Paeonol attenuates nonalcoholic steatohepatitis by regulating intestinal flora and AhR/NLRP3/Caspase-1 metabolic pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Non-alcoholic steatohepatitis (NASH) is a common metabolic liver injury disease that is closely associated with obesity and metabolic disorders. Paeonol, an active ingredient found in Moutan Cortex, a traditional Chinese medicine which exhibits significant therapeutic effect on liver protection, has shown promising effects in treating liver diseases, particularly NASH. However, the specific intervention mechanism of paeonol on NASH is still unknown. AIM OF THE STUDY: Our objective is to elucidate the pharmacological mechanism of paeonol in intervening NASH at the in vivo level, focusing on the impact on intestinal flora, tryptophan-related targeted metabolome, and related Aryl hydrocarbon receptor (AhR) pathways. MATERIALS AND METHODS: Here, we explored the intervention effect of paeonol on NASH by utilizing the NASH mouse model. The Illumina highthroughput sequencing technology was preformed to determine the differences of gut microbiota of model and paeonol treatment group. The concentration of Indoleacetic acid is determined by ELISA. The intervention effect of NASH mouse and AhR/NLRP3/Caspase-1 metabolic pathway is analyzed by HE staining, oil red O staining, Immunohistochemistry, Immunofluorescence, Western blot and qRT-PCR assays. Fecal microbiota transplantation experiment also was performed to verify the intervention effect of paeonol on NASH by affecting gut microbiota. RESULTS: Firstly, we discovered that paeonol effectively reduced liver pathology and blood lipid levels in NASH mice, thereby intervening in the progression of NASH. Subsequently, through 16S meta-analysis, we identified that paeonol can effectively regulate the composition of intestinal flora in NASH mice, transforming it to resemble that of normal mice. Specifically, paeonol decreased the abundance of certain Gram-negative tryptophan-metabolizing bacteria. Moreover, we discovered that paeonol significantly increased the levels of metabolites Indoleacetic acid, subsequently enhancing the expression of AhR-related pathway proteins. This led to the inhibition of the NOD-like receptor protein 3 (NLRP3) inflammasome production and inflammation generation in NASH. Lastly, we verified the efficacy of paeonol in intervening NASH by conducting fecal microbiota transplantation experiments, which confirmed its role in promoting the AhR/NLRP3/cysteinyl aspartate specific proteinase (Caspase-1) pathway. CONCLUSIONS: Our findings suggest that paeonol can increase the production of Indoleacetic acid by regulating the gut flora, and promote the AhR/NLRP3/Caspase-1 metabolic pathway to intervene NASH.

Hydroxy-α-sanshool from the fruits of Zanthoxylum bungeanum Maxim. promotes browning of white fat by activating TRPV1 to induce PPAR-γ deacetylation.

BACKGROUND: Accumulating evidence suggested increasing energy expenditure is a feasible strategy for combating obesity, and browning of white adipose tissue (WAT) to promote thermogenesis might be one of the attractive ways. Hydroxy-α-sanshool (HAS), a natural amide alkaloid extracted from the fruits of Zanthoxylum bungeanum Maxim, possesses lots of benefits in lipid metabolism regulation. METHODS: The anti-obesity effect of HAS was investigated by establishing an animal model of obesity and a 3T3-L1 differentiation cell model. Effects of HAS on the whole-body fat and liver of obese mice, and the role of HAS in inducing browning of white fat were studied by Micro CT, Metabolic cage detection, Cell mitochondrial pressure detection, transmission electron microscopy and cold exposure assays. Furthermore, the Real-time PCR (qPCR), digital PCR (dPCR), western blot, Co-immunoprecipitation (Co-IP), molecular docking, drug affinity responsive target stability (DARTS), Cellular thermal shift assay (CETSA) and other methods were used to investigate the target and mechanisms of HAS. RESULTS: We found that treatment with HAS helped mice combat obesity caused by a high fat diet (HFD) and improve metabolic characteristics. In addition, our results suggested that the anti-obesity effect of HAS is related to increase energy consumption and thermogenesis via induction of browning of WAT. The further investigations uncovered that HAS can up-regulate UCP-1 expression, increase mitochondria number, and elevate the cellular oxygen consumption rates (OCRs) of white adipocytes. Importantly, the results indicated that browning effects of HAS is closely associated with SIRT1-dependent PPAR-γ deacetylation through activating the TRPV1/AMPK pathway, and TRPV1 is the potential drug target of HAS for the browning effects of WAT. CONCLUSIONS: Our results suggested the HAS can promote browning of WAT via regulating AMPK/SIRT-1/PPARγ signaling, and the potential drug target of HAS is the membrane receptor of TRPV1.

