

Recently, amino acids other than glycine and taurine were found to be conjugated with bile acids by the gut microbiome in mouse and human. As potential diagnostic markers for inflammatory bowel disease and farnesoid X receptor agonists, their physiological effects and mechanisms, however, remain to be elucidated. A tool for the rapid and comprehensive annotation of such new metabolites is required. Thus, we developed a semi-empirical MS/MS library for bile acids conjugated with 18 common amino acids, including alanine, arginine, asparagine, aspartate, glutamine, glutamate, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. To investigate their fragmentation rules, these amino acids were chemically conjugated with lithocholic acid, deoxycholic acid, and cholic acid, and their accurate-mass MS/MS spectra were acquired. The common fragmentation patterns from the amino acid moieties were combined with 10 general bile acid skeletons to generate a semi-empirical MS/MS library of 180 structures. Software named BAFinder 2.0 was developed to combine the semi-empirical library in negative mode and the characteristic fragments in positive mode for automatic unknown identification. As a proof of concept, this workflow was applied to the LC-MS/MS analysis of the feces of human, beagle dogs, and rats. In total, 171 common amino acid-conjugated bile acids were annotated and 105 of them were confirmed with the retention times of synthesized compounds. To explore other potential bile acid conjugates, user-defined small molecules were in-silico conjugated with bile acids and searched in the fecal dataset. Four novel bile acid conjugates were discovered, including D-Ala-D-Ala, Lys(iso)-Gly, L-2-aminobutyric acid, and ornithine.

PARP-1 inhibitor alleviates liver lipid accumulation of atherosclerosis via modulating bile acid metabolism and gut microbes.

Background: The DNA damage repair enzyme, poly(ADP-ribose) polymerase 1 (PARP1), is crucial for lipid and glucose metabolism. However, no evidence has been presented on the relationship between liver lipid accumulation and the PARP1 inhibitor, 3-aminobenzamide (3-AB), in atherosclerosis. Methods: ApoE-/- mice were used to explore the effect of 3-AB on atherosclerotic liver lipid accumulation, and the experiment of Sprague Dawley (SD) rats was designed to determine if the lowering of liver lipid levels by 3-AB was linked to gut bacteria. The levels of bile acid metabolism-related targets were assessed by ELISA, western blotting, and RT-qPCR. The relative abundances of gut microbes and biomarkers were determined using 16S rRNA sequencing analysis. Bile acid levels in the liver and ileum were examined by ultra-performance liquid chromatography-tandem mass spectrometry. The relationship between gut microbes and bile acids was assessed by Spearman's correlation analysis. Results: 3-AB significantly reduced the formation of aortic plaques in apoE-/- mice, according to gross oil red staining. H & E and Oil Red O staining revealed that 3-AB significantly reduced the hepatic lipid droplet area in ApoE-/- mice and SD rats. Compared with the atherosclerosis (ATH) group, 3-AB dramatically decreased the levels of total cholesterol (TC), triglyceride (TG), and low-density lipoprotein-cholesterol (LDL-C) in the serum of SD rats and apoE-/- mice, and the levels of TC, TG, and LDL-C in the serum and liver of apoE-/- mice. Furthermore, in apoE-/- mice and SD rats, 3-AB increased the mRNA and protein levels of farnesoid X receptor (FXR) and bile salt export pump (BSEP) in the liver, while inhibiting the mRNA and protein levels of FXR and fibroblast growth factor 15 (FGF15) in the ileum, respectively. 3-AB clearly inhibited the mRNA and protein levels of PARP1 in the liver and ileum of apoE-/- mice and rats. Following treatment with 3-AB, the levels of conjugated bile acids decreased in the liver of apoE-/- mice and increased in the ileum of SD rats, according to targeted metabolomic analysis. Microbiome sequencing analysis revealed that 3-AB reduced the relative abundance of Lactobacillus, Bifidobacterium, Listeria, Clostridium, Bacillus, and Staphylococcus in the feces of apoE-/- mice, and the relative abundance of Blautia, Clostridium, and Listeria in the feces of SD rats, eventually decreasing the total abundance of 10 bile salt hydrolase-associated gut microbes. According to the correlation analysis, 3-AB regulates bile acid metabolism, which is primarily related to Bifidobacterium. Conclusion: 3-AB alleviated atherosclerosis by modulating the bile acid metabolism and bile salt hydrolase-related gut microbes.

