Mn(iii)-mediated carbon-centered radicals generate an enhanced immunotherapeutic effect.

A strategy for designing cancer therapeutic nanovaccines based on immunogenic cell death (ICD)-inducing therapeutic modalities is particularly attractive for optimal therapeutic efficacy. In this work, a highly effective cancer therapeutic nanovaccine (denoted as MPL@ICC) based on immunogenic photodynamic therapy (PDT) was rationally designed and fabricated. MPL@ICC was composed of a nanovehicle of MnO2 modified with a host-guest complex using amino pillar[6]arene and lactose-pyridine, a prodrug of isoniazid (INH), and chlorine e6 (Ce6). The nanovaccine exhibited excellent biosafety, good targeting ability to hepatoma cells and enrichment at tumor sites. Most importantly, it could modulate the tumor microenvironment (TME) to facilitate the existence of Mn(iii) and Mn(iii)-mediated carbon-centered radical generation with INH released from the prodrug in situ to further strengthen ICD. This is the first report on Mn(iii)-mediated generation of carbon-centered radicals for successful anti-tumor immunotherapy using ICD, which provides a novel strategy for designing highly efficient cancer therapeutic nanovaccines.

Structural characterization and immunomodulatory effect of a starch-like Grifola frondosa polysaccharides on cyclophosphamide-induced immunosuppression in mice.

In this study, a pure Grifola frondosa polysaccharide (GFP-1) was extracted and purified from Grifola frondosa. By HPLC, GC-MS, FT-IR, and NMR analysis, GFP-1 was determined to be a starch-like polysaccharide with an average molecular weight of 3370 kDa. It included three monosaccharides, i.e., glucose, galactose, and mannose. The backbone of GFP-1 consisted of →4)-α-Glcp-(1→ and →4,6)-α-Glcp-(1 â†’ . The side branches were composed of →6)-α-Galp-(1→, α-Glcp-(1→, and a small amount of α-Manp-(1 â†’ . By using a cyclophosphamide (CTX)-induced immunosuppressed mice model, we evaluated the immunomodulatory activity of GFP-1. The results showed that GFP-1 increased the thymic and spleen indices, promoted the level of IgG and IgA in serum, and activated the mitogen-activated protein kinase (MAPK) pathway in CTX-induced mice. Also, GFP-1 significantly promoted the mRNA expression of intestinal barrier factors and protected intestinal structural integrity in immunosuppressed mice. In conclusion, the data presented here suggested that GFP-1 might be a potential immune-enhancing supplement.

Bi Xie Fen Qing Yin decoction alleviates potassium oxonate and adenine induced-hyperuricemic nephropathy in mice by modulating gut microbiota and intestinal metabolites.

This study aimed to evaluate the preventive effect of Bi Xie Fen Qing Yin (BXFQY) decoction on hyperuricemic nephropathy (HN). Using an HN mouse model induced by oral gavage of potassium oxonate and adenine, we found that BXFQY significantly reduced plasma uric acid levels and improved renal function. Further study shows that BXFQY suppressed the activation of the NLRP3 inflammasome and decreased the mRNA expressions of pro-inflammatory and fibrosis-associated factors in renal tissues of HN mice. Also, BXFQY prevented the damage to intestinal tissues of HN mice, indicative of suppressed colonic inflammation and increased gut barrier integrity. By 16 S rDNA sequencing, BXFQY significantly improved gut microbiota dysbiosis of HN mice. On the one hand, BXFQY down-regulated the abundance of some harmful bacteria, like Desulfovibrionaceae, Enterobacter, Helicobacter, and Desulfovibrio. On the other hand, BXFQY up-regulated the contents of several beneficial microbes, such as Ruminococcaceae, Clostridium sensu stricto 1, and Streptococcus. Using gas or liquid chromatography-mass spectrometry (GC/LC-MS) analysis, BXFQY reversed the changes in intestinal bacterial metabolites of HN mice, including indole and BAs. The depletion of intestinal flora from HN or HN plus BXFQY mice confirmed the significance of gut microbiota in BXFQY-initiated treatment of HN. In conclusion, BXFQY can alleviate renal inflammation and fibrosis of HN mice by modulating gut microbiota and intestinal metabolites. This study provides new insight into the underlying mechanism of BXFQY against HN.

