Anemoside B4 attenuates necrotic enteritis of laying hens induced by Clostridium perfringens via inhibiting NF-κB and PI3K/Akt/mTOR signalling pathways.

Poultry necrotic enteritis is an important enteric disease which might be controlled by antibiotics. However, with the excessive use of antibiotics, the phenomenon of drug resistance of Clostridium perfringens is becoming increasingly prominent. Anemoside B4 exhibits important anti-inflammatory, antioxidant and immunomodulatory effects. This study was performed to estimate the effect of Anemoside B4 on chicken necrotic enteritis induced by C. perfringens in vivo and in vitro. In the in vivo experiment we investigated the efficacy of Anemoside B4 on the growth curve, biofilm formation, haemolytic activity, virulence-related gene expression and NF-κB and PI3K/AKT/mTOR activation in Caco-2 cells induced by C. perfringens. The results showed that 12.5-50 µg/mL Anemoside B4 had no antibacterial activity but could inhibit biofilm formation, attenuate haemolytic activity and virulence-related gene expression of C. perfringens and weaken NF-κB and PI3K/Akt/mTOR activation triggered by C. perfringens in Caco-2 cells. In the in vivo experiment, 60 17-day-old healthy White Leghorns were randomly divided into six groups. The growing laying hens of the control group were fed a basic diet, and those of the five challenged groups were fed a basic diet (infection group), added 0.43 g/kg Anemoside B4 (0.43 g/kg Ane group), 0.86 g/kg Anemoside B4 (0.86 g/kg Ane group), 1.72 g/kg Anemoside B4 (1.72 g/kg Ane group) and 40 mg/kg lincomycin (lincomycin group), respectively. All challenged laying hens were infected with 1 × 109 CFU C. perfringens from day 17-20. Blood and intestinal samples were obtained, and the data demonstrated that Anemoside B4 improved the blood biochemical parameters, attenuated jejunum tissue injury, increased the spleen, thymus, bursa of fabricius index, and decreased lesion scores of the jejunum and the ileum. In the jejunum, Anemoside B4 and lincomycin downregulated the expression of IL-1ß, IL-6, IL-10, TNF-α and IFN-γ at mRNA levels. Moreover, Anemoside B4 significantly enhanced both mRNA and protein levels of tight junctions ZO-1, Claudin-1 and MUC-2 in the jejunum. Anemoside B4 weakened p-P65, p-PI3K, p-Akt and p-mTOR protein expression in the jejunum infected by C. perfringens. Diets supplemented with Anemoside B4 alleviated C. perfringens-induced necrotic enteritis in laying hens by inhibiting NF-κB and PI3K/Akt/mTOR signalling pathways and improving intestinal barrier functions.

Anemoside B4 attenuates abdominal aortic aneurysm by limiting smooth muscle cell transdifferentiation and its mediated inflammation.

Abdominal aortic aneurysm (AAA) is a degenerative disease characterized by local abnormal dilation of the aorta accompanied by vascular smooth muscle cell (VSMC) dysfunction and chronic inflammation. VSMC dedifferentiation, transdifferentiation, and increased expression of matrix metalloproteinases (MMPs) are essential causes of AAA formation. Previous studies from us and others have shown that Anemoside B4 (AB4), a saponin from Pulsatilla chinensis, has anti-inflammatory, anti-tumor, and regulatory effects on VSMC dedifferentiation. The current study aimed to investigate whether AB4 inhibits AAA development and its underlying mechanisms. By using an Ang II induced AAA model in vivo and cholesterol loading mediated VSMC to macrophage transdifferentiation model in vitro, our study demonstrated that AB4 could attenuate AAA pathogenesis, prevent VSMC dedifferentiation and transdifferentiation to macrophage-like cells, decrease vascular inflammation, and suppress MMP expression and activity. Furthermore, KLF4 overexpression attenuated the effects of AB4 on VSMC to macrophage-like cell transition and VSMC inflammation in vitro. In conclusion, AB4 protects against AAA formation in mice by inhibiting KLF4 mediated VSMC transdifferentiation and inflammation. Our study provides the first proof of concept of using AB4 for AAA management.

Anemoside B4 inhibits SARS-CoV-2 replication in vitro and in vivo.