Salidroside intensifies mitochondrial function of CoCl2-damaged HT22 cells by stimulating PI3K-AKT-MAPK signaling pathway.

BACKGROUND: Salidroside (Sal), an active component from Rhodiola crenulata, has been confirmed to exert neuroprotective effects against hypoxia. However, its molecular mechanisms of intensifying mitochondrial function still largely unknown. In the present study, we aimed to explore the mechanisms by which Sal heightened mitochondrial function in CoCl2-induced HT22 hypoxic injury. METHODS: The hypoxic condition of HT22 cells was performed by CoCl2 stimulus. We then investigated the effects of Sal on the viability of hypoxic HT22 cells by cell counting kit-8. The contents of lactate dehydrogenase (LDH) release in cultured supernatant were detected by using commercial biochemical kit. Superoxide free radical scavenging activity, total antioxidant capacity assay kit with ferric reducing ability of plasma and 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) methods were employed to detect the free radical scavenging ability and antioxidant capacity of Sal. Meanwhile, intracellular reactive oxygen species (ROS), Ca2+ and mitochondrial membrane potential (MMP) were determined by corresponding specific labeled probes. Mitochondrial morphology was tested by Mito-tracker green with confocal microscopy. Hoechst 33342 and Annexin V-FITC/propidium iodide staining were also employed to evaluate the effect of Sal on cell apoptosis. Oxygen consumption rate (OCR), real-time ATP production and proton efflux rate were measured using a Seahorse analyzer. Additionally, the potential interactions of Sal with PI3K-AKT signaling pathway-related proteins were predicted and tested by molecular docking, molecular dynamics simulation (MDS) and localized surface plasmon resonance (LSPR) techniques, respectively. Furthermore, the protein levels of p-PI3K, PI3K, p-AKT, AKT, p-JNK, JNK, p-p38 and p38 were estimated by western blot analysis. RESULTS: Sal alleviated CoCl2-induced hypoxic injury in HT22 cells as evidenced by increased cell viability and decreased LDH release. In vitro antioxidant test confirmed that Sal had marvelous antioxidant abilities. The protected mitochondrial function by Sal treatment was illustrated by the decrease of ROS, Ca2+, mitochondrial fragment and the increase of MMP. In addition, Sal ameliorated the apoptosis of HT22 cells by decreasing Hoechst 33342 positive cells and the rate of apoptotic cells. Enhancement of energy metabolism in HT22 by Sal was demonstrated by increased OCR, real-time ATP generation and proton efflux rate. The molecular docking confirmed the potential binding of Sal to PI3K, AKT and CaMK II proteins with calculated binding energy of -1.32, -4.21 and -4.38 kcal/mol, respectively. The MDS test revealed the average hydrogen bond of complex Sal-PI3K and Sal-AKT were 0.79 and 4.46, respectively. The results of LSPR verified the potential binding of Sal to proteins PI3K, AKT and HIF-1α with affinity values of 5.20 × 10 - 3, 2.83 × 10 - 3 and 3.97 × 10 - 3 KD, respectively. Western blot analysis further argued that Sal consolidated the levels of p-PI3K and p-AKT. Meanwhile, Sal could downregulate the proteins expression of p-JNK and p-p38. CONCLUSION: Collectively, our findings suggested that Sal can intensify mitochondrial function of CoCl2-simulated hypoxia injury in HT22 cells by stimulating PI3K-AKT-MAPK signaling pathway. Sal is a potential agent for mitochondrial protection against hypoxia with the underlying molecular mechanisms of energy metabolism being further elucidated.

Essential oil from the roots of Paeonia lactiflora pall. has protective effect against corticosterone-induced depression in mice via modulation of PI3K/Akt signaling pathway.

For thousands of years, the roots of Paeonia lactiflora Pall (PLP) has been considered by traditional Chinese medicine as a drug that can improve mental or emotional disorders, including depression, anxiety and affective disorders. Unfortunately, the research on the mechanism of action and active ingredients of this beneficial drug is not comprehensive. This study focused on the activity of essential oil from PLP (EOP), systematically studied the antidepressant effect of EOP for the first time, and discussed the potential mechanism of its antidepressant effect. In this study, we used a mouse model of corticosterone (CORT)-induced depression, and found that EOP had a significant antidepressant effect on the symptoms of CORT-induced depression in mice, and significantly down-regulated the levels of CRH, ACTH and cortisol in the brain tissues of mice. In addition, we found that EOP treatment alleviated CORT-induced hippocampal neuron injury in mice In vitro experiments. It was also found that EOP could inhibit CORT-induced apoptosis and improve the proliferation ability and cell viability of PC12 cells. Further, with the help of network analysis, it was revealed that PI3K-Akt might be one of the main signaling pathways of EOP against CORT-induced hippocampal neuron apoptosis. In this study, we also found that EOP up-regulated the phosphorylation of PI3K and Akt in CORT-induced mouse hippocampal neurons and PC12 cells, and promoted the nuclear transcription of Nrf2 in CORT-induced PC12 cells. In conclusion, with the integrated approach, we demonstrated that EOP exerted anti-apoptotic effects on hippocampal neurons through PI3K/Akt/Nrf2 signaling pathway.