Tannic Acid Suppresses HBV Replication via the Regulation of NF-κB, MAPKs, and Autophagy in HepG2.2.15 Cells.

Hepatitis B virus (HBV) infection is a serious global health problem that threatens the health of human. Tannic acid (TA), a natural polyphenol in foods, fruits, and plants, exhibits a variety of bioactive functions. In our research, we decide to explore the pharmacological mechanism of TA against HBV replication. Our results showed that TA effectively reduced the content of HBV DNA and viral antigens (HBsAg and HBeAg) in HepG2.2.15 cells. Meanwhile, TA significantly decreased the mRNA expression of HBV RNA, which include total HBV RNA, HBV pregenomic RNA, and HBV precore mRNA. Besides, TA evidently downregulated the activity of HBV promoters in HepG2.2.15 cells. Furthermore, we found that TA upregulated the expression of IL-8, TNF-α, IFN-α, and IFN-α-mediated antiviral effectors in HepG2.2.15 cells. On the contrary, TA downregulated the expression of IL-10 and hepatic nuclear factor 4 (HNF4α). In addition, TA activated the NF-κB and MAPK pathways that contributed to the inhibition of HBV replication. Finally, TA treatment led to the occurrence of autophagy, which accelerated the elimination of HBV components in HepG2.2.15 cells. Taken together, our results elucidated the suppressive effect of TA on HBV replication and provided inspiration for its clinical application in HBV treatment.

Structural characterization and anti-inflammatory activity of polysaccharides from Astragalus membranaceus.

In this study, two homogeneous polysaccharides (APS-A1 and APS-B1) were isolated from Astragalus membranaceus by DEAE-52 cellulose and Sephadex G-100 column chromatography. Their chemical structures were characterized by molecular weight distribution, monosaccharide composition, infrared spectrum, methylation analysis, and NMR. The results revealed that APS-A1 (2.62 × 106 Da) was a 1,4-α-D-Glcp backbone with a 1,4,6-α-D-Glcp branch every ten residues. APS-B1 (4.95 × 106 Da) was a heteropolysaccharide composed of glucose, galactose, and arabinose (75.24:17.27:19.35). Its backbone consisted of 1,4-α-D-Glcp, 1,4,6-α-D-Glcp, 1,5-α-L-Araf and the sidechains composed of 1,6-α-D-Galp and T-α/ß-Glcp. Bioactivity assays showed that APS-A1 and APS-B1 had potential anti-inflammatory activity. They could inhibit the production of inflammatory factors (TNF-α, IL-6, and MCP-1) in LPS-stimulated RAW264.7 macrophages via NF-κB and MAPK (ERK, JNK) pathways. These results suggested that the two polysaccharides could be potential anti-inflammatory supplements.

Changes of serum metabolomics and gut microbiota reveal specific characteristics of children with febrile seizures.

