LncRNA-mediated ceRNA network reveals the mechanism of action of Saorilao-4 decoction against pulmonary fibrosis.

Introduction: Pulmonary fibrosis (PF), a type of interstitial pneumonia with complex etiology and high mortality, is characterized by progressive scarring of the alveolar interstitium and myofibroblastic lesions. In this study, we screened for potential biomarkers in PF and clarified the role of the lncRNA-miRNA-mRNA ceRNA network in the inhibitory effect of SRL-4 on PF. Methods: Healthy male SPF SD rats were randomly divided into three groups, namely, CON, MOD, and SRL-4. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed to determine the biological functions of the target genes. A visualized lncRNA-miRNA-mRNA ceRNA network was constructed using Cytoscape, while key genes in the network were identified using the cytoNCA plugin. Results: Seventy-four differentially expressed lncRNAs and 118 differentially expressed mRNAs were identified. Gene Ontology analysis revealed that the target genes were mainly enriched in the cell membrane and in response to organic substances, while Kyoto Encyclopedia of Genes and Genomes analysis showed that the target genes were mainly enriched in the AMPK, PPAR, and cAMP signaling pathways. We elucidated a ceRNA axis, namely, Plcd3-OT1/rno-miR-150-3p/Fkbp5, with potential implications in PF. Key genes, such as AABR07051308.1-201, F2rl2-OT1, and LINC3337, may be important targets for the treatment of PF, while the AMPK, PPAR, and cAMP signaling pathways are potential key targets and important pathways through which SRL-4 mitigates PF. Conclusion: Our findings suggest that SRL-4 improves PF by regulating the lncRNA-miRNA-mRNA network.

Assessment of progression of pulmonary fibrosis based on metabonomics and analysis of intestinal microbiota.

The main purpose of this study was to explore the changes of biomarkers in different developmental stages of bleomycin-induced pulmonary fibrosis (PF) in rats via comprehensive pathophysiology, UPLC-QTOF/MS metabonomic technology, and 16S rRNA gene sequencing of intestinal microbiota. The rats were randomly divided into normal control and 1-, 2- and 4-week model group. The rat model of PF was established by one-time intratracheal instillation of bleomycin. The levels of inflammatory and fibrosis-related factors such as hydroxyproline (HYP), type III procollagen (COL-III), type IV collagen (COL-IV), hyaluronidase (HA), laminin (LN), interleukin (IL)-1ß, IL-6, malondialdehyde (MDA) increased and superoxide dismutase (SOD) decreased as the PF cycle progressed. In the 1-, 2- and 4-week model group, 2, 19 and 18 potential metabolic biomarkers and 3, 16 and 12 potential microbial biomarkers were detected, respectively, which were significantly correlated. Glycerophospholipid metabolism pathway was observed to be an important pathway affecting PF at 1, 2 and 4 weeks; arginine and proline metabolism pathways significantly affected PF at 2 weeks. Linoleic acid metabolism pathway exhibited clear metabolic abnormalities at 2 and 4 weeks of PF, and alpha-linolenic acid metabolism pathway significantly affected PF at 4 weeks.

In this study, metabolomics technology and intestinal microbiota 16S rRNA gene sequencing were used to search for biomarkers with significant differences in each stage of pulmonary fibrosis. Finally, the variation characteristics of each stage of the disease were discussed. The hope is to provide new insights into the development of diagnostic biomarkers and potential therapeutic targets at all stages.

Identification of Central Genes and Regulatory Pathways Associated with Hyperlipidemia in Rats.