Objective: Anemoside B4 (AB4), the most abundant triterpenoidal saponin isolated from Pulsatilla chinensis, inhibited influenza virus FM1 or Klebsiella pneumoniae-induced pneumonia. However, the anti-SARS-CoV-2 effect of AB4 has not been unraveled. Therefore, this study aimed to determine the antiviral activity and potential mechanism of AB4 in inhibiting human coronavirus SARS-CoV-2 in vivo and in vitro. Methods: The cytotoxicity of AB4 was evaluated using the Cell Counting Kit-8 (CCK8) assay. SARS-CoV-2 infected HEK293T, HPAEpiC, and Vero E6 cells were used for in vitro assays. The antiviral effect of AB4 in vivo was evaluated by SARS-CoV-2-infected hACE2-IRES-luc transgenic mouse model. Furthermore, label-free quantitative proteomics and bioinformatic analysis were performed to explore the potential antiviral mechanism of action of AB4. Type I IFN signaling-associated proteins were assessed using Western blotting or immumohistochemical staining. Results: The data showed that AB4 reduced the propagation of SARS-CoV-2 along with the decreased Nucleocapsid protein (N), Spike protein (S), and 3C-like protease (3CLpro) in HEK293T cells. In vivo antiviral activity data revealed that AB4 inhibited viral replication and relieved pneumonia in a SARS-CoV-2 infected mouse model. We further disclosed that the antiviral activity of AB4 was associated with the enhanced interferon (IFN)-ß response via the activation of retinoic acid-inducible gene I (RIG-1) like receptor (RLP) pathways. Additionally, label-free quantitative proteomic analyses discovered that 17 proteins were significantly altered by AB4 in the SARS-CoV-2 coronavirus infections cells. These proteins mainly clustered in RNA metabolism. Conclusion: Our results indicated that AB4 inhibited SARS-CoV-2 replication through the RLR pathways and moderated the RNA metabolism, suggesting that it would be a potential lead compound for the development of anti-SARS-CoV-2 drugs.

Anemoside B4 alleviates arthritis pain via suppressing ferroptosis-mediated inflammation.

Chronic pain is the key manifestations of rheumatoid arthritis. Neuroinflammation in the spinal cord drives central sensitization and chronic pain. Ferroptosis has potentially important roles in the occurrence of neuroinflammation and chronic pain. In the current study, mouse model of collagen-induced arthritis was established by intradermal injection of type II collagen in complete Freund's adjuvant (CFA) solution. CFA inducement resulted in swollen paw and ankle, mechanical and spontaneous pain, and impaired motor coordination. The spinal inflammation was triggered, astrocytes were activated, and increased NLRP3-mediated inflammatory signal was found in CFA spinal cord. Oxidative stress and ferroptosis in the spinal cord were manifested. Meanwhile, enhancive spinal GSK-3ß activity and abnormal phosphorylated Drp1 were observed. To investigate the potential therapeutic options for arthritic pain, mice were intraperitoneally injected with AB4 for three consecutive days. AB4 treatment reduced pain sensitivity and increased the motor coordination. In the spinal cord, AB4 treatment inhibited NLRP3 inflammasome-mediated inflammatory response, increased antioxidation, decreased mitochondrial reactive oxygen species and ferroptosis. Furthermore, AB4 decreased GSK-3ß activity by binding with GSK-3ß through five electrovalent bonds. Our findings indicated that AB treatment relieves arthritis pain by inhibiting GSK-3ß activation, increasing antioxidant capability, reducing Drp1-mediated mitochondrial dysfunction and suppressing neuroinflammation.

Exploration of the immuno-adjuvant effect and mechanism of Anemoside B4 through network pharmacology and experiment verification.

BACKGROUND: Extensive investigation has been undertaken about the utilization of saponin adjuvants in vaccines intended for veterinary and human applications. AB4 is the main constituent of the traditional Chinese medicine, Pulsatilla chinensis (Bunge) Regel, and has immunomodulatory activity. However, there is a paucity of reports on AB4 as a potential adjuvant. PURPOSE: The objective of this work was to clarify the adjuvant role of AB4 and the molecular mechanisms that underlie its immunomodulatory actions. STUDY DESIGN AND METHODS: The immunomodulatory effects of AB4 were investigated using network pharmacological analyses. These effects were validated by evaluating the developmental status of the immune organs and by using the following techniques: ELISA for the quantification of serum-specific antibodies to determine immune-related cytokine levels; the MTS method for the assessment of proliferative activity of splenic lymphocytes; flow cytometry to analyze lymphocyte and dendritic cell activation status; and western blotting for mechanistic analysis at the protein level. RESULTS: The network pharmacological analysis predicted a total of 52 targets and 12 pathways for AB4 to exert immunomodulatory effects. In a mouse model with immunity to OVA, the introduction of AB4 resulted in the enhancement of immunological organ growth and maturation, elevation of blood antibodies targeting OVA, and amplification of the production of cytokines associated with Th1 and Th2 immune responses. Additionally, the administration of AB4 resulted in a notable augmentation of lymphocyte proliferation and an elevation in the CD4+/CD8+ T lymphocyte ratios. Furthermore, the administration of AB4 enhanced the maturation process of DCs in the draining LNs and increased the production of co-stimulatory factors and MHC II molecules. AB4 induces the upregulation of TLR4 and IKK proteins, as well as the phosphorylation of NF-κB p65 protein within the TLR4/NF-κB signaling cascade, while concurrently suppressing the expression of IκBα protein. CONCLUSION: The specific immunoadjuvant effects of AB4 have been demonstrated to modulate the growth and maturation of immune organs and enhance the secretion and cellular activity of pertinent immune molecules. The utilization of network pharmacology, combined within and in vivo vitro assays, clarified the adjuvant function of AB4, which potentially involves the regulation of the TLR4/NF-κB signaling pathway.