BACKGROUND AND PURPOSE: Febrile seizures (FS) pose a severe threat to the neurological development of children. Probing the abnormality of host metabolism is essential for the prevention, diagnosis, and treatment of FS. METHODS: Based on clinically collected serum and fecal samples, we used nontargeted metabolomics and 16S rDNA sequencing to explore the relationship of serum metabolite levels and gut microbiota community with the occurrence of FS. RESULTS: Metabolomic analysis revealed abnormalities in multiple metabolic pathways in serum of FS patients, such as tryptophan metabolism and steroid hormone biosynthesis. Intestinal flora analysis indicated that the α-diversity of gut microbiota in FS patients was significantly reduced. In addition, the relative abundance of a variety of bacteria at the phylum level was remarkably changed in patients with FS, including decreased Firmicutes and Verrucomicrobia. Eleven serum metabolites were identified to be biomarker candidates for FS diagnosis. With the help of a panel biomarker strategy combining four biomarkers as a cluster, four bacteria (i.e., Rothia, Coprococcus, Lactobacillus, and Oscillospira) in a defined panel displayed perfect differentiation of subtypes of FS. CONCLUSIONS: Combining metabolomic and intestinal flora analysis revealed specific characteristics of children with FS, and provided new clues for the diagnosis of FS and the classification of seizure types. In summary, these findings may provide new insights into revealing the significance of serum metabolites and gut microbiota in the pathogenesis of FS.

Poria cocos oligosaccharides ameliorate dextran sodium sulfate-induced colitis mice by regulating gut microbiota dysbiosis.

Poria cocos, a widely accepted function food in China, has multiple pharmacological activities. This study aimed to investigate the therapeutic effect and molecular mechanism of Poria cocos oligosaccharides (PCOs) against dextran sodium sulfate (DSS)-induced mouse colitis. In this study, BALB/c mice were treated with 3% (w/v) DSS for seven days to establish a colitis model. The results showed that oral administration of PCOs (200 mg per kg per day) significantly reversed the changes in the physiological indices in colitis mice, including body weight, disease activity index scores (DAI), spleen index, and colon length. From the qRT-PCR assay, it was observed that PCOs suppressed the mRNA expression of pro-inflammatory cytokines, such as Tnf-α, Il-1ß, and Il-6. In addition, PCOs protected the intestinal barrier from damage by promoting the expression of mucins and tight junction proteins at both mRNA and protein levels. Upon 16S rDNA sequencing, it was observed that PCO treatment partly reversed the changes in the gut microbiota of colitis mice by selectively regulating the abundance of specific bacteria. And Odoribacter, Muribaculum, Desulfovibrio, Oscillibacter, Escherichia-Shigella, and Turicibacter might be the critical bacteria in improving colitis via PCOs. Finally, using antibiotic mixtures to destroy the intestinal bacteria, we documented that PCO fermentation broth (PCO FB) instead of PCOs prevented the occurrence of colitis in gut microbiota-depleted mice. In conclusion, PCOs showed a protective effect on colitis by reversing gut microbiota dysbiosis. Our study sheds light on the potential application of PCOs as a prebiotic for treating colitis.

Dihydromyricetin inhibits Hepatitis B virus replication by activating NF-κB, MAPKs, and autophagy in HepG2.2.15 cells.

BACKGROUND: Hepatitis B virus (HBV) infection is a severe global health problem, and there has been no effective method to eliminate HBV. This study was designed to explore the pharmacological mechanism of Dihydromyricetin (DHM) treatment on HBV replication in vitro. METHODS AND RESULTS: DHM is a flavonoid compound from Ampelopsis grossedentata. Using HepG2.2.15 cells, which can stably express HBV in vitro, we demonstrated that DHM treatment dramatically reduced HBV replication and secretions of HBsAg and HBeAg. Meanwhile, DHM inhibited mRNA expression of HBV RNAs in HepG2.2.15 cells, including Total HBV RNA, HBV pregenomic RNA (pgRNA), and HBV precore mRNA (pcRNA). Also, DHM elevated the mRNA expressions of inflammatory cytokines and antiviral effectors. In contrast, DHM decreased the mRNA level of HNF4α, which positively correlated with HBV replication. Further studies show that the activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathway played a critical role in DHM-initiated inhibition of HBV replication in HepG2.2.15 cells. Besides, activated autophagy was another contributor that may accelerate the clearance of HBV components. CONCLUSION: In summary, DHM could suppress HBV replication by activating NF-κB, MAPKs, and autophagy in HepG2.2.15 cells. Our studies shed light on the future application of DHM for the clinical treatment of HBV infection.