Oxysophocarpine inhibits airway inflammation and mucus hypersecretion through JNK/AP-1 pathway in vivo and in vitro.

Asthma is a high-incidence disease in the world. Oxysophocarpine (OSC), a quinolizidine alkaloid displays various pharmacological functions including anti-inflammation, neuroprotective, anti-virus and antioxidant. Here, we established mice and cell asthmatic model to explore the effects of OSC for asthma treatment. Mice were sensitized and challenged with ovalbumin (OVA) and treated with OSC before challenge. Enzyme-linked immuno sorbent assay (ELISA), hematoxylin and eosin (H&E), periodic acid-schiff (PAS), tolonium chloride staining and immunohistochemical assay were performed. OSC treatment inhibited inflammatory cell infiltration and mucus secretion in the airway, reduced IgE level in mouse serum and decreased IL-4, IL-5 production in bronchoalveolar lavage fluid (BALF). OSC also reduced the spleen index to regulate immune function. Meanwhile, NCI-H292 cells were induced by lipopolysaccharide (LPS) to simulate airway epithelial injury. OSC pretreatment decreased the IL-6 and IL-8 cytokine levels, mucin 5 AC expression, and mucin 5 AC mRNA level in the cell model. Further, OSC suppressed the phosphorylation of c-Jun N-terminal kinase (JNK), and activator protein 1 (AP-1, Fos and Jun). These findings revealed that OSC alleviated bronchial asthma associated with JNK/AP-1 signaling pathway.

Rhodiola crenulate alleviates hypobaric hypoxia-induced brain injury via adjusting NF-κB/NLRP3-mediated inflammation.

BACKGROUND: Rhodiola crenulate (R. crenulate), a famous Tibetan medicine, has been demonstrated to possess superiorly protective effects in high-altitude hypoxic brain injury (HHBI). However, its mechanisms on HHBI are still largely unknown. METHODS: Herein, the protective effects and underlying mechanisms of R. crenulate on HHBI of BABL/c mice were explored through in vivo experiments. The mice model of HHBI was established using an animal hypobaric and hypoxic chamber. R. crenulate extract (RCE) (0.5, 1.0 and 2.0 g/kg) was given by gavage for 7 days. Pathological changes and neuronal viability of mice hippocampus and cortex were evaluated using H&E and Nissl staining, respectively. The brain water content (BWC) in mice was determined by calculating the ratio of dry to wet weight of brain tissue. And serum of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH-Px) and lactate dehydrogenase (LDH) were detected via commercial biochemical kits. Synchronously, the contents of total antioxidant capacity (T-AOC), lactic acid (LA), adenosine triphosphate (ATP), succinate dehydrogenase (SDH), pyruvate kinase (PK), Ca2+-Mg2+-ATPcase, Na+-K+-ATPcase, TNF-α, IL-1ß and IL-6 in brain tissue were quantitative analysis by corresponding ELISA assay. Subsequently, NLRP3, ZO-1, claudin-5, occluding, p-p65, p65, ASC, cleaved-caspase-1, caspase-1 and IL-18 were determined by immunofluorescent and western blot analyses. RESULTS: The results demonstrated that RCE remarkably alleviated pathological damage, BWC, as well enhanced neuronal viability. Furthermore, the oxidative stress injuries were reversely abrogated after RCE treatment, evidenced by the increases of SOD, GSH-Px and T-AOC, while the decreases of MDA and LDH contents. Marvelously, the administration of RCE rectified and balanced the abnormal energy metabolism via elevating the levels of ATP, SDH, PK, Ca2+-Mg2+-ATPcase and Na+-K+-ATPcase, and lowering LA. Simultaneously, the expression of tight junction proteins (ZO-1, claudin-5 and occludin) was enhanced, illustrating RCE treatment might maintain the integrity of blood-brain barrier (BBB). Additionally, RCE treatment confined the contents of IL-6, IL-1ß and TNF-α, and attenuated fluorescent signal of NLRP3 protein. Concurrently, the results of western blot indicated that RCE treatment dramatically restrained p-p65/p65, ASC, NLRP3, cleaved-caspase-1/caspase-1 and IL-18 protein expressions in brain tissues of mice. CONCLUSION: RCE may afford a protectively intervention in HHBI of mice through suppressing the oxidative stress, improving energy metabolism and the integrity of BBB, and subsiding inflammatory responses via the NF-κB/NLRP3 signaling pathway. As a promising agent for the treatment of mice HHBI, the deep-crossing molecular mechanisms of R. crenulate still needs to be further elucidated to identify novel core hub targets.