Hyperlipidemia is an independent risk factor for cardiovascular and cerebrovascular diseases. The transcriptomic data and the gene regulatory networks of hyperlipidemia are largely unclear. We analyzed the changes in liver gene expression and the serum levels of biochemical indicators in rats with hyperlipidemia induced by high-fat diet (HFD). The body weight, liver weight, and the serum levels of TG, TC, HDL-C, LDL-C, ALT, and AST were significantly higher in the hyperlipidemic rats compared to the healthy controls (P < 0.05). In addition, HFD feeding decreased the antioxidant capacity of the liver tissues and significantly increased the arteriosclerosis index (AI) (P < 0.05). There were 584 differentially expressed genes (DEGs) in the hyperlipidemia model compared to the control, with |log2FC|≥ 1 and P-adjust ≤ 0.05 as the thresholds. GO analysis of the DEGs revealed significant enrichment of 382 biological processes (BP), 18 cellular components (CC), and 40 molecular functions (MF). In addition, pathways related to bile secretion, cholesterol metabolism, and steroid hormone biosynthesis were significantly associated with hyperlipidemia. The key genes potentially involved in the blood lipid changes were Agt, Src, Gnai3, Cyp2c7, Cyp2c11, Cyp2c22, Apoa1, Apoe, and Srebf1. The genes and pathways identified in this study are potential intervention targets for hyperlipidemia and warrant further investigation.

Effects of N-butanol extract of Amygdalus mongolica on rats with bleomycin-induced pulmonary fibrosis based on metabolomics.

Pulmonary fibrosis (PF) is a major public health issue with limited treatment options. As the active ingredient of the n-butanol extract of Amygdalus mongolica (BUT), amygdalin inhibits PF. However, its mechanisms of action are unclear and need further verification. Therefore, the purpose of the present studies was to investigate the anti-fibrotic effects of BUT on PF by serum metabolomics and the transforming growth factor ß (TGF-ß) pathway. Sixty male Sprague-Dawley rats were randomly divided into control, untreated PF, prednisone-treated (5 mg/kg), and BUT-treated (1.75, 1.25, 0.75 g/kg) groups, and the respective drugs were administered intragastrically for 21 days. The serum metabolomics profiles were determined by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) and metabolism network analysis. The expression of TGF-ß1, Smad-3, Smad-7, and α-smooth muscle actin (α-SMA) was measured using a real-time polymerase chain reaction in the lung tissue. BUT significantly alleviated fibrosis by reducing the mRNA expressions of TGF-ß1 (from 1.73 to 1.13), Smad-3 (from 2.01 to 1.19), and α-SMA (from 2.14 to 1.19) and increasing that of Smad7 (from 0.17 to 0.62). Twenty-eight potential biomarkers associated with PF were identified. In addition, four key biomarkers were restored to baseline levels following BUT treatment, with the lowest dose showing optimal effect. Furthermore, A. mongolica BUT was found to improve PF by the pentose phosphate pathway and by taurine, hypotaurine, and arachidonic acid metabolism. These findings revealed the mechanism of A. mongolica BUT antifibrotic effects and metabolic activity in PF rats and provided the experimental basis for its clinical application.

Effects of N-butanol extract of Amygdalus mongolica on rats with bleomycin‐induced pulmonary fibrosis based on metabolomics

Pulmonary fibrosis (PF) is a major public health issue with limited treatment options. As the active ingredient of the n-butanol extract of Amygdalus mongolica (BUT), amygdalin inhibits PF. However, its mechanisms of action are unclear and need further verification. Therefore, the purpose of the present studies was to investigate the anti-fibrotic effects of BUT on PF by serum metabolomics and the transforming growth factor β (TGF-β) pathway. Sixty male Sprague-Dawley rats were randomly divided into control, untreated PF, prednisone-treated (5 mg/kg), and BUT-treated (1.75, 1.25, 0.75 g/kg) groups, and the respective drugs were administered intragastrically for 21 days. The serum metabolomics profiles were determined by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) and metabolism network analysis. The expression of TGF-β1, Smad-3, Smad-7, and α-smooth muscle actin (α-SMA) was measured using a real-time polymerase chain reaction in the lung tissue. BUT significantly alleviated fibrosis by reducing the mRNA expressions of TGF-β1 (from 1.73 to 1.13), Smad-3 (from 2.01 to 1.19), and α-SMA (from 2.14 to 1.19) and increasing that of Smad7 (from 0.17 to 0.62). Twenty-eight potential biomarkers associated with PF were identified. In addition, four key biomarkers were restored to baseline levels following BUT treatment, with the lowest dose showing optimal effect. Furthermore, A. mongolica BUT was found to improve PF by the pentose phosphate pathway and by taurine, hypotaurine, and arachidonic acid metabolism. These findings revealed the mechanism of A. mongolica BUT antifibrotic effects and metabolic activity in PF rats and provided the experimental basis for its clinical application.