Natural sporopollenin microcapsules: biological evaluation and application in regulating hepatic toxicity of diclofenac sodium in vivo.

Diclofenac sodium (DIC) is a pain reliever and anti-nociceptive medication. Significant limitations of DIC treatment stem from its adverse effects. This study investigates the feasibility of using natural Lycopodium clavatum sporopollenin (LCS) microcapsules loaded with DIC to mitigate the hepatotoxicity associated with DIC treatment. In addition, LCS microcapsules were tracked in the blood, stomach, small intestine, and feces of rats to demonstrate their morphological integrity and uptake behavior. Four groups (6 per group) of adult male albino rats were administered normal saline (control), empty LCS (30 mg kg-1), plain DIC (10 mg kg-1), and DIC-loaded LCS (40 mg kg-1) orally for seven consecutive days. The first comprehensive histological examination of the rat stomach demonstrated the robustness and bioadhesion ability of LCS under severe conditions. The findings suggested that these versatile microcapsules are unlikely to be digested in the gastrointestinal tract (GIT). The administration of DIC-loaded LCS was found to play a potential protective role in regulating DIC-induced substantially increased serum levels of transaminases, alkaline phosphatase, total bilirubin, and pro-inflammatory cytokines. In addition, DIC-loaded LCS restored the antioxidant enzymes, DNA damage, and liver histological architecture abnormalities caused by DIC. Microencapsulation of DIC into pollen-derived biomaterials could be employed as an efficient platform with enough safety coverage on rat liver, pending further clinical studies.

Effects of electroacupuncture at "Zhongliao" and "Xialiao" on serotonin signaling system and short-chain fatty acids in slow transit constipation rats

To observe the effect of electroacupuncture (EA) at "Zhongliao" (BL33) and "Xialiao" (BL34) on the 5-hydroxytryptamine (5-HT) signaling system in colon tissue and short-chain fatty acids in feces of rats with slow transit constipation (STC), so as to explore the underlying mechanisms of EA in the treatment of STC. A total of 32 SD rats were randomly divided into normal, model, drug control and EA groups, with 8 rats in each group. The STC model was established by intragastric administration of loperamide for 14 days. The EA stimulation (2 Hz/15 Hz) was performed at bilateral BL33 and BL34 for 30 min, once a day for 14 days. The first black stool de-fecation time and fecal water content were detected after treatment. The expressions of 5-hydroxytryptamine 4 receptor (5-HT4R), tryptophan hydroxylase 1 (TPH1) and 5-HT transporter (SERT) in colon tissues were detected by Western blot. The contents of substance P (SP) and vasoactive intestinal peptide (VIP) in serum were detected by ELISA. The contents of 5-HT in colon tissue and short chain fatty acid (SCFA) in feces were detected by mass spectrometry. Compared with the normal group, the fecal water content, the expressions of 5-HT, 5-HT4R, TPH1 and SERT in colon tissue, the content of serum SP were significantly decreased (P<0.05), the first black stool de-fecation time, and the content of serum VIP was significantly increased (P<0.05), the contents of SCFA in feces were significantly decreased except isobutyric acid (P<0.05) in the model group. Compared with the model group, the fecal water content, the expressions of 5-HT, 5-HT4R, TPH1 and SERT in colon tissues, the contents of acetic acid and butyrate in feces were significantly increased (P<0.05) in the EA and drug control groups, the first black stool defecation time was decreased (P<0.05) in the EA and drug control groups, and the content of serum SP was increased and the content of serum VIP was decreased (P<0.05) in the EA group. Compared with the drug control group, the content of serum VIP was significantly decreased (P<0.05), and the expressions of TPH1 and SERT in colon tissue were significantly increased (P<0.05) in the EA group. EA at BL33 and BL34 can promote intestinal motility by intervening multiple links of 5-HT signaling system in treating STC.

Toxic mechanism of the Mongolian medicine "Hunqile-7" based on metabonomics and the metabolism of intestinal flora.