Ursolic acid-enriched kudingcha extract enhances the antitumor activity of bacteria-mediated cancer immunotherapy.

BACKGROUND: Bacteria-mediated cancer immunotherapy (BCI) robustly stimulates the immune system and represses angiogenesis, but tumor recurrence and metastasis commonly occur after BCI. The natural product Ilex kudingcha C. J Tseng enriched with ursolic acid has anti-cancer activity and could potentially augment the therapeutic effects of BCI. The objective of the present study was to determine potential additive effects of these modalities. METHODS: We investigated the anti-cancer activity of KDCE (Kudingcha extract) combined with S.t△ppGpp in the mice colon cancer models. RESULTS: In the present study, KDCE combined with S.t△ppGpp BCI improved antitumor therapeutic efficacy compared to S.t△ppGpp or KDCE alone. KDCE did not prolong bacterial tumor-colonizing time, but enhanced the antiangiogenic effect of S.t△ppGpp by downregulatingVEGFR2. We speculated that KDCE-induced VEGFR2 downregulation is associated with FAK/MMP9/STAT3 axis but not AKT or ERK. CONCLUSIONS: Ursolic acid-enriched KDCE enhances the antitumor activity of BCI, which could be mediated by VEGFR2 downregulation and subsequent suppression of angiogenesis. Therefore, combination therapy with S.t△ppGpp and KDCE is a potential cancer therapeutic strategy.

Salidroside, a phenyl ethanol glycoside from Rhodiola crenulata, orchestrates hypoxic mitochondrial dynamics homeostasis by stimulating Sirt1/p53/Drp1 signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhodiola crenulata is clinically used to combat hypobaric hypoxia brain injury at high altitude with the function of invigorating Qi and promoting blood circulation in Tibetan medicine. Salidroside (Sal), an active compound identified from Rhodiola species, has been shown to exert neuroprotective effects against hypoxic brain injury. However, its mitochondrial protective mechanisms remain largely unknown. AIM OF THE STUDY: The present study aimed to explore the mitochondrial protection of Sal and the involved mechanisms related to mitochondrial dynamics homeostasis on hypoxia-induced injury of HT22 cells. MATERIALS AND METHODS: Hypoxic condition was performed as cells cultured in a tri-gas incubator with 1% O2, 5% CO2 and 94% N2. We firstly investigated the effects of different concentrations of Sal on the viability of normal or hypoxic HT22 cells. Whereafter, the levels of lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), adenosine triphosphate (ATP) and Na+-K+-ATPase were tested by commercial kits. Meanwhile, mitochondrial superoxide, intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were determined by specific labeled probes. Mitochondrial morphology was detected by mito-tracker green with confocal microscopy. Additionally, the potential interactions of Sal with Sirt1/p53/Drp1 signaling pathway-related proteins were predicted and tested by molecular docking and localized surface plasmon resonance (LSPR) techniques, respectively. Furthermore, the protein levels of Sirt1, p53, ac-p53, Drp1, p-Drp1(s616), Fis1 and Mfn2 were estimated by western blot analysis. RESULTS: Sal alleviated hypoxia-induced oxidative stress in HT22 cells as evidenced by increased cell viability and SOD activity, while decreased LDH release and MDA content. The protected mitochondrial function by Sal treatment was indicated by the increases of ATP level, Na+-K+-ATPase activity and MMP. Miraculously, Sal reduced hypoxia-induced mitochondrial fission, while increased mitochondrial tubular or linear morphology. The results of molecular docking and LSPR confirmed the potential binding of Sal to proteins Sirt1, p53, Fis1 and Mfn2 with affinity values 1.38 × 10-2, 5.26 × 10-3, 6.46 × 10-3 and 7.26 × 10-3 KD, respectively. And western blot analysis further demonstrated that Sal memorably raised the levels of Sirt1 and Mfn2, while decreased the levels of ac-p53, Drp1, p-Drp1 (s616) and Fis1. CONCLUSION: Collectively, our data confirm that Sal can maintain mitochondrial dynamics homeostasis by activating the Sirt1/p53/Drp1 signaling pathway.