The intervention effect of Amygdalus mongolica oil on the metabolomics and intestinal flora in pulmonary fibrosis.

Amygdalus mongolica oil is rich in unsaturated fatty acids such as inoleic acid (47.11%) and oleic acid (23.81%). Our research demonstrates that it exerts a protective effect on rat models of pulmonary fibrosis, however, little is known regarding the underlying mechanism of action. This study aimed to characterize the therapeutic mechanism of action of A. mongolica oil on bleomycin-induced pulmonary fibrosis in rats. A. mongolica oil appears to regulate the levels of potential key serum biomarkers which include tetrahydrobiopterin, L-serine, citrulline and estradiol to participate in folate biosynthesis, glycine, serine and threonine metabolism, arginine biosynthesis and steroid hormone biosynthesis. And it also enriched intestinal microbial abundance, homogeneity and modulated the abundance of Duncaniell, Desulfovibrio, Peptococcaceae_unclassified, Dubosiella, Tyzzerella, Lachnospiraceae_NK4A136_group, Lactobacillus, Clostridiales_unclassified to exert a protective effect against pulmonary fibrosis. A. mongolica oil appears to confer protective effects against pulmonary fibrosis by affecting the level of pulmonary fibrosis metabolites and the abundance of related intestinal flora through multiple targets, as evidenced by our untargeted LC-MS/MS metabonomics evaluation and 16S rDNA sequencing technology.

Screening and identification of key microRNAs and regulatory pathways associated with the renal fibrosis process.

Renal fibrosis (RF) is a chronic and fatal disease related to the gradual deterioration of kidney function. MicroRNAs (miRNAs) play a key role in cellular functions and several of them related to the pathogenesis of RF have been identified, although the underlying mechanisms are unclear. In order to explore the miRNAs involved in RF progression, we established a model in rats by the unilateral ureteral ligation method. The animals were randomly divided into the control group, and the 2 week, 4 week and 6 week model groups. The indices of renal function were measured using routine biochemical assays. The differentially expressed miRNAs (DE-miRNAs) between the sham-operated and modelled rats were screened, and their putative target genes were identified using the miRanda software and functionally annotated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The expression of transforming growth factor ß1 (TGF-ß1), Smad3 and Smad7 was confirmed by RT-PCR. Compared to the sham-operated group, the model groups showed a decrease in SOD activity, along with the increased renal coefficient, and higher MDA, HYP, Scr, BUN and ALB levels. In addition, TGF-ß1, Smad3 and Smad7 were also upregulated in the RF groups. We identified 274 known and 11 novel DE-miRNAs in the 2 week, 114 known and 6 novel DE-miRNAs in the 4 week, and 41 known and 1 novel DE-miRNAs in the 6 week model groups. The putative target genes of these DE-miRNAs were enriched in metabolic processes, apoptosis, pyrimidine metabolism, and TNF and VEGF signalling pathways. Based on our findings, we surmise that miR-146a-3p, miR-148a-3p, miR-130a-5p, miR-362-3p and miR-122-5p are likely to be involved in the occurrence and development of RF, and miR-122-5p may play an inhibitory role. The underlying mechanisms need further investigation.

Metabolomics reveal the mechanism for anti-renal fibrosis effects of an n-butanol extract from Amygdalus mongolica.

