Andrographolide regulates H3 histone lactylation by interfering with p300 to alleviate aortic valve calcification.

BACKGROUND AND PURPOSE: Our previous studies have found that andrographolide (AGP) alleviates calcific aortic valve disease (CAVD), but the underlying mechanism is unclear. This study explores the molecular target and signal mechanisms of AGP in inhibiting CAVD. EXPERIMENTAL APPROACH: The anti-calcification effects of the aortic valve with AGP treatment were evaluated by alizarin red staining in vitro and ultrasound and histopathological assessment of a high-fat (HF)-fed ApoE-/- mouse valve calcification model. A correlation between the H3 histone lactylation (H3Kla) and calcification was detected. Molecular docking and surface plasmon resonance (SPR) experiments were further used to confirm p300 as a target for AGP. Overexpression (oe) and silencing (si) of p300 were used to verify the inhibitory effect of AGP targeting p300 on the H3Kla in vitro and ex vivo. KEY RESULTS: AGP significantly inhibited calcium deposition in valve interstitial cells (VICs) and ameliorated aortic valve calcification. The multi-omics analysis revealed the glycolysis pathway involved in CAVD, indicating that AGP interfered with lactate production by regulating lactate dehydrogenase A (LDHA). In addition, lactylation, a new post-translational modification, was shown to have a role in promoting aortic valve calcification. Furthermore, H3Kla and H3K9la site were shown to correlate with Runx2 expression inhibition by AGP treatment. Importantly, we found that p300 transferase was the molecular target of AGP in inhibiting H3Kla. CONCLUSIONS AND IMPLICATIONS: Our findings, for the first time, demonstrated that AGP alleviates calcification by interfering with H3Kla via p300, which might be a powerful drug to prevent CAVD.

Atractylodes lancea Rhizome Polysaccharide Alleviates Immunosuppression and Intestinal Mucosal Injury in Mice Treated with Cyclophosphamide.

Plant-derived polysaccharides, such as Atractylodes lancea rhizome polysaccharide (ALP), are good immune regulators. However, the immune regulatory mechanism of the ALP is unknown. This study aimed to evaluate the effects of ALP on the intestinal mucosal barrier and intestinal mucosal immunity of immunosuppressed mice. We also compared the activity of raw Atractylodes lancea rhizome polysaccharide (SALP) with wheat bran processed bran-fried Atractylodes lancea rhizome polysaccharide (FALP; both at 1.2 g/kg/d for mice). Our results showed that ALP effectively increased the immune organ index and blood cell count, stimulated the secretion of cytokines, and promoted the expression of occludin and zonula occludens-1 (ZO-1). ALP also promoted the expression of T cells and the secretion of sIgA. Furthermore, ALP alleviated the gut microbiota disorder in Cy-treated mice and increased the relative abundances of Lactobacillus and Faecalibaculum. ALP reversed the decrease in the level of SCFAs and promoted the expression of G protein-coupled receptor 43 (GPR43). To our knowledge, this study was the first to explore how the ALP protects the intestinal mucosal barrier and enhances intestinal mucosal immunity by alleviating the gut microbiota imbalance and metabolic disorders of SCFAs. FALP was more therapeutic than SALP, suggesting that FALP could be developed as a promising functional food component.

Royal jelly acid suppresses hepatocellular carcinoma tumorigenicity by inhibiting H3 histone lactylation at H3K9la and H3K14la sites.

BACKGROUND AND PURPOSE: Human hepatocellular carcinoma (HCC) features include enhanced glycolysis and elevated lactate concentrations. Accumulation of lactate during metabolism provides a precursor for histone lysine modification. This study was designed to determine whether royal jelly acid (RJA) acts against HCC through the lactate modification pathway. EXPERIMENTAL APPROACH: The effects of RJA on Hep3B and HCCLM3 cell invasion, migration, proliferation, and apoptosis were investigated using cell scratching, colony formation assay, flow cytometry, western blotting, and real-time qPCR, gas chromatography, and RNA sequencing to determine the pathways and molecular targets involved. Tumor xenografts were used to evaluate the anti-HCC effects of RJA in vivo. In-cell Western blotting and expression correlation analysis were applied to confirm the associations between H3 histone lactylation and the antitumor effects of RJA. KEY RESULTS: RJA has good antitumor effects in vivo and in vitro. Multi-omics analysis with metabolome and transcriptome determined that the glycolytic metabolic pathway provided the principle antitumor effect of RJA. Further mechanistic studies showed that RJA inhibited HCC development by interfering with lactate production and inhibiting H3 histone lactylation at H3K9la and H3K14la sites. CONCLUSIONS AND IMPLICATIONS: This study first demonstrated that RJA exerts antitumor effects by affecting the glycolytic pathway. RJA could regulate the lactylation of H3K9la and H3K14la sites on H3 histone using lactate as a clue in the glycolytic pathway. Therefore, the lactylation of H3 histone is vital in exerting the antitumor effect of RJA, providing new evidence for screening and exploring antitumor drug mechanisms in the later stage.