Structural characterization of Poria cocos oligosaccharides and their effects on the hepatic metabolome in high-fat diet-fed mice.

In this study, novel Poria cocos oligosaccharides (PCO) were prepared by enzymatic degradation, and their polymerization degree was determined to be 2-6 by LC-MS analysis. By monosaccharide composition analysis, methylation assay, FT-IR, and NMR analysis, PCO were deduced to contain the sugar residues of (1 → 2)-ß-D-Glcp, (1 → 2)-α-D-Glcp, and (1 → 4)-α-D-Glcp. Using an HFD-fed mouse model with dyslipidemia, PCO could significantly suppress lipid metabolism disorders, characterized by the reduction of lipid accumulation and inflammatory responses in the blood and liver tissues. Based on the non-targeted metabolomic analysis and Spearman's correlation analysis, we presume that the preventive effect of PCO on dyslipidemia might contribute to the reversal of changed metabolic pathways, which were related to the metabolisms of glycerophospholipids, unsaturated fatty acids, amino acids, choline, bile acids, tryptophan, sphingolipids, and glutathione. Our research shed light on the potential application of PCO for the treatment of dyslipidemia.

Bletilla striata Oligosaccharides Improve Ulcerative Colitis by Regulating Gut Microbiota and Intestinal Metabolites in Dextran Sulfate Sodium-Induced Mice.

This study aimed to elucidate the mechanism of Bletilla striata oligosaccharides (BO) in the treatment of ulcerative colitis (UC). A UC mouse model was induced by 3% Dextran sodium sulfate (DSS), and BO (200 mg/kg/d) were administered for intervention. The results show that BO effectively inhibited the release of intestinal inflammatory cytokines such as IL-6, TNF-α, and IL-1ß. Also, BO profoundly elevated the secretion of mucins and the expression of tight junction (TJ) proteins to attenuate dysfunction of the intestinal barrier. The 16S rDNA sequencing and liquid chromatography/gas chromatography-mass spectrometer (LC/GC-MS) analysis of mouse feces revealed that BO regulated the disturbance of gut microbiota and intestinal metabolites. By using the in vitro fermentation broth of BO and gut microbiota-depleted mice treated with antibiotics, we confirmed the protection of BO against UC. In conclusion, BO played a role in improving UC by modulating gut microbial composition and intestinal metabolites, which provided new therapeutic strategies for UC treatment.

Regulation of gut microbiota and intestinal metabolites by Poria cocos oligosaccharides improves glycolipid metabolism disturbance in high-fat diet-fed mice.

In this study, we aimed to explore the effect of Poria cocos oligosaccharides (PCO) on glucolipid metabolism disorder. Based on a high-fat diet (HFD)-induced obese mouse model, we demonstrated that PCO ameliorated glucose intolerance and insulin resistance, decreased the levels of blood glucose (187.8±19.8 mg/dL) and insulin (566.3±53.34 ng/L) in HFD-fed mice compared to the Ctrl group (140.4±7.942 mg/dL for glucose, 423.2±19.56 ng/L for insulin). Moreover, PCO treatment suppressed the mRNA expressions of fatty acid synthesis regulators (decreases of 68.8%, 62.8%, and 32.0% for G6Pase, FASN, and DGAT, respectively, vs. HFD group) and pro-inflammatory cytokines in epididymal fat (decreases of 71.9%, 81.5%, 76.0%, 29.3%, and 63.9% for TNF-α, IL-1ß, IL-6, COX-5b, and MCP-1, respectively, vs. HFD group). Also, PCO treatment alleviated damage to the intestinal barrier of HFD-fed mice. By 16S rDNA gene sequencing, PCO partly restored the imbalance of gut microbiota in HFD-fed mice, accompanied by the reversal of several intestinal metabolites, including bile acids (BAs), short-chain fatty acids (SCFAs), and tryptophan metabolites. By Spearman's correlation analysis, we found that the changed gut microbiota and their metabolites were significantly correlated with the alteration of metabolic markers. Finally, the significance of gut microbiota in PCO-mediated improvement on glucolipid metabolism disorder was confirmed by an antibiotic depletion experiment and fecal microbiota transplantation. In summary, PCO may be used as a novel prebiotic in the treatment of glucolipid disorders by reshaping intestinal bacteria structure. Our studies also point towards the potential of Poria cocos as a healthy food in the clinical application to metabolic diseases in the future.