Apoptosis Exerts a Vital Role in the Treatment of Colitis-Associated Cancer by Herbal Medicine.

Colitis-associated cancer (CAC) is known as inflammatory bowel disease (IBD)-developed colorectal cancer, the pathogenesis of which involves the occurrence of apoptosis. Western drugs clinically applied to CAC are often single-targeted and exert many adverse reactions after long-term administration, so it is urgent to develop new drugs for the treatment of CAC. Herbal medicines commonly have multiple components with multiple targets, and most of them are low-toxicity. Some herbal medicines have been reported to ameliorate CAC through inducing apoptosis, but there is still a lack of systematic review. In this work, we reviewed articles published in Sci Finder, Web of Science, PubMed, Google Scholar, CNKI, and other databases in recent years by setting the keywords as apoptosis in combination with colitis-associated cancer. We summarized the herbal medicine extracts or their compounds that can prevent CAC by modulating apoptosis and analyzed the mechanism of action. The results show the following. (1) Herbal medicines regulate both the mitochondrial apoptosis pathway and death receptor apoptosis pathway. (2) Herbal medicines modulate the above two apoptotic pathways by affecting signal transductions of IL-6/STAT3, MAPK/NF-κ B, Oxidative stress, Non-canonical TGF-ß1, WNT/ß-catenin, and Cell cycle, thereby ameliorating CAC. We conclude that following. (1) Studies on the role of herbal medicine in regulating apoptosis through the Ras/Raf/ERK, WNT/ß-catenin, and Cell cycle pathways have not yet been carried out in sufficient depth. (2) The active constituents of reported anti-CAC herbal medicine mainly include polyphenols, terpenoids, and saccharide. Also, we identified other herbal medicines with the constituents mentioned above as their main components, aiming to provide a reference for the clinical use of herbal medicine in the treatment of CAC. (3) New dosage forms can be utilized to elevate the targeting and reduce the toxicity of herbal medicine.

Chlorogenic Acid-Enriched Extract of Ilex kudingcha C.J. Tseng Inhibits Angiogenesis in Zebrafish.

Kudingcha is a particularly bitter tasting tea that has been widely used in China to eliminate fever and itching eyes, and to clear blood toxins. Kudingcha is considered of value for its potential anticancer effects that are attributed to the presence of characteristic bioactive ingredients. The chlorogenic acid (CGA) derivatives 3-0-caffeoylquinic acid, 5-0-caffeoylquinic acid, 3,5-0-dicaffeoylquinic acid, and 4,5-0-dicaffeoylquinic acid were separated from Ilex kudingcha C.J. Tseng extract by high-performance liquid chromatography (HPLC)-photodiode array detector (PDA) and HPLC-nuclear magnetic resonance (NMR). In Tg(flk1:EGFP) zebrafish embryos at 52 hours postfertilization (hpf), angiogenesis was significantly inhibited by kudingcha extract (KDCE) at concentrations of 400 and 500 µg/mL and CGA also showed significant inhibition in embryos treated with 80, 100, and 130 µg/mL. Endothelial cell apoptosis showed a dose-dependent increase in response to KDCE and CGA. CGA derivatives from KDCE could have potential as anticancer agents against tumor angiogenesis.

Effect of Salmonella treatment on an implanted tumor (CT26) in a mouse model.

The use of bacteria has contributed to recent advances in targeted cancer therapy especially for its tumor-specific accumulation and proliferation. In this study, we investigated the molecular events following bacterial therapy using an attenuated Salmonella Typhimurium defective in ppGpp synthesis (ΔppGpp), by analyzing those proteins differentially expressed in tumor tissues from treated and untreated mice. CT26 murine colon cancer cells were implanted in BALB/c mice and allowed to form tumors. The tumor-bearing mice were treated with the attenuated Salmonella Typhimurium. Tumor tissues were analyzed by 2D-PAGE. Fourteen differentially expressed proteins were identified by mass spectrometry. The analysis revealed that cytoskeletal components, including vimentin, drebrin-like protein, and tropomyosin-alpha 3, were decreased while serum proteins related to heme or iron metabolism, including transferrin, hemopexin, and haptoglobin were increased. Subsequent studies revealed that the decrease in cytoskeletal components occurred at the transcriptional level and that the increase in heme and iron metabolism proteins occurred in liver. Most interestingly, the same pattern of increased expression of transferrin, hemopexin, and haptoglobin was observed following radiotherapy at the dosage of 14 Gy.