The traditional Mongolian medicine Hunqile-7 (HQL-7), which is mainly used to relieve pain in clinic, has certain toxicity. Therefore, toxicological investigation of HQL-7 is of great significance to its safety assessment. In this study, the toxic mechanism of HQL-7 was explored based on a combination of metabolomics and intestinal flora metabolism. UHPLC-MS was used to analyze the serum, liver and kidney samples of rats after intragastric administration of HQL-7. The decision tree and K Nearest Neighbor (KNN) model were established based on the bootstrap aggregation (bagging) algorithm to classify the omics data. After samples were extracted from rat feces, the high-throughput sequencing platform was used to analyze the 16s rRNA V3-V4 region of bacteria. The experimental results confirm that the bagging algorithm improved the classification accuracy. The toxic dose, toxic intensity, and toxic target organ of HQL-7 were determined in toxicity tests. Seventeen biomarkers were identified and the metabolism dysregulation of these biomarkers may be responsible for the toxicity of HQL-7 in vivo. Several kinds of bacteria was demonstrated to be closely related to the physiological indices of renal and liver function, indicating liver and kidney damage induced by HQL-7 may be related to the disturbance of these intestinal bacteria. Overall, the toxic mechanism of HQL-7 was revealed in vivo, which not only provides a scientific basis for the safe and rational clinical use of HQL-7, but also opens up a new field of research on big data for Mongolian medicine.

Modified Baihu decoction therapeutically remodels gut microbiota to inhibit acute gouty arthritis.

Background: Acute gouty arthritis (AGA) is the most common first symptom of gout, and the development of gout as a metabolic and immune inflammatory disease is also correlated with the gut microbiota. However, the mechanism of the effect of changes in the gut microbiota on AGA remains unclear. The intestinal flora can not only affect purine metabolism or regulate inflammation, but also influence the therapeutic effect of drugs on AGA. The aim of this study was to investigate the exact mechanism of modified Baihu decoction (MBD) in the treatment of AGA and whether it is related to the regulation of the structure of the intestinal flora. Methods: On the 21st day of MBD administration by continuous gavage, a rat acute gouty arthritis model was constructed using sodium urate (0.1mL/rat, 50mg/mL), and the ankle joint swelling was measured before and 4h, 8h, 24h, and 48h after the injection of sodium urate. After 48h of sodium urate injection, serum, liver, kidney, ankle synovial tissue and feces were collected from rats. The collected samples were examined and analyzed using H&E, Elisa, Immunohistochemistry, Histopathology, 16S rDNA, and Biochemical analysis. To investigate the mechanism of MBD to alleviate AGA using pro-inflammatory factors and intestinal flora. Results: MBD (5.84, 35g/kg) was administered orally to AGA rats and diclofenac sodium tablets (DS-tablets) were used as standard treatment control. Serum biochemical assessment confirmed that MBD is a safe drug for the treatment of AGA. In addition, our findings confirmed that MBD relieved AGA-related symptoms, such as toe swelling. Lowering serum levels of uric acid, IL-1β, and TGF-β1 immunohistochemical results also confirmed that MBD reduced the expression of inflammatory elements such as IL-1β, NLRP3, ASC, and Caspase-1 in synovial tissue.Furthermore, compared with control group, the 16s rDNA sequencing of AGA rat faeces revealed an increase in the relative abundance of Lachnospiraceae, Muribaculaceae, and Bifidobacteriaceae species. While the relative abundance of Lactobacillaceae, Erysipelotrichaceae, Ruminococcaceae, Prevotellaceae and Enterobacteriaceae showed a relative decrease in species abundance. Of these, the reduction in species abundance of Enterobacteriaceae was associated with a reduction in amino acid metabolism and environmental perception. After MBD therapeutic intervention, the disturbance of the intestinal flora caused by AGA was restored. Conclusion: In summary, MBD is an effective agent for the treatment of AGA, with the potential mechanism being the regulation of intestinal flora to control inflammation. This would help to promote the therapeutic effect of MBD on AGA.

P2Y1 receptor in the colonic submucosa of rats and its association with opioid‑induced constipation.