Anemoside B4 ameliorates dextran sulfate sodium (DSS)-induced colitis through inhibiting NLRP3 inflammasome and modulating gut microbiota.

Ulcerative colitis (UC) has been recognized as a chronic and relapsing inflammatory disease of the gastrointestinal tract. Clinically, aminosalicylates, immunosuppressants and biological agents are commonly used to treat UC at different stages of the disease. However, these drugs often have side effects. Here, we investigated the anti-UC activity of Anemoside B4 (AB4) in mice with dextran sulfate sodium (DSS) induced colitis. Colon tissues, serum, and colonic contents were collected for assessment of intestinal barrier function, inflammatory cytokines production and microenvironment of intestinal microbiota. Results showed that AB4 alleviated colon shortening, weight lossing and histopathological damage in DSS-induced mice. In addition, we demonstrated both in vivo and in vitro that AB4 remarkably ameliorated colonic inflammation through suppressing NLRP3 pathway. Moreover, AB4 strengthened the intestinal epithelial barrier by regulating myosin light chain kinase (MLCK)-phosphorylated myosin light chain 2 (pMLC2) signaling pathway. Furthermore, we performed 16 S rRNA gene sequencing and fecal microbiome transplantation (FMT) experiments to demonstrate that AB4 alleviated colitis through regulating dysbiosis of intestinal microbiota. These results revealed that AB4 effectively ameliorate experimental UC mainly through regulating MLCK/pMLC2 pathway, NLRP3 pathway and dysbiosis of microbiota, provided new insights into the development of novel anti-UC drugs.

Anemoside B4 inhibits SARS-CoV-2 replication in vitro and in vivo / 中草药·英文版

OBJECTIVE@#Anemoside B4 (AB4), the most abundant triterpenoidal saponin isolated from Pulsatilla chinensis, inhibited influenza virus FM1 or Klebsiella pneumoniae-induced pneumonia. However, the anti-SARS-CoV-2 effect of AB4 has not been unraveled. Therefore, this study aimed to determine the antiviral activity and potential mechanism of AB4 in inhibiting human coronavirus SARS-CoV-2 in vivo and in vitro.@*METHODS@#The cytotoxicity of AB4 was evaluated using the Cell Counting Kit-8 (CCK8) assay. SARS-CoV-2 infected HEK293T, HPAEpiC, and Vero E6 cells were used for in vitro assays. The antiviral effect of AB4 in vivo was evaluated by SARS-CoV-2-infected hACE2-IRES-luc transgenic mouse model. Furthermore, label-free quantitative proteomics and bioinformatic analysis were performed to explore the potential antiviral mechanism of action of AB4. Type I IFN signaling-associated proteins were assessed using Western blotting or immumohistochemical staining.@*RESULTS@#The data showed that AB4 reduced the propagation of SARS-CoV-2 along with the decreased Nucleocapsid protein (N), Spike protein (S), and 3C-like protease (3CLpro) in HEK293T cells. In vivo antiviral activity data revealed that AB4 inhibited viral replication and relieved pneumonia in a SARS-CoV-2 infected mouse model. We further disclosed that the antiviral activity of AB4 was associated with the enhanced interferon (IFN)-β response via the activation of retinoic acid-inducible gene I (RIG-1) like receptor (RLP) pathways. Additionally, label-free quantitative proteomic analyses discovered that 17 proteins were significantly altered by AB4 in the SARS-CoV-2 coronavirus infections cells. These proteins mainly clustered in RNA metabolism.@*CONCLUSION@#Our results indicated that AB4 inhibited SARS-CoV-2 replication through the RLR pathways and moderated the RNA metabolism, suggesting that it would be a potential lead compound for the development of anti-SARS-CoV-2 drugs.

Anemoside B4, a new pyruvate carboxylase inhibitor, alleviates colitis by reprogramming macrophage function.