To reveal the mechanism of anti-renal fibrosis effects of an n-butanol extract from Amygdalus mongolica, renal fibrosis was induced with unilateral ureteral obstruction (UUO) and then treated with an n-butanol extract (BUT) from Amygdalus mongolica (Rosaceae). Sixty male Sprague-Dawley rats were randomly divided into the sham-operated, renal fibrosis (RF) model, benazepril hydrochloride-treated model (1.5 mg kg-1) and BUT-treated (1.75, 1.5 and 1.25 g kg-1) groups and the respective drugs were administered intragastrically for 21 days. Related biochemical indices in rat serum were determined and histopathological morphology observed. Serum metabolomics was assessed with HPLC-Q-TOF-MS. The BUT reduced levels of blood urea nitrogen, serum creatinine and albumin and lowered the content of malondialdehyde and hydroxyproline in tissues. The activity of superoxide dismutase in tissues was increased and an improvement in the severity of RF was observed. Sixteen possible biomarkers were identified by metabolomic analysis and six key metabolic pathways, including the TCA cycle and tyrosine metabolism, were analyzed. After treatment with the extract, 8, 12 and 9 possible biomarkers could be detected in the high-, medium- and low-dose groups, respectively. Key biomarkers of RF, identified using metabolomics, were most affected by the medium dose. A. mongolica BUT extract displays a protective effect on RF in rats and should be investigated as a candidate drug for the treatment of the disease.

A metabolomic approach to elucidate the inhibitory effects of baicalin in pulmonary fibrosis.

CONTEXT: Baicalin, a major flavonoid extracted from Scutellaria baicalensis Georgi (Lamiaceae), has been shown to exert therapeutic effects on pulmonary fibrosis (PF). OBJECTIVE: To use serum metabolomics combined with biochemical and histopathological analyses to clarify anti-PF mechanisms of baicalin on metabolic pathways and the levels of potential biomarkers. MATERIALS AND METHODS: Forty male Sprague-Dawley rats were randomly divided into the control, PF model, prednisolone acetate-treated (4.2 mg/kg/day) and baicalin-treated (25 and 100 mg/kg/day) groups. A rat model of PF was established using a tracheal injection of bleomycin, and the respective drugs were administered intragastrically for 4 weeks. Histomorphology of lung tissue was examined after H&E and Masson's trichrome staining. Biochemical indicators including SOD, MDA and HYP were measured. Serum-metabonomic analysis based on UPLC-Q-TOF/MS was used to clarify the changes in potential biomarkers among different groups of PF rats. RESULTS: Both doses of baicalin effectively alleviated bleomycin-induced pathological changes, and increased the levels of SOD (from 69.48 to 99.50 and 112.30, respectively), reduced the levels of MDA (from 10.91 to 5.0 and 7.53, respectively) and HYP (from 0.63 to 0.41 and 0.49, respectively). Forty-eight potential biomarkers associated with PF were identified. Meanwhile, the metabolic profiles and fluctuating metabolite levels were normalized or partially reversed after baicalin treatment. Furthermore, baicalin was found to improve PF potentially by the regulation of four key biomarkers involving taurine and hypotaurine metabolism, glutathione metabolism, and glycerophospholipid metabolism. CONCLUSIONS: These findings revealed the anti-fibrotic mechanisms of baicalin and it may be considered as an effective therapy for PF.

Metabolomics reveals the renoprotective effect of n-butanol extract and amygdalin extract from Amygdalus mongolica in rats with renal fibrosis.

Renal fibrosis (RF) is a pathological process of progression from chronic kidney disease to end-stage renal disease. Amygdalus mongolica is a traditional Chinese medicine, and our previous studies demonstrated that the n-butanol extract (BUT) and amygdalin extract (AMY) from its seeds can prevent RF. However, the underlying mechanism remains unclear. The present study investigated the exact mechanism of the protective effect of A. mongolica on RF. A renal fibrosis rat model was induced with unilateral ureteral obstruction. Biochemical indicators were measured and combined with histopathology of renal tissue to evaluate the anti-RF effects. A serum metabonomic method was used to clarify the changes in the metabolic profile. The tubulointerstitial damage and fibrosis were significantly improved and metabolic perturbations were restored after treatment with BUT and AMY. Thirty-eight metabolites associated with RF progression and related to the regulation of arginine and proline metabolism, nicotinate and nicotinamide metabolism, and histidine metabolism were identified. They were restored to levels similar to those in controls after treatment. Moreover, no significant differences in efficacy were observed between the BUT and AMY groups. This study reveals and compares the potential mechanisms of the renoprotective effects after treatment with BUT and AMY from a metabolomic perspective.

Metabolomic assessment of mechanisms underlying anti-renal fibrosis properties of petroleum ether extract from Amygdalus mongolica.