Atractylenolide-1 affects glycolysis/gluconeogenesis by downregulating the expression of TPI1 and GPI to inhibit the proliferation and invasion of human triple-negative breast cancer cells.

Atractylenolide-1 (AT-1) is a major octanol alkaloid isolated from Atractylodes Rhizoma and is widely used to treat various diseases. However, few reports have addressed the anticancer potential of AT-1, and the underlying molecular mechanisms of its anticancer effects are unclear. This study aimed to assess the effect of AT-1 on triple-negative breast cancer (TNBC) cell proliferation and migration and explore its potential molecular mechanisms. Cell invasion assays confirmed that the number of migrating cells decreased after AT-1 treatment. Colony formation assays showed that AT-1 treatment impaired the ability of MDA-MB-231 cells to form colonies. AT-1 inhibited the expression of p-p38, p-ERK, and p-AKT in MDA-MB-231 cells, significantly downregulated the proliferation of anti-apoptosis-related proteins CDK1, CCND1, and Bcl2, and up-regulated pro-apoptotic proteins Bak, caspase 3, and caspase 9. The gas chromatography-mass spectroscopy results showed that AT-1 downregulated the metabolism-related genes TPI1 and GPI through the glycolysis/gluconeogenesis pathway and inhibited tumor growth in vivo. AT-1 affected glycolysis/gluconeogenesis by downregulating the expression of TPI1 and GPI, inhibiting the proliferation, migration, and invasion of (TNBC) MDA-MB-231 cells and suppressing tumor growth in vivo.

Atractylodin targets GLA to regulate D-mannose metabolism to inhibit osteogenic differentiation of human valve interstitial cells and ameliorate aortic valve calcification.

Atractylodin (ATL) has been reported to exert anti-inflammatory effects. Osteogenic changes induced by inflammation in valve interstitial cells (VICs) play a key role in the development of calcified aortic valve disease (CAVD). This study aimed to investigate the anti-calcification effects of ATL on aortic valves. Human VICs (hVICs) were exposed to osteogenic induction medium (OM) containing ATL to investigate cell viability, osteogenic gene and protein expression, and anti-calcification effects. Gas chromatography-mass spectroscopy (GC-MS) metabolomics analysis was used to detect changes in the metabolites of hVICs stimulated with OM before and after ATL administration. The compound-reaction-enzyme-gene network was used to identify drug targets. Gene interference was used to verify the targets. ApoE-/- mice fed a high-fat (HF) diet were used to evaluate the inhibition of aortic valve calcification by ATL. Treatment with 20 µM ATL in OM prevented calcified nodule accumulation and decreases in the gene and protein expression levels of ALP, RUNX2, and IL-1ß. Differential metabolite analysis showed that D-mannose was highly associated with the anti-calcification effect of ATL. The addition of D-mannose prevented calcified nodule accumulation and inhibited succinate-mediated HIF-1α activation and IL-1ß production. The target of ATL was identified as GLA. Silencing of the GLA gene (si-GLA) reversed the anti-osteogenic differentiation of ATL. In vivo, ATL ameliorated aortic valve calcification by preventing decreases in GLA expression and the up-regulation of IL-1ß expression synchronously. In conclusion, ATL is a potential drug for the treatment of CAVD by targeting GLA to regulate D-mannose metabolism, thereby inhibiting succinate-mediated HIF-1α activation and IL-1ß production.

The role of the gut microbiota and probiotics associated with microbial metabolisms in cancer prevention and therapy.