OBJECTIVES: Colitis is a global disease usually accompanied by intestinal epithelial damage and intestinal inflammation, and an increasing number of studies have found natural products to be highly effective in treating colitis. Anemoside B4 (AB4), an abundant saponin isolated from Pulsatilla chinensis (Bunge), which was found to have strong anti-inflammatory activity. However, the exact molecular mechanisms and direct targets of AB4 in the treatment of colitis remain to be discovered. METHODS: The anti-inflammatory activities of AB4 were verified in LPS-induced cell models and 2, 4, 6-trinitrobenzene sulfonic (TNBS) or dextran sulfate sodium (DSS)-induced colitis mice and rat models. The molecular target of AB4 was identified by affinity chromatography analysis using chemical probes derived from AB4. Experiments including proteomics, molecular docking, biotin pull-down, surface plasmon resonance (SPR), and cellular thermal shift assay (CETSA) were used to confirm the binding of AB4 to its molecular target. Overexpression of pyruvate carboxylase (PC) and PC agonist were used to study the effects of PC on the anti-inflammatory and metabolic regulation of AB4 in vitro and in vivo. RESULTS: AB4 not only significantly inhibited LPS-induced NF-κB activation and increased ROS levels in THP-1 cells, but also suppressed TNBS/DSS-induced colonic inflammation in mice and rats. The molecular target of AB4 was identified as PC, a key enzyme related to fatty acid, amino acid and tricarboxylic acid (TCA) cycle. We next demonstrated that AB4 specifically bound to the His879 site of PC and altered the protein's spatial conformation, thereby affecting the enzymatic activity of PC. LPS activated NF-κB pathway and increased PC activity, which caused metabolic reprogramming, while AB4 reversed this phenomenon by inhibiting the PC activity. In vivo studies showed that diisopropylamine dichloroacetate (DADA), a PC agonist, eliminated the therapeutic effects of AB4 by changing the metabolic rearrangement of intestinal tissues in colitis mice. CONCLUSION: We identified PC as a direct cellular target of AB4 in the modulation of inflammation, especially colitis. Moreover, PC/pyruvate metabolism/NF-κB is crucial for LPS-driven inflammation and oxidative stress. These findings shed more light on the possibilities of PC as a potential new target for treating colitis.

Anemoside B4 attenuates RANKL-induced osteoclastogenesis by upregulating Nrf2 and dampens ovariectomy-induced bone loss.

Increased numbers and functional overactivity of osteoclasts are the pathological basis for bone loss diseases such as osteoporosis, which are characterized by cortical bone thinning, decreased trabecular bone quantity, and reduced bone mineral density. Effective inhibition of osteoclast formation and bone resorption are important means of treating such skeletal diseases. Anemoside B4 (AB4), the main active component of Pulsatilla chinensis, possesses a wide range of anti-inflammatory and immunoregulatory effects. However, its effect and mechanism in osteoclast differentiation remain unclear. In this study, we found through tartrate-resistant acidic phosphatase (TRAcP) staining and immunofluorescence staining that AB4 inhibited the differentiation, fusion, and bone-resorption functions of osteoclasts induced by receptor activator of nuclear factor κB ligand (RANKL) in vitro. Additionally, real time PCR (RT-qPCR) and western blot analysis showed AB4 downregulated the expression of osteoclast marker genes, including Nfatc1, Fos, and Ctsk, while upregulating Nrf2 expression. AB4 (5 mg/kg) alleviated bone loss in ovariectomized mice by inhibiting osteoclast formation. Furthermore, the knockout of Nrf2 weakened the inhibitory effects of AB4 on osteoclast formation and related gene expression. In summary, the results suggest AB4 can inhibit osteoclast differentiation and function by activating Nrf2 and indicate AB4 may be a candidate drug for osteoporosis.

Effects of Anemoside B4 on Plasma Metabolites in Cows with Clinical Mastitis.

Anemoside B4 has a good curative effect on cows with CM; however, its impact on their metabolic profiles is unclear. Based on similar somatic cell counts and clinical symptoms, nine healthy dairy cows and nine cows with CM were selected, respectively. Blood samples were collected from cows with mastitis on the day of diagnosis. Cows with mastitis were injected with anemoside B4 (0.05 mL/kg, once daily) for three consecutive days, and healthy cows were injected with the same volume of normal saline. Subsequently, blood samples were collected. The plasma metabolic profiles were analyzed using untargeted mass spectrometry, and the concentrations of interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α (TNF-α) in serum were evaluated via ELISA. The cows with CM showed increased concentrations of IL-1ß, IL-6, and TNF-α (p < 0.05). After treatment with anemoside B4, the concentrations of IL-1ß, IL-6, and TNF-α were significantly decreased (p < 0.01). Untargeted metabolomics analysis showed that choline, glycocholic acid, PC (18:0/18:1), 20-HETE, PGF3α, and oleic acid were upregulated in cows with CM. After treatment with anemoside B4, the concentrations of PC (16:0/16:0), PC (18:0/18:1), linoleic acid, eicosapentaenoic acid, phosphorylcholine, and glycerophosphocholine were downregulated, while the LysoPC (14:0), LysoPC (18:0), LysoPC (18:1), and cis-9-palmitoleic acid were upregulated. This study indicated that anemoside B4 alleviated the inflammatory response in cows with CM mainly by regulating lipid metabolism.