CONTEXT: The petroleum ether extract (PET) of Amygdalus mongolica (Maxim.) Ricker (Rosaceae) has an ameliorative effect on renal fibrosis (RF). OBJECTIVE: To evaluate the antifibrotic effects of A. mongolica seeds PET on RF by serum metabolomics, biochemical and histopathological analyses. MATERIALS AND METHODS: Sixty male Sprague-Dawley rats were randomly divided into the sham-operated, RF model, benazepril hydrochloride-treated model (1.5 mg/kg) and PET-treated (1.75, 1.25, 0.75 g/kg) groups, and the respective drugs were administered intragastrically for 21 days. Biochemical indicators including BUN, Scr, HYP, SOD, and MDA were measured. Haematoxylin and eosin and Masson staining were used for histological examination. The serum metabolomic profiles were determined by UPLC-Q-TOF/MS and metabolism network analysis. Acute toxicity test was performed to validate biosafety. RESULTS: The PET LD50 was >23.9 g/kg in rats. PET significantly alleviated fibrosis by reducing the levels of Scr (from 34.02 to 32.02), HYP (from 403.67 to 303.17) and MDA (from 1.84 to 1.73), and increasing that of SOD (from 256.42 to 271.85). Metabolomic profiling identified 10 potential biomarkers, of which three key markers were significantly associated with RF-related pathways including phenylalanine, tyrosine and tryptophan biosynthesis, amino sugar and nucleotide sugar metabolism and tyrosine metabolism. In addition, three key biomarkers were restored to baseline levels following PET treatment, with the medium dose showing optimal effect. CONCLUSIONS: These findings revealed the mechanism of A. mongolica PET antifibrotic effects for RF rats on metabolic activity and provided the experimental basis for the clinical application.

Protective effects of Amygdalus mongolica on rats with renal fibrosis based on serum metabolomics.

ETHNOPHARMACOLOGICAL RELEVANCE: Renal fibrosis (RF) is a common outcome of various progressive chronic kidney diseases (CKDs) and, thus, seriously endangers human health. As the active ingredient of Amygdalus mongolica, amygdalin inhibits RF. Furthermore, our previous studies demonstrated that n-butanol extract (BUT) and petroleum ether extract (PET), which are effective components of A. mongolica, have an anti-renal fibrosis effect. However, their potential mechanisms of action are unclear and need further verification. AIMS OF THE STUDY: The aims of this study were to further investigate the effects and potential mechanisms of A. mongolica extracts in the treatment of RF. MATERIALS AND METHODS: The animals were divided into the control group, RF model group, PET group and BUT group. The RF rat model was established through unilateral ureteral obstruction (UUO). Biochemical indicators, including blood urea nitrogen (BUN), serum creatinine (Scr), and hydroxyproline (HYP, a routine marker of fibrosis), and the antioxidant index (including superoxide dismutase (SOD) and malondialdehyde (MDA)) were measured to evaluate the anti-RF effects of the extracts of A. mongolica. The histomorphology of renal tissue was observed and scored by HE and Masson staining. A serum metabonomic analysis based on UPLC-Q-TOF/MS was performed to assess the changes in the metabolic profile among the different groups. RESULTS: The results showed that PET and BUT significantly improved tubulointerstitial damage and fibrosis by reducing the levels of Scr, BUN, HYP, and MDA and increasing the level of SOD. Moreover, no significant differences in efficacy were observed between the BUT and PET groups. According to the metabolomics analysis, seventy-four potential biomarkers were identified, and eight crucial biomarkers were further selected. These key biomarkers significantly contributed to RF progression by participating in six metabolic pathways, including pathways involved in arginine and proline metabolism, histidine metabolism, nicotinamide metabolism, pentose and glucuronate interconversion, ascorbate and aldarate metabolism, and amino sugar and nucleotide sugar metabolism. In addition, eight key biomarkers and six crucial biomarkers were restored to levels similar to those observed in controls following the treatment with PET and BUT, respectively. CONCLUSIONS: The outcomes of these studies demonstrate the renoprotective effects of A. mongolica extracts in rats with RF and revealed the mechanism underlying these antifibrotic effects on metabolic activity for the first time.