Cancer is the second leading cause of elevated mortality worldwide. Thus, the development of drugs and treatments is needed to enhance the survival rate of the cancer-affected population. Recently, gut microbiota research in the healthy development of the human body has garnered widespread attention. Many reports indicate that changes in the gut microbiota are strongly associated with chronic inflammation-related diseases, including colitis, liver disease, and cancer within the intestine and the extraintestinal tract. Different gut bacteria are vital in the occurrence and development of tumors within the gut and extraintestinal tract. The human gut microbiome has significant implications for human physiology, including metabolism, nutrient absorption, and immune function. Moreover, diet and lifestyle habits are involved in the evolution of the human microbiome throughout the lifetime of the host and are involved in drug metabolism. Probiotics are a functional food with a protective role in cancer development in animal models. Probiotics alter the gut microbiota in the host; thus, beneficial bacterial activity is stimulated, and detrimental activity is inhibited. Clinical applications have revealed that some probiotic strains could reduce the occurrence of postoperative inflammation among cancer patients. An association network was constructed by analyzing the previous literature to explore the role of probiotics from the anti-tumor perspective. Therefore, it provides direction and insights for research on tumor treatment.

Atractylenolide-1 Targets FLT3 to Regulate PI3K/AKT/HIF1-α Pathway to Inhibit Osteogenic Differentiation of Human Valve Interstitial Cells.

Atractylenolide-1 (AT-1), a natural active ingredient extracted from Atractylodes macrocephala, was reported to have good anti-fibrotic and anti-inflammatory effects. Osteogenic changes induced by the inflammation of valve interstitial cells (VICs) play a role in the development of calcified aortic valve disease (CAVD). This study aimed to investigate the anti-osteogenic effects of AT-1 in human VICs. Human VICs were exposed to osteogenic induction medium (OM) containing AT-1 to analyze cell viability, as well as protein and osteogenic gene expression. Anti-calcification tests were also performed. mRNA transcriptome sequencing was performed to identify differential genes and pathways regulated by AT-1. Western blotting was used to verify the enrichment pathway, protein-protein interaction (PPI) analysis was conducted to identify drug targets. Finally, molecular docking and inhibitors are used to verify the drug targets. Treatment of VICs with 20 µM AT-1 resulted in no significant cytotoxicity. The addition of AT-1 to OM prevented the accumulation of calcified nodules, and decreases in the level of (Alkaline Phosphatase) ALP and RUNX2 gene and protein expression were observed. Atractylenolide-1 can target FLT3 protein and inhibit the phosphorylation of FLT3, thereby blocking PI3K/AKT pathway activation, reducing the production of Hypoxia inducible factor(HIF)1-α, and inhibiting the osteogenic differentiation of VICs. These results suggest AT-1 as a potential drug for treating calcified aortic valve disease.

Preventive effect of Atractylodis Rhizoma extract on DSS-induced acute ulcerative colitis through the regulation of the MAPK/NF-κB signals in vivo and in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Atractylodes lancea (Thunb.) DC. is traditionally used as a folk medicine for treating gastrointestinal diseases in China. Nevertheless, the effect and mechanisms of its anti-inflammatory activity on dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) have not yet been fully investigated. AIM OF THE STUDY: This study aimed to explore the therapeutic effect and underlying molecular mechanisms of Ethanolic Extract of Atractylodis Rhizoma (EEAR) on DSS-induced UC mice and LPS-induced RAW264.7 cells. MATERIALS AND METHODS: The EEAR was obtained and then analyzed by HPLC analysis. The protective effect of EEAR on DSS-induced UC was evaluated by weight loss, disease activity index (DAI) score, spleen index, goblet cell loss, colon length shortening, myeloperoxidase (MPO) activity and pathological changes. The level of inflammatory cytokines were detected by immunohistochemistry (IHC) and RT-PCR analysis. The expressions of the tight junction (TJ, such as ZO-1, Occludin) proteins and the target proteins in mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) were determined by western blotting analysis. RESULTS: EEAR significantly attenuated the symptoms of UC, suppressed the colon MPO activity, and increased the goblet cell loss. In addition, EEAR could significantly increase the expression of TJs in UC mice. Meanwhile, EEAR treatment could reduce the levels of inflammatory cytokines and inhibit the phosphorylation of MAPK and NF-κB signaling pathways in UC mice and in LPS-induced RAW264.7 cells. CONCLUSION: Our results indicated that EEAR ameliorated DSS-induced UC by inhibiting the inflammatory response and maintaining the intestinal barrier function via modulation of MAPK/NF-κB pathways, thus, EEAR might be a promising therapeutic candidate for UC therapy.

Daidzin inhibits hepatocellular carcinoma survival by interfering with the glycolytic/gluconeogenic pathway through downregulation of TPI1.