[Anemoside B4 regulates fatty acid metabolism reprogramming in mice with colitis-associated cancer].

The study aimed to investigate the effect of anemoside B4(B4) on fatty acid metabolism in mice with colitis-associated cancer(CAC). The CAC model was established by azoxymethane(AOM)/dextran sodium sulfate(DSS) in mice. Mice were randomly divided into a normal group, a model group, and low-, medium-, and high-dose anemoside B4 groups. After the experiment, the length of the mouse colon and the size of the tumor were measured, and the pathological alterations in the mouse colon were observed using hematoxylin-eosin(HE) staining. The slices of the colon tumor were obtained for spatial metabolome analysis to analyze the distribution of fatty acid metabolism-related substances in the tumor. The mRNA levels of SREBP-1, FAS, ACCα, SCD-1, PPARα, ACOX, UCP-2, and CPT-1 were determined by real-time quantitative PCR(RT-qPCR). The results revealed that the model group showed decreased body weight(P<0.05) and colon length(P<0.001), increased number of tumors, and increased pathological score(P<0.01). Spatial metabolome analysis revealed that the content of fatty acids and their derivatives, carnitine, and phospholipid in the colon tumor was increased. RT-qPCR results indicated that fatty acid de novo synthesis and ß-oxidation-related genes, such as SREBP-1, FASN, ACCα, SCD-1, ACOX, UCP-2, and CPT-1 mRNA expression levels increased considerably(P<0.05, P<0.001). After anemoside B4 administration, the colon length increased(P<0.01), and the number of tumors decreased in the high-dose anemoside B4 group(P<0.05). Additionally, spatial metabolome analysis showed that anemoside B4 could decrease the content of fatty acids and their derivatives, carnitine, and phospholipids in colon tumors. Meanwhile, anemoside B4 could also down-regulate the expression of FASN, ACCα, SCD-1, PPARα, ACOX, UCP-2, and CPT-1 in the colon(P<0.05, P<0.01, P<0.001). The findings of this study show that anemoside B4 may inhibit CAC via regulating fatty acid metabolism reprogramming.

Pharmacokinetic and metabolite profile of orally administered anemoside B4 in rats with an improved exposure in formulations of rectal suppository.

ETHNOPHARMACOLOGICAL RELEVANCE: Pulsatilla chinensis (Bunge) Regel is a traditional Chinese herbal medicine used to treat intestinal amebiasis, malaria, vaginal trichomoniasis, and bacterial infections. Anemoside B4 (AB4), a pentacyclic triterpenoid saponin, is one of the primary bioactive substances in Pulsatilla chinensis (Bunge) Regel, and gavage administration of AB4 to animals has been demonstrated to exhibit anticancer, anti-inflammatory, and antiviral actions. However, AB4 exposure in plasma is very low after oral administration, and the biotransformation of AB4 in vivo after oral administration remains unknown. AIM OF THE STUDY: The reason for conducting this research was to explore at the metabolite profile of AB4 in rats following oral administration. Additionally, we aimed to develop an appropriate extravascular formulation to increase the exposure and duration of AB4 in vivo. MATERIALS AND METHODS: A well-validated HPLC-QQQ-MS/MS method was used for the quantification of AB4 in plasma and was further applied to evaluate and compare the pharmacokinetic properties of AB4 dissolved in a saline solution and AB4 formulations in a rectal suppository or enteric capsule. Reliable UHPLC coupled to Q-Exactive Plus high-resolution MS was used to identify the metabolites in rat plasma, bile, urine, and faeces. RESULTS: AB4 was extensively metabolized, and a total of 29 metabolites were identified. The primary metabolic routes included deglycosylation, oxidation, dehydrogenation, reduction, sulfation, hydration, acetylation, and glucuronidation. The pharmacokinetic comparison showed that both the rectal suppository and enteric capsule increased the exposures of AB4 and one of its active metabolites, 23-hydroxybetulinic acid (23-HA). Notably, rectal suppositories increased systemic AB4 exposure (AUC0-∞) by approximately 49 and 28 times higher than that of the AB4 saline solution and enteric capsules, respectively. The t1/2 of AB4 was extended to approximately 7 h after rectal administration compared to 2 h after oral administration. CONCLUSION: Overall, our study demonstrated that the mismatched exposure-response relationship of AB4 could result from extensive metabolism in the gastrointestinal and circulatory systems. Thus, a rectal suppository could be an alternative formulation of AB4 to obtain both higher and longer exposure.

Anemoside B4 Exerts Hypoglycemic Effect by Regulating the Expression of GLUT4 in HFD/STZ Rats.