The aim of the present study was to explore the expression changes of P2Y purinergic receptor 1 (P2Y1) in the distal colonic submucosa of opioid-induced constipation (OIC) rats and its association with the occurrence of OIC, an OIC rat model was generated by intraperitoneal injection of loperamide hydrochloride, a selective agonist of µ-opioid receptors (MORs). At 7 days post-treatment, the model was assessed by analyzing stool scores and calculating the gastrointestinal (GI) transit ratio of rats. The distribution of P2Y1-expressing neurons in the colonic submucosal plexus was demonstrated by immunofluorescence (IF). Western blotting was performed to evaluate the expression changes of MOR, P2Y1 and ATP synthase subunit β (ATPB) proteins in the colonic submucosa, while reverse transcription-quantitative PCR (RT-qPCR) analysis was performed to determine the relative mRNA expression of MOR and P2Y1. After 7 days, the feces of OIC rats exhibited an appearance of sausage-shaped pieces and both the stool weight and GI transit ratio of OIC rats were significantly decreased. IF revealed co-expression of P2Y1 and calbindin and MOR and ATPB in the nerve cells of the distal colonic submucosal plexus. Moreover, RT-qPCR analysis showed that the MOR mRNA levels were significantly increased in the distal colonic submucosa of OIC rats, while mRNA levels of P2Y1 were decreased. WB showed that in the distal colonic submucosa of OIC rats, MOR protein expression was increased, whereas that of P2Y1 was significantly decreased. GI transit ratio analysis suggested that the P2Y agonist ATP significantly relieved constipation symptoms in rats, while the P2Y inhibitor MRS2179 aggravated these symptoms. Finally, P2Y1 expression change was shown to be associated with the occurrence of OIC, while expression of MOR and P2Y1 was associated with OIC development in rats.

Gut Microbiota Alterations in Trace Amine-Associated Receptor 9 (TAAR9) Knockout Rats

Trace amine-associated receptors (TAAR1-TAAR9) are a family of G-protein-coupled monoaminergic receptors which might have great pharmacological potential. It has now been well established that TAAR1 plays an important role in the central nervous system. Interestingly, deletion of TAAR9 in rats leads to alterations in the periphery. Previously, we found that knockout of TAAR9 in rats (TAAR9-KO rats) decreased low-density lipoprotein cholesterol levels in the blood. TAAR9 was also identified in intestinal tissues, and it is known that it responds to polyamines. To elucidate the role of TAAR9 in the intestinal epithelium, we analyzed TAAR9-co-expressed gene clusters in public data for cecum samples. As identified by gene ontology enrichment analysis, in the intestine, TAAR9 is co-expressed with genes involved in intestinal mucosa homeostasis and function, including cell organization, differentiation, and death. Additionally, TAAR9 was co-expressed with genes implicated in dopamine signaling, which may suggest a role for this receptor in the regulation of peripheral dopaminergic transmission. To further investigate how TAAR9 might be involved in colonic mucosal homeostasis, we analyzed the fecal microbiome composition in TAAR9-KO rats and their wild-type littermates. We identified a significant difference in the number of observed taxa between the microbiome of TAAR9-KO and wild-type rats. In TAAR9-KO rats, the gut microbial community became more variable compared with the wild-type rats. Furthermore, it was found that the family Saccharimonadaceae, which is one of the top 10 most abundant families in TAAR9-KO rat feces, is almost completely absent in wild-type animal fecal samples. Taken together, these data indicate a role of TAAR9 in intestinal function.

Metabolite Profiling of Tartary Buckwheat Extracts in Rats Following Co-Administration of Ethanol Using UFLC-Q-Orbitrap High-Resolution Mass Spectrometry