Comparative analysis of bovine maternal corpus luteum microRNAs with aberrant and normal developed cloned fetus at late gestation.

BACKGROUND: The development efficiency of cloned cattle is extremely low (< 5%), most of them were aborted at late gestation. Based on our previous studies, some recipient cows with a cloned fetus would present as engorged uterine vessels and enlarged umbilical vessels randomly. Abortion involves both maternal and fetal factors. OBJECTIVE: Our aim was to explore this phenomenon by microRNAs expression profile analysis of maternal corpus luteum (CL), which was related to pregnancy maintenance. METHODS: The present study provided the comparison of maternal CL miRNAs expression of abnormally and normally developed cloned bovine fetus at late gestation (~ 210 days) using RNA-Seq technology. RESULTS: We selected two abnormally pregnant cows (abnormal group, AG) and three normally pregnant cows (normal group, NG) and acquired valid reads of 9317,261-12,327,185 (~ 84.53-91.28%) from five libraries. In total, we identified 981 conserved miRNAs and 223 novel miRNAs. 1052 miRNAs were co-expressed, 124 miRNAs were uniquely expressed in AG, and 93 miRNAs were uniquely expressed in the NG. Compared with NG, 11 were significantly overexpressed, and 22 were downregulated (p < 0.05) at AG among 1052 co-expressed miRNAs. The differentially expressed miRNAs-targeted genes were further analyzed by Gene Ontology and KEGG pathway analysis. Notably, the steroid biosynthesis pathway was a significantly enriched term (p < 0.01), which may affect the secretion of progesterone. CONCLUSION: Our research suggested that abnormal miRNAs expression of bovine maternal CL may affect the pregnant status at late gestation.

Isolation and identification of bovine nasopharyngeal mucosal epithelial cells and establishment of cell models of acute infection by foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) commonly occurs via the respiratory tract, and bovine nasopharyngeal mucosal epithelial cells are the primary infection cells in cattle. The aim of the present study was to isolate and culture epithelial cells from the bovine nasopharyngeal mucosa in vitro using a mechanical separation method. The cells were expanded, established in continuous cell culture, and used for immunofluorescence cytochemistry and establishment of infection models. We detected pan-cytokeratin markers of bovine nasopharyngeal mucosal epithelial cells by immunofluorescence. Bovine nasopharyngeal mucosal epithelial cells were then infected with foot-and-mouth disease virus (FMDV) serum type O. RT-PCR demonstrated the successful establishment of acute FMDV infection in the cell models. This infection model provides the basis for clarification of the interaction between FMDV and host bovine nasopharyngeal mucosal epithelial cells in vitro.

[The analysis of the content of bile acids in ox-bile powder and goat gall powder of Mongolian medicine].

The present study was to detect and compare the content of bile acids in ox bile powder and goat gall powder of Mongolia medicine by UV. Cholic acid with sulphuric acid were heated and dehydrated, and they produced conjugated double bond. The conjugated bond showed the same absorption peak in the ultraviolet range. The method of ultraviolet spectrophotometry can be used to detect and compare the content of bile acids in ox bile powder and goat gall powder. The result showed that the linear range was 0.003 3-0.016 7 mg x mL(-1) (r = 0.999 7). The average recovery (n = 5) of standard addition method of ox bile powder and goat gall powder was 98.48% (RSD = 1.79%) and 96.46% (RSD = 2.50%) respectively. The result of determination of five different samples showed that the content of bile acids in ox bile powder and goat gall powder was 40.85%-43.03% and 30.88%-32.64% respectively. The RSD of the analysis of ox bile powder and goat ball powder was 2.40% and 2.92% respectively, the RSD of stationary test of ox bile powder and goat ball powder in eight hours was 0.55% and 0.59% respectively, and the RSD of reproducibility of the analysis of ox bile powder and goat ball powder was 2.11% and 2.68% respectively. The method was simple, accurate, fast and easy to generalize and apply in many fields. It can be used to control the quality of ox bile powder and goat gall powder.