Anemoside B4 (B4) is a saponin that is extracted from Pulsatilla chinensis (Bge.), and Regel exhibited anti-inflammatory, antioxidant, antiviral, and immunomodulatory activities. However, its hypoglycemic activity in diabetes mellitus has not been evaluated. Here, we explored the effect of B4 on hyperglycemia and studied its underlying mechanism of lowering blood glucose based on hyperglycemic rats in vivo and L6 skeletal muscle cells (L6) in vitro. The rats were fed a high-fat diet (HFD) for one month, combined with an intraperitoneal injection of 60 mg/kg streptozotocin (STZ) to construct the animal model, and the drug was administrated for two weeks. Blood glucose was detected and the proteins and mRNA were expressed. Our study showed that B4 significantly diminished fasting blood glucose (FBG) and improved glucose metabolism. In addition, B4 facilitated glucose utilization in L6 cells. B4 could enhance the expression of glucose transporter 4 (GLUT4) in rat skeletal muscle and L6 cells. Mechanistically, B4 elevated the inhibition of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways. Furthermore, we confirmed the effect of B4 on glucose uptake involved in the enhancement of GLUT4 expression in part due to PI3K/AKT signaling by using a small molecule inhibitor assay and constructing a GLUT4 promoter plasmid. Taken together, our study found that B4 ameliorates hyperglycemia through the PI3K/AKT pathway and promotes GLUT4 initiation, showing a new perspective of B4 as a potential agent against diabetes.

Anemoside B4 regulates fatty acid metabolism reprogramming in mice with colitis-associated cancer / 中国中药杂志

The study aimed to investigate the effect of anemoside B4(B4) on fatty acid metabolism in mice with colitis-associated cancer(CAC). The CAC model was established by azoxymethane(AOM)/dextran sodium sulfate(DSS) in mice. Mice were randomly divided into a normal group, a model group, and low-, medium-, and high-dose anemoside B4 groups. After the experiment, the length of the mouse colon and the size of the tumor were measured, and the pathological alterations in the mouse colon were observed using hematoxylin-eosin(HE) staining. The slices of the colon tumor were obtained for spatial metabolome analysis to analyze the distribution of fatty acid metabolism-related substances in the tumor. The mRNA levels of SREBP-1, FAS, ACCα, SCD-1, PPARα, ACOX, UCP-2, and CPT-1 were determined by real-time quantitative PCR(RT-qPCR). The results revealed that the model group showed decreased body weight(P<0.05) and colon length(P<0.001), increased number of tumors, and increased pathological score(P<0.01). Spatial metabolome analysis revealed that the content of fatty acids and their derivatives, carnitine, and phospholipid in the colon tumor was increased. RT-qPCR results indicated that fatty acid de novo synthesis and β-oxidation-related genes, such as SREBP-1, FASN, ACCα, SCD-1, ACOX, UCP-2, and CPT-1 mRNA expression levels increased considerably(P<0.05, P<0.001). After anemoside B4 administration, the colon length increased(P<0.01), and the number of tumors decreased in the high-dose anemoside B4 group(P<0.05). Additionally, spatial metabolome analysis showed that anemoside B4 could decrease the content of fatty acids and their derivatives, carnitine, and phospholipids in colon tumors. Meanwhile, anemoside B4 could also down-regulate the expression of FASN, ACCα, SCD-1, PPARα, ACOX, UCP-2, and CPT-1 in the colon(P<0.05, P<0.01, P<0.001). The findings of this study show that anemoside B4 may inhibit CAC via regulating fatty acid metabolism reprogramming.

Preparation and cellular uptake study of anemoside B4 and PD-L1 siRNA co-delivered cRGD-modified targeting liposomes / 中国药房

OBJECTIVE To prepare anemoside B4 （AB4） and programmed cell death ligand 1 （PDL1） siRNA （siP） co- delivered cRGD-modified targeting liposomes （AB4/siP-c-L）， and to study the cellular uptake in vitro. METHODS The cRGD- modified AB4-loaded targeted liposomes （AB4-c-L） were prepared by ethanol injection. AB4-c-L was mixed with 20 nmol/L siP in the same volume and AB4/siP-c-L was obtained through electrostatic adsorption. The particle size， Zeta potential， morphology， encapsulation efficiency and drug content， in vitro release behavior and serum stability of AB4/siP-c-L were investigated by laser scattering particle size tester， transmission electron microscopy， ultrafiltration centrifugation， dialysis and agar-gel electrophoresis block test. Cellular uptake of AB4/siP-c-L by Lewis lung cancer cells LLC and its intracellular localization were evaluated by flow cytometry and confocal laser scan technique. RESULTS The average particle size of AB4/siP-c-L was （187.4±3.1） nm， and the Zeta potential was （33.5±1.4） mV. AB4/siP-c-L was spheroidal in shape. The encapsulation efficiency and content of AB4 were （95.2±0.4） % and （1.0±0.2） mg/mL， respectively. AB4/siP-c-L could better package siP， and exhibited good serum stability， obvious pH sensitivity and sustained release property. The uptake rate of AB4/siP-c-L by LLC cells was significantly higher than that of free drug， and was able to accumulate in cytoplasm. CONCLUSIONS AB4/siP-c-L can effectively realize the co-loading of AB4 and gene drug siP， which has certain in vitro targeting to LLC cells.