Tartary buckwheat, a gluten-free pseudocereal, has received considerable attention owing to its unique nutritional ingredients and beneficial health effects such as anti-tumor, anti-oxidation, anti-inflammation and hepatoprotective activities. Pharmacokinetic and metabolite profiling have been preliminarily assessed for Tartary buckwheat extracts. However, its metabolites have not yet been characterized in vivo after co-administration with ethanol when Tartary buckwheat extracts are used for the treatment of alcoholic liver disease. In this paper, a Q-Exactive orbitrap high-resolution mass spectrometer was employed to identify the metabolites of Tartary buckwheat extracts in rat biological samples. Compared with previous metabolite profiling results, a total of 26 novel metabolites were found in rat biological samples, including 11, 10, 2 and 5 novel metabolites in rat plasma, bile, urine and feces, respectively, after oral co-administration of 240 mg/kg Tartary buckwheat extracts with ethanol (42%, v/v). The major metabolic pathways of the constituents in Tartary buckwheat extracts involved hydroxylation, methylation, glucuronidation, acetylation and sulfation. Quercetin and its metabolites may be the pharmacological material basis of Tartary buckwheat for the protective effect against alcoholic liver injury. The research enriched in vivo metabolite profiling of Tartary buckwheat extracts, which provided experimental data for a comprehensive understanding and rational use of Tartary buckwheat against alcoholic liver disease.

Excretion of Cynanchum auriculatum extract in urine and feces of normal and functional dyspepsia rats

In the present study, the excretion of four active components(qingyangshengenin, deacylmetaplexigenin, baishouwu benzophenone, and scopoletin) in Cynanchum auriculatum extract in the urine and feces of normal and functional dyspepsia(FD) rats was investigated. Rats were divided into a normal group and an FD model group. The FD model was induced by oral administration of ice hydrochloric acid combined with irregular feeding. The C. auriculatum extract was administered orally at a dose of 1 g·kg~(-1). The rat urine and feces were collected at 4, 8, 12, 24, 36, 48, 60, 72, and 84 h for UPLC-ESI-MS/MS analysis. The differences in excretion of the four components were compared between normal and FD rats. The results showed that except for the baishouwu benzophenone in the feces, the components such as qingyangshengenin in the urine and feces did not reach the plateau value within 84 h. Qingyangshengenin was mainly excreted through defecation, and the cumulative excretion rates in the normal state and FD were 0.32% and 0.66%, respectively. Deacylmetaplexigenin was mainly excreted through urination, and the cumulative excretion rates in the normal state and FD were 6.70% and 7.56%, respectively. Baishouwu benzophenone was mainly excreted through defecation in the normal state, but mainly excreted through urination in the FD state, with cumulative excretion rates of 0.41% and 0.52%, respectively. Scopoletin was mainly excreted through urination, with cumulative excretion rates of 0.83% and 2.13% in the normal state and FD, respectively. In general, the components were mainly excreted in the urine in the FD state. Compared with the normal group, the FD group showed decreased cumulative excretion rates of qingyangshengenin, baishouwu benzophenone, and scopoletin in the urine(P&lt;0.05). Therefore, FD had a certain influence on the excretion of the main components of C. auriculatum extract, and the excretion of each component through urination and defecation was low, suggesting that there might be a wide range of metabolic pathways after oral administration and components were mainly excreted in the form of metabolites. This experiment provides a reference for the new drug development and clinical application of C. auriculatum.

Salvia miltiorrhiza extract may exert an anti-obesity effect in rats with high-fat diet-induced obesity by modulating gut microbiome and lipid metabolism