Daidzin (DDZ) is a natural brassin-like compound extracted from the soybean, and has been found to have therapeutic potential against tumors in recent years. This study investigates the therapeutic effect of DDZ on hepatocellular carcinoma cells and elucidates the possible mechanisms of action. The viability of HCCLM3 and Hep3B cells was detected by MTT assay. Western blots and qPCR were used to detect the protein and mRNA levels of proliferation and apoptosis related genes. Gas chromatography-mass spectrometry (GC-MS) was used for metabolome analysis. In vivo antitumor effects were assessed in nude mice engrafted with HCC cell lines. Our results show that DDZ treatment dose-dependently inhibited cell viability, migration, and survival. The expressions of CDK1, BCL2, MYC, and survivin were reduced, while the expressions of BAX and PARP were increased in DDZ treated cells. The differentially expressed metabolites detected in DDZ treated cultures are associated with glycolysis/gluconeogenesis pathways. Bioinformatic analysis identified TPI1, a gene in the glycolysis pathway with prognostic value for hepatocellular carcinoma (HCC), and DDZ treatment downregulated this gene. In vivo experiments show that DDZ significantly reduced the tumor volume and weight, and inhibited Ki67 expression within tumors. This study shows that DDZ interfered with the survival and migration of hepatocellular carcinoma cells, likely via TPI1 and the gluconeogenesis pathway.

Atractylenolide-1 targets SPHK1 and B4GALT2 to regulate intestinal metabolism and flora composition to improve inflammation in mice with colitis.

BACKGROUND: Atractylenolide-1, an active component of Atractylodes Lancea, which is widely used to improve the gastrointestinal function. However, the efficacy and mechanism remain unclear in treating ulcerative colitis (UC). PURPOSE: This study aimed to investigate the efficacy and the underlying mechanism of atractylenolide-1in UC. METHODS: A dextran sulfate sodium (DSS)-induced UC mouse model was used to investigate the efficacy of atractylenolide-1. 16S DNA sequencing, GC-MS technique and transcriptome sequencing were used to detect the composition of mouse intestinal flora, the changes of metabolites and gene expression in mouse intestine. Compound-reaction-enzyme-gene network was used to find drug targets. Recombinant plasmid overexpression was used to verify drug targets in DSS mouse models. RESULTS: The results showed that Atractylenolide-1 could significantly improve weight loss, diarrhea, blood in the stool, shortening of the colon, the loss of colonic goblet cells, reduction in mucoprotein MUC2, and tight junction proteins (zo-1, occludin) in mice with colitis. It reduced the inflammatory factors TNF-α, IL-6, IL-1ß as well. The 16S sequencing showed that Atractylenolide-1 regulated the diversity and abundance of the intestinal flora in mice with colitis, and the analysis of flora enrichment indicated that the regulation of intestinal flora by atractylenolide-1 may be related to the regulation of metabolism. Correlation analysis of metabolomics and transcriptome showed that two genes SPHK1 and B4GALT2 related to the metabolism of fructose and galactose were regulated by atractylenolide-1. Further verification showed that atractylenolide-1 significantly inhibited the aberrance of SPHK1 and B4GALT2 in the colon with colitis. Meanwhile, it inhibited the activation of the PI3K-AKT pathway. SPHK1 and B4GALT2 overexpressing reversed the therapeutic effect of atractylenolide-1 in mice with colitis. CONCLUSION: Atractylenolide-1 is a potential drug for the treatment of colitis by suppressing inflammation via the SPHK1/PI3K/AKT axis and by targeting SPHK1 and B4GAT2 to regulate fructose/galactose-related metabolism, thereby regulating the composition of the intestinal flora.

Atractylodes lancea Rhizoma Attenuates DSS-Induced Colitis by Regulating Intestinal Flora and Metabolites.