Anemoside B4 protects against IgE-dependent allergic responses by suppressing the PLC/IP3 and JAK/STAT3 pathways.

Anemoside B4 (AB4) is a natural triterpenoid abundant in the roots of Pulsatilla chinensis. Although various biological activities have been widely attributed to AB4, few studies have focused on its antiallergic effects. In this study the inhibitory effects of AB4 on immunoglobulin E (IgE)-mediated allergic responses were investigated, both in vitro and in vivo, and the mechanism of its effects. IgE-mediated passive cutaneous anaphylaxis was used to elucidate the antiallergic effects of AB4 in vivo. The degranulation assay, calcium imaging, and cytokine and chemokine release in the laboratory of allergic disease 2 (LAD2) cell line were used to evaluate the antiallergic effect of AB4 in vitro. Pathological staining was performed to analyze angiectasis. Western blot analysis was performed to investigate the downstream signaling pathways. AB4 dose-dependently attenuated ovalbumin/IgE-induced paw swelling in mice, and reduced the serum concentrations of tumor necrosis factor-alpha and C-C motif chemokine 2. In addition, AB4 suppressed IgE-mediated LAD2 cell degranulation, calcium influx, and PLC/IP3 and JAK/STAT3 phosphorylation. Our results suggest that AB4 inhibits allergic reactions through the PLC/IP3 and JAK/STAT3 pathways.

Anemoside B4 prevents chronic obstructive pulmonary disease through alleviating cigarette smoke-induced inflammatory response and airway epithelial hyperplasia.

BACKGROUND: Cigarette smoke (CS) is one of the major risk factors for chronic obstructive pulmonary disease (COPD) and increases the risk of lung cancer (LC). Anemoside B4 (B4) is the main bioactive ingredient in Pulsatilla chinensis (P. chinensis), a traditional medicinal herb for various diseases. It has a wide range of anti-inflammatory, anti-oxidation and anti-cancer activities. However, in recent years, there is no relevant literature report on the therapeutic effect of B4 on COPD, and the anti-inflammatory and inhibitory effects of anemoside B4 on basal cell hyperplasia in CS-induced COPD have not been clearly established. PURPOSE: In the present study, we investigated whether anemoside B4 could alleviate CS or cigarette smoke extract (CSE) induced inflammation of COPD and further prevent basal cell hyperplasia, hoping to find its possible mechanism. METHODS: In this study, a COPD mouse model was established in C57BL mice by CS exposure 3 months. Bronchial pathology and basal cell hyperplasia were observed by HE staining and immunostaining. The contents of glutathione peroxidase catalase (GSH-PX), malondialdehyde (MDA) and superoxide dismutase (MPO) were determined by GSH-PX, MDA and SOD assay kits, respectively. 16HBE cells were cultured with 5% CSE with or without treatment with B4 (1, 10, 100 µM) or DEX (20 µM) in vitro. Cell viability was assessed by a cell counting kit 8 (CCK-8). Reactive oxygen species (ROS) generation was tested by DCFH-DA. Moreover, anti-inflammatory mechanism of anemoside B4 was further determined by pro-inflammatory cytokines production using RT-PCR. Protein expression levels of MAPK/AP-1/TGF-ß signaling pathway were measured by western blot. RESULTS: Anemoside B4 improved the lung function of mice, relieved lung inflammation and reduced the MDA, MPO and GSH-Px in the plasma. At the same time, B4 repressed the oxidative stress response and played a role in balancing the levels of protease and anti-protease. During the process of bronchial basal cell hyperplasia, B4 alleviated the degree of cell hyperplasia, and prevented further deterioration of hyperplasia through increased P53 and inhibited FHIT protein. In addition, B4 reduced ROS levels in human bronchial epithelial cells stimulated by CSE in vitro study. Meanwhile, B4 treatment also significantly attenuated increased IL-1ß, TGF-ß, IL-8 and TNF-α from CSE treated human bronchial epithelial cells. The expression of p-P38, AP-1(c-fos, and c-Jun), TGF-ß proteins in MAPK/AP-1/TGF-ß signaling pathway were decreased and the signal cascade reaction was blocked. CONCLUSION: Anemoside B4 protects against CS-induced COPD. These findings indicated that B4 may have therapeutic potential for the prevention and treatment of COPD.

Anemoside B4 protects against chronic relapsing colitis in mice by modulating inflammatory response, colonic transcriptome and the gut microbiota.