Stewed Rhubarb Decoction Ameliorates Adenine-Induced Chronic Renal Failure in Mice by Regulating Gut Microbiota Dysbiosis.

This study aimed to investigate the protective effect of Stewed Rhubarb (SR) decoction on chronic renal failure (CRF) through the regulation of gut microbiota. Using a CRF mouse model induced by a 0.2% adenine diet, we proved that SR decoction (2.0 g crude SR/kg) significantly reduced the levels of urea and creatinine in plasma of CRF mice, accompanied by the improvement of renal fibrosis and tubular atrophy, amelioration of inflammation, and inhibition of aquaporins damage. Also, SR decoction alleviated gut barrier damage, indicative of the elevated mRNA expression of intestinal mucins and tight junctions. By 16S rDNA sequencing, SR decoction reshaped the imbalanced gut microbiota in CRF mice by statistically reversing the abundance changes of a wide range of intestinal bacteria at family and genus levels, which further led to balance in the production of intestinal metabolites, including short-chain fatty acids (acetic acid, propionic acid, and valeric acid), indole, and bile acids (TUDCA and CDCA). Inversely, SR decoction failed to repress the occurrence of CRF in mice with gut microbiota depletion, confirming the essential role of gut microbiota in SR decoction-initiated protection against CRF. In summary, SR decoction can improve adenine-induced CRF in mice by remolding the structure of destructed gut microbiota community. Our findings shed light on the clinical application of SR decoction in nephropathy treatment.

Structural characterization and preventive effect on non-alcoholic fatty liver disease of oligosaccharides from Bletilla striata.

In this study, Bletilla striata polysaccharides were degraded into oligosaccharides. The structural features were analyzed by HPLC, HPLC-MS, FT-IR, and NMR spectroscopy. The results indicated that Bletilla striata oligosaccharides (BOs) were composed of mannose and glucose with a molar ratio of 5.2 : 1, and the main backbones of BOs contained (1→4)-linked-α-D-Man, (1→2)-linked-α-D-Man, and (1→2)-linked-α-D-Glc. By using a high-fat diet (HFD)-induced mouse model, we demonstrated that BOs had an improving effect on non-alcoholic fatty liver disease (NAFLD). Using the metabolomics assay, we found that BOs significantly regulated the hepatic metabolism of fatty acids, arachidonic acid, and other related metabolites in HFD-fed mice, accompanied by the reduction of lipid accumulation and fibrosis in liver tissues. In summary, BOs displayed high potential for the treatment of NAFLD as a functional food.

Plasma metabolomic profiles reveal regulatory effect of chitosan oligosaccharides on loperamide-induced constipation in mice.

Chitosan oligosaccharides (COS) can improve the symptoms of constipation. In this study, we further explored the regulator effect of COS on aberrant plasma metabolomics in constipated mice. Using untargeted metabolomic analysis by ultra-performance liquid chromatography-mass spectrometer (UPLC-MS), we identified several most significantly changed metabolic pathways in plasma of constipated mice induced by loperamide, including those correlated with the metabolisms of sphingolipid, glycerophospholipid, tryptophan, bile acids, unsaturated fatty acids, and amino acids. The changes in these metabolic pathways were reversed by COS treatment largely. Furthermore, the mRNA levels of some key target genes related to the above metabolic pathways in colon samples were detected by reverse transcription-polymerase chain reaction analysis. We showed that COS significantly suppressed the abnormal expression of these genes, including ceramide glucosyltransferase (CGT), sphingolipid 4-desaturase (DEGS2), alkaline ceramidase (ACER1), sphingosine kinase 2 (SPHK2), lysophosphatidylcholine acyltransferase (LPCAT1), and aromatic-L-amino-acid (DDC). These data provide insight into the mechanisms by which COS ameliorates loperamide-induced constipation in mice.