Studies have shown that a high-fat diet (HFD) can alter gut microbiota (GM) homeostasis and participate in lipid metabolism disorders associated with obesity. Therefore, regulating the construction of GM with the balance of lipid metabolism has become essential for treating obesity. Salvia miltiorrhiza extract (Sal), a common traditional Chinese medicine, has been proven effective against atherosclerosis, hyperlipidemia, obesity, and other dyslipidemia-related diseases. To investigate the anti-obesity effects of Sal in rats with HFD-induced obesity, and explore the underlying mechanism by focusing on GM and lipid metabolism. Obesity was induced in rats with an HFD for 7 wk, and Sal (0.675 g/1.35 g/2.70 g/kg/d) was administered to treat obese rats for 8 wk. The therapeutic effect was evaluated by body weight, body fat index, waistline, and serum lipid level. Lipid factors (cAMP, PKA, and HSL) in liver and fat homogenates were analyzed by ELISA. The effect of Sal on GM and lipid metabolism was assessed by 16S rRNA-based microbiota analysis and untargeted lipidomic analysis (LC-MS/MS), respectively. Sal treatment markedly reduced weight, body fat index, serum triglycerides (TG), total cholesterol (TC), low-density lipoprotein, glucose, free fatty acid, hepatic lipid accumulation, and adipocyte vacuolation, and increased serum high-density lipoprotein (HDL-C) in rats with HFD-induced obesity. These effects were associated with increased concentrations of lipid factors such as cAMP, PKA, and HSL in the liver and adipose tissues, enhanced gut integrity, and improved lipid metabolism. GM analysis revealed that Sal could reverse HFD-induced dysbacteriosis by promoting the abundance of Actinobacteriota and Proteobacteria, and decreasing the growth of Firmicutes and Desulfobacterita. Furthermore, LC-MS/MS analysis indicated that Sal decreased TGs (TG18:2/18:2/20:4, TG16:0/18:2/22:6), DGs (DG14:0/22:6, DG22:6/22:6), CL (18:2/ 18:1/18:1/20:0), and increased ceramides (Cers; Cer d16:0/21:0, Cer d16:1/24:1), (O-acyl)-ω-hydroxy fatty acids (OAHFAs; OAHFA18:0/14:0) in the feces of rats. Spearman's correlation analysis further indicated that TGs, DGs, and CL were negatively related to the abundance of Facklamia and Dubosiella, and positively correlated with Blautia and Quinella, while OAHFAs and Cers were the opposite. Sal has an anti-obesity effect by regulating the GM and lipid metabolism.

Gender differences pharmacokinetics, bioavailability, hepatic metabolism and metabolism studies of Pinnatifolone A, a sesquiterpenoid compound, in rats by LC-MS/MS and UHPLC-Q-TOF-MS/MS

BackgroundPinnatifolone A is a typical sesquiterpenoid and the primary active ingredient of Syringa oblata Lindl., has potent anti-inflammatory activity. However, Pinnatifolone A pharmacokinetic and metabolites analysis investigations in male and female rats, as well as its in vitro stability in male and female rat liver microsomes, have not been evaluated and compared. PurposeTo investigate preclinical pharmacokinetic and metabolite in both genders, confirm gender differences, and provide usable information for the development of clinical applications. MethodsA quick, precise, and sensitive LC-MS/MS method was created and effectively used to determine the pharmacokinetics of oral (140 mg/kg) and intravenous (6.3 mg/kg) Pinnatifolone A in male and female rats, in vitro Pinnatifolone A elimination studies in male and female rat liver microsomes. Following that, a UHPLC-Q-TOF-MS/MS technique was established to identify the metabolic profiles of Pinnatifolone A obtained from rat plasma and excreta. ResultsIn the current study, we established for the first time an LC-MS/MS method for the quantitation of Pinnatifolone A with acceptable linearity and selectivity, recovery and matrix effect, accuracy and precision. The absolute oral bioavailability of Pinnatifolone A was approximately 30.36% in female rats, the clearance (CL) was 20.99±3.33 l/h/kg in female rats and 472.37±437.31 l/h/kg in male rats. This difference in rat genders may pertain to the sex-specific expression of hepatic enzymes as demonstrated in the metabolic stability evaluation in the present research; the male rats exhibited higher CLint(mic) (158.83±9.57 μl/min/mg protein) than female rats (76.47±7.90 μl/min/mg protein) liver microsomes, indicating higher Pinnatifolone A clearance in male rats. Twenty-four metabolites were detected and identified in female and male rats; N-acetylcysteine conjugation metabolite was the most abundant metabolites in both rat feces and urine. Furthermore, male and female rats had significantly different levels of the N-acetylcysteine conjugation metabolite. Hydrogenation metabolite was particular to female rats both in rat fecal and urine. Glucuronide conjugation metabolite was the predominant metabolite in rat plasma, and its amount in female rats was double that of male rats. ConclusionsThe present research is the first to report the preclinical pharmacokinetics and metabolites of Pinnatifolone A in male and female rats, confirming the gender-based differences. The findings provide a comprehensive overview for further understanding of the pharmacokinetic and metabolic characteristics of Pinnatifolone A and serve as a guide for its future development and utilization.

Metabolism, pharmacokinetics, and bioavailability of yuanhuacine in rat using LC-MS.