BACKGROUND: Anemoside B4 (AB4) is reported to prevent acute colitis when given via intraperitoneal injection by two recent studies. However, whether oral AB4 protects against chronic colitis which resembles the clinical phenotype of ulcerative colitis (UC) and its mechanism of action are largely unknown. PURPOSE: To systemically investigate the effects of oral AB4 against chronic colitis and illustrate the underlying mechanism of action. METHODS: The preventive, therapeutic, and dose-dependent effects of AB4 against UC were examined in mice with acute or chronic relapsing colitis induced by dextran sulfate sodium (DSS). The inflammatory responses, colonic transcriptome, and 16S rDNA sequencing of the intestinal content of mice were analyzed. RESULTS: Oral administration of AB4 alleviated disease severity and colon shortening in mice with chronic relapsing colitis in a dose-dependent manner. The effects of AB4 were comparable to those of two positive-control compounds: tofacitinib and berberine. Unlike tofacitinib, AB4 did not have a deleterious effect on DSS-induced splenic swelling and anemia. Furthermore, AB4 inhibited the inflammatory responses of colitis, as evidenced by in-vivo, ex-vivo, and in-vitro studies. Transcriptomics revealed that AB4 treatment reversed the DSS-mediated decrease in the expression of colonic Pelo, B3gat2 and Mir8010. In addition, AB4 reversed DSS-induced alterations in the intestinal microbiome in mice. Through fecal microbiota transplantation, we proved that AB4 partially exerted its anti-colitis effects by modulating the gut microbiota. CONCLUSIONS: We demonstrated for the first time that AB4 has dose-dependent therapeutic effects against chronic relapsing colitis by modulating the inflammatory response, colonic gene expression, and intestinal microbiota.

Anemoside B4 ameliorates experimental autoimmune encephalomyelitis in mice by modulating inflammatory responses and the gut microbiota.

BACKGROUND: Anemoside B4 (AB4) is a representative component of Pulsatilla decoction that is used in traditional Chinese medicine for treating inflammatory conditions. It is not known whether AB4 has beneficial effects on multiple sclerosis (MS). METHODS: In the present study, we examined the preventative and therapeutic effects of AB4, and the possible mechanism by which it protects female mice against experimental autoimmune encephalomyelitis (EAE). RESULTS: Preventative treatment with AB4 (given orally at 100 and 200 mg/kg for 18 days) reduced the clinical severity of EAE significantly (from 3.6 ± 1.3 to 1.8 ± 1.5 and 1.6 ± 0.6, respectively), and inhibited demyelination and inflammatory infiltration of the spinal cord. In the therapeutic protocol, oral administration of 200 mg/kg AB4 for 21 days after initiation of EAE significantly alleviated disease severity (from 2.6 ± 1.3 to 0.9 ± 0.6) and was as effective as the clinically used drug fingolimod (0.3 ± 0.6). Furthermore, both doses of AB4 significantly inhibited mRNA expression of TNF-α, IL-6, and IL-17, and STAT3 activation, in the spinal cord; and the ex vivo and iv vitro AB4 treatment markedly inhibited secretion of the three cytokines from lymphocytes of EAE mice upon in vitro restimulation. In addition, AB4 reversed the changes in the composition of the intestinal microbiome observed in EAE mice. CONCLUSION: We reveal for the first time that AB4 protects against EAE by modulating inflammatory responses and the gut microbiota, demonstrating that AB4 may have potential as a therapeutic agent for treating MS in humans.

Liposomal Co-delivery of PD-L1 siRNA/Anemoside B4 for Enhanced Combinational Immunotherapeutic Effect.

Combination therapy has gained a lot of attention thanks to its superior activity against cancer. In the present study, we report a cRGD-targeted liposomal preparation for co-delivery of programmed cell death ligand 1 (PD-L1) small interfering RNA (siRNA) and anemoside B4 (AB4)─AB4/siP-c-L─and evaluate its anticancer efficiency in mouse models of LLC and 4T1 tumors. AB4/siP-c-L showed a particle size of (180.7 ± 7.3) nm and a ζ-potential of (32.8 ± 1.5) mV, with high drug encapsulation, pH-sensitive release properties, and good stability in serum. AB4/siP-c-L demonstrated prolonged blood circulation and increased tumor accumulation. Elevated cellular uptake was dependent on the targeting ligand cRGD. This combination induced significant tumor inhibition in LLC xenograft tumor-bearing mice by downregulating PD-L1 protein expression and modulating the immunosuppressive microenvironment. Liposomes favored the antitumor T-cell response with long-term memory, without obvious toxicity. A similar tumor growth inhibition was also demonstrated in the 4T1 tumor model. In summary, our results indicate that cRGD-modified and AB4- and PD-L1 siRNA-coloaded liposomes have potential as an antitumor preparation, and this approach may lay a foundation for the development of a new targeted drug delivery system.