Bile acid profiles in bile and feces of obese mice by a high-performance liquid chromatography-tandem mass spectrometry.

Bile acids (BAs) play a pivotal role in manipulating the development of metabolic diseases. However, due to the compositional complexity and functional variation of BAs, it remains unclear about the changes in BA pool for individuals with obesity or metabolic syndrome. We established a high-performance liquid chromatography-mass spectrometer detection system for the simultaneous analysis of both unconjugated and conjugated BAs in the bile and feces of mice. Ten BAs were completely separated, identified, and quantified with low limit of detection (0.5 ng/mL) and inter/intraday precision (relative standard deviation < 12%). By using this method, these BAs in bile and feces of mice were quantified. The result showed that taurochenodeoxycholic acid, taurine-conjugated α-muricholic acids, and taurine-conjugated ß-muricholic acids were the dominated BAs in bile, whereas deoxycholic acid and chenodeoxycholic acid predominated in feces. Further, most of the BA levels were significantly elevated in either bile or fecal samples of high-fat diet-fed mice as compared with those in normal chow diet-fed mice, indicating that excessive production of BAs was closely associated with the occurrence of lipid metabolism disorders. In summary, the present method is practicable for analysis of BAs in bile and fecal samples of patients with obesity.

Integrative analysis of hepatic metabolomic and transcriptomic data reveals potential mechanism of nonalcoholic steatohepatitis in high-fat diet-fed mice.

BACKGROUND: Due to the complex pathogenesis, the molecular mechanism of nonalcoholic steatohepatitis (NASH) remains unclear. In this study, we aimed to reveal the comprehensive metabolic and signaling pathways in the occurrence of NASH. METHODS: C57BL/6 mice were treated with high-fat diet for 4 months to mimic the NASH phenotype. After the treatment, the physiochemical parameters were evaluated, and the liver tissues were prepared for untargeted metabolomic analysis with ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Then, three relevant Gene Expression Omnibus (GEO) datasets were selected for integrative analysis of differentiated messenger RNA and metabolites. RESULTS: The levels of phosphatidylethanolamine (PE) (16:1(9Z)/20:4(5Z,8Z,11Z,14Z)), oleic acid, and sphingomyelin (SM) (d18:0/12:0) were significantly increased, and the content of adenosine was severely reduced in NASH mice. The integrated interpretation of transcriptomic and metabolomic data indicated that the glycerophospholipid metabolism and necroptosis signaling were evidently affected in the development of NASH. The high level of SM (d18:0/12:0) may be related to the expression of acid sphingomyelinase (ASMase), and the elevated arachidonic acid was coordinated with the upregulation of cytosol phospholipase A2 (cPLA2) in the necroptosis pathway. CONCLUSIONS: In summary, the inflammatory response, necroptosis, and glycerophospholipid may serve as potential targets for mechanistic exploration and clinical practice in the treatment of NASH.

Bletilla striata oligosaccharides improve metabolic syndrome through modulation of gut microbiota and intestinal metabolites in high fat diet-fed mice.