Yuanhuacine is a Daphne-type diterpene ortho-ester and is one of the main active ingredients of genkwa flos. Anticancer activity of yuanhuacine has been well investigated in various tumor cells and animal models, but information on metabolism and pharmacokinetics is limited. The aims of the present study were to investigate the metabolic and pharmacokinetic profiles of yuanhuacine in rat. The metabolic profile of yuanhuacine was obtained from rat plasma, urine, and feces using ultra-high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. A total of seven metabolites were detected, and the proposed metabolic pathways involved oxidation and glucuronidation. A simple and sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry method was developed for the determination of yuanhuacine in rat plasma. The linear range of yuanhuacine was 1-1000 ng/ml (R2 =0.998). The intra- and inter-precision (coefficient of variation %) of the assay was 3.86-6.18% and 2.65-5.75%, respectively, and the intra- and inter-accuracy (relative error %) was -3.83-4.77% and -3.03-5.11%, respectively. The extraction recovery, matrix effect, stability, and incurred sample reanalysis of yuanhuacine were within acceptable levels. The established method was validated and successfully applied to the preclinical pharmacokinetic study of yuanhuacine. The absolute oral bioavailability of yuanhuacine was calculated as 1.14%, and it reached the maximum plasma concentration of 28.21 ± 2.79 ng/ml in rat plasma at 2h in the oral dosing group. The apparent volume of distribution of intravenous and intragastric administrations was 26.07 ±6.45 and 21.83 ±3.54L/kg, respectively. The half-life of elimination of yuanhuacine was 9.64 ± 1.53 h in the intravenous dosing group.

A pre-column derivatization high-performance liquid chromatography method for simultaneous determination of short-chain and medium-chain fatty acids in a fecal sample.

Short-chain and medium-chain fatty acids have plentiful biological functions, which play a crucial role in the diagnosis and therapy of many diseases. Herein, a new method for simultaneous quantifying 17 short-chain and medium-chain fatty acids with high-performance liquid chromatography coupled with an ultraviolet detector was developed and the pre-column derivatization by indole-3-acetic acid hydrazide was performed to improve the separation and detection. The conditions of the derivatization reaction were systematically investigated. Subsequently, the method was validated and the results showed a satisfactory linearity (linear regression coefficients > 0.9969), the limit of detection (4.0×10-3 -1.9×10-2 μmol/L), precision (0.9%-7.3% for intra-day and 2.0%-9.8% for inter-day), recovery (90.0%-109.1% with relative standard deviation <7.7%) and stability (0.1%-3.3% for standard solution and 0.2%-3.9% for fecal sample). Finally, the established method was successfully applied to quantify short-chain and medium-chain fatty acids in the feces of healthy control and diabetic rats. Eleven kinds of short-chain and medium-chain fatty acids were detected and six of them showed a significant difference between the control group and the model group.

CHARACTERISTICS OF RATS AND ECTOPARASITES ON ANIMAL QUARANTINE HEALTH IN PORTS

Rats are wild animals that can cause disease. Diseases that can be caused by Toxoplasma caused by parasites. One that can be transmitted from rat urine, saliva, feces and fleas. This research is a qualitative research that uses a descriptive observational approach. Identification was carried out at the Kendari Class II KKP Laboratory. The disease is directly bitten by ectoparasites in the body of mice (fleas, fleas, ticks and mites). The study was conducted by observing at the same time (point time approach) which included all rats in the Perimeter area of the People's Harbor Area, Nusantara Port, Ferry Port and Bungkutoko Port. 100 traps were set and placed scattered around in the Perimeter area. The catch was carried out in the afternoon and taken in the morning. The rats and ectoparasites obtained were then identified. In the trap taking in April obtained as many as 30 tails. The rats found were Norvegicus, Mus Muscular and Hundred diardi species. The number of ectoparasites found was 29 types of pinja. In the trap taking in June, 18 individuals were found. Ectoparasites were found with Norvegicus species with 156 fleas, Hundred diardi with 2 fleas. Conclusion The rats in the people's port and Ferry had the highest number of rats and had the highest number of extoparsites compared to the ports of Nusantara and Bungkutoko..