As traditional Chinese medicine, Bletilla striata has been widely applied to clinical treatment for its unique pharmacological profiles. This study aimed to investigate the beneficial role of Bletilla striata oligosaccharides (BO) in improving the metabolic syndrome by regulation of gut microbiota and intestinal metabolites. Treatment of HFD-fed mice with BO prevented weight gain, reversed the glucose intolerance and insulin resistance, and inhibited adipocyte hypertrophy. BO-treated mice also suppressed chronic inflammation and protected intestinal barrier from damage. These effects were linked to the reversal of gut microbiota dysbiosis, which contributed to the homeostasis of intestinal metabolites including bile acids, short-chain fatty acids and tryptophan catabolites. The depletion and reconstitution of intestinal flora from BO- or HFD-treated mice confirmed the significance of gut microbiota in regulation of HFD-induced metabolic disorders. We demonstrated for the first time that BO improved metabolic syndrome through the regulation of gut microbiota and intestinal metabolites. The modulation initiated by BO represents a promising strategy for treatment of obesity and related metabolic diseases.

Effect of Chitosan oligosaccharides on ischemic symptom and gut microbiota disbalance in mice with hindlimb ischemia.

This study was designed to explore the effect of Chitosan oligosaccharides (COS) on mouse hindlimb ischemia by femoral artery ligation. Here, we demonstrated that COS treatment statistically promoted the blood perfusion and neovascularization in ischemic hindlimb of mice, accompanied by the suppression of inflammation and oxidative stress. By 16S rDNA gene sequencing, the disbalanced gut microbiota was observed in ischemic mice, while COS treatment, at least in part, restored the abundance changes of some intestinal bacteria at either phylum or genus levels. Based on metabolomics analysis on mouse plasma by UPLC-QTOF-MS, we screened 20 metabolites with the largest responses to ischemia, several of which were markedly reversed by COS. By Spearman's correlation analysis, the changed metabolites might act as a bridge between improved intestinal bacterial structure and alleviated hindlimb ischemia of mice treated by COS. Our studies point towards a potential role of COS in treatment of peripheral ischemia diseases.

Serum metabolomic profiles reveal the impact of BuZangTongLuo formula on metabolic pathways in diabetic mice with hindlimb ischemia.

ETHNOPHARMACOLOGICAL RELEVANCE: BuZangTongLuo Formula (BZTLF) was the decoction of eight traditional Chinese medicines including Astragalus membranaceus, Dioscorea opposita, Salvia miltiorrhiza, Scrophularia ningpoensis, Ophiopogon japonicus, Panax ginseng, Fritillariae cirrhosae and Whitmania pigra. This formula has been used as an effective remedy for treatment of diabetic ischemia clinically. AIM OF THE STUDY: In previous study, we have reported the therapeutic effect of BZTLF on diabetic vascular dysfunction. However, it remains obscure about the role of metabolic pathways in BZTLF-initiated improvement on hindlimb ischemia. MATERIALS AND METHODS: Diabetic mice with hindlimb ischemia were orally administrated with BZTLF by gavage. The serum samples were prepared for untargeted metabolomic analysis by ultra-performance liquid chromatography-mass spectrometer. The metabolic network was built by integrating metabolite data with the Gene Expression Omnibus (GEO) dataset (GSE3313). Further, quantitative PCR was used to confirm the key target genes. RESULTS: BZTLF treatment remarkably led to the reversal of changed metabolite levels in serum of diabetic mice with hindlimb ischemia, which mainly derived from bacteria, plant and signaling molecules. Also, BZTLF reshaped the metabolic pathways, especially those responsible for metabolism of lipid, gluthanine and tryptophan. In addition, BZTLF led to the reduction of lysophosphatidic acids (LPAs) and increment of triglycerides (TGs) conjugation with non-saturated fatty acids in serum. BZTLF significantly restored the down-regulation of vascular endothelial growth factor receptor 2 (VEGFR2) and endothelial nitric oxide synthase (eNOS) or the up-regulation of interleukin 4-induced 1 (IL4I1) and cytochrome P450 family 1 subfamily B member 1 (CYP1B1) at mRNA level, which were key regulatory genes located in metabolic pathways of glutamate and tryptophan. CONCLUSIONS: BZTLF improved hindlimb ischemia in diabetic mice by the positive regulation of metabolome changes in serum.