Atractylodes lancea (Thunb.) DC. is a herb widely used traditionally for the treatment of gastrointestinal diseases such as gastric ulcer, spleen deficiency, and diarrhea. In China, people fry raw A. lancea (SCZ) together with wheat bran to make bran-fried A. lancea (FCZ). Ancient Chinese texts have documented that FCZ can enhance the function of regulating the intestines and stomach. Nevertheless, the effect and mechanism of SCZ and FCZ on ulcerative colitis (UC) are still unclear. The aim of this study was to compare the therapeutic effects of SCZ and FCZ and their mechanisms on dextran sulfate sodium (DSS)-induced UC in mice. The chemical constituents of SCZ and FCZ were analyzed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with six reference compounds. The effects of SCZ and FCZ were investigated based on their effects on weight loss, disease activity index (DAI) score, colon length shortening, goblet cell loss, and pathological changes using the colons from a mouse model of DSS-induced UC. The effects of SCZ and FCZ on levels of the inflammatory cytokines (tumor necrosis factor-[Formula: see text], interleukin-6, interleukin-1[Formula: see text], mucoprotein (MUC2), tight protein (ZO-1, occludin), and the activation of macrophages were determined using immunohistochemistry (IHC) and immunofluorescence (IF). 16s RNA sequencing technology was used to detect the composition of the intestinal flora in each group. Nontargeted metabonomics was used to detect the serum metabolite levels of mice in each group. Pearson analysis was used to determine the correlation between the intestinal flora, metabolites, and pathological indices. Reverse transcription-polymerase chain reaction was used to detect the genes of different metabolite-related enzymes. A pseudogerm free (PGF) mouse model was used to verify whether the effect of SCZ and FCZ in UC depends on the regulation of intestinal flora. SCZ and FCZ could inhibit weight loss and decrease the DAI score, colon length shortening, goblet cell loss, and the extent of pathological changes in the colons of mice with DSS-induced colitis. Moreover, SCZ and FCZ inhibited the decrease in MUC2, ZO-1, occludin, production of pro-inflammatory factors, and activation of pro-inflammatory macrophages in colonic tissue. The effect of FCZ was better than that of SCZ. SCZ and FCZ not only inhibited the abundance of harmful bacteria and increased the abundance of beneficial bacteria, but also regulated the metabolism of disease-related metabolites such as amino acid and cholesterol metabolism. Both preparations inhibited the gene expression (Slc6A7, PRODH, Sdsl, HMGCR, SREBP-2) of different metabolite-related enzymes. In the PGF mouse model, the above effects were not observed. Rhizoma Atractylodes was effective in alleviating DSS-induced UC in mice, and FCZ was found to be superior to SCZ. The mechanism of action of FCZ and SCZ is mainly related to the regulation of intestinal flora and their associated metabolites.

Cardamonin inhibits osteogenic differentiation of human valve interstitial cells and ameliorates aortic valve calcification via interfering in the NF-κB/NLRP3 inflammasome pathway.

Cardamonin (CDM) is a natural chalcone with strong anti-inflammatory properties. Inflammation-induced osteogenic changes in valve interstitial cells (VICs) play crucial roles in the development of calcific aortic valve disease (CAVD), a degenerative disease characterized by degeneration, thickening, fibrosis, and calcification of the heart valve tissues. To investigate the anti-osteogenic differentiation role of CDM in human valve interstitial cells (hVICs), which consequently reverses the calcification of the aortic valve, human VICs were exposed to osteogenic induction medium (OM) with CDM for further cell viability, osteogenic gene and protein expression analyses, and anti-calcification testing. mRNA sequencing was utilized to analyze the differentially expressed genes (DEGs) and related signaling pathways as potential molecular targets involved in CDM's anti-calcification activity. Human aortic valve leaflet ex vivo calcific cultures were used to investigate the CDM inhibition of osteogenic differentiation of hVICs at the tissue level. ApoE-/- mice fed with a high-fat (HF) diet were used to evaluate the effect of CDM on aortic valve calcification. No significant CDM cytotoxicity was seen in the hVICs at 10 µM. The addition of CDM to OM prevented calcified nodule accumulation, and a decrease in the gene/protein expression levels of BMP2, RUNX2, SPP1, TNF-α, and COL1A2 was observed. Venn diagram analysis of the DEGs identified 666 common DEGs and highlighted the NOD-like receptor signaling pathway (ko04621) as an anti-calcification target of CDM. CDM also repressed the activation of p-AKT, p-ERK1/2, and p-IκBα, and prevented the OM-induced nuclear transcription of NF-κB p65. In the in vitro and ex vivo calcific conditional culture experiments, CDM exhibited anti-inflammatory and anti-calcification effects by suppressing the activation of the NLRP3 inflammasome and downregulating IL-1ß expression. In vivo, CDM ameliorated aortic valve calcification by interfering with NLRP3 expression. Our study demonstrated that CDM inhibited the phenotypical calcific transformation of hVICs by mediating the inactivation of the NF-κB/NLRP3 inflammasome. Therefore, it is considered to be a promising natural compound for use in preventing the progression of heart valve calcification disease.

Atractylodin Attenuates Dextran Sulfate Sodium-Induced Colitis by Alleviating Gut Microbiota Dysbiosis and Inhibiting Inflammatory Response Through the MAPK Pathway.

In this study, we investigated the therapeutic effects and mechanism of atractylodin (ATL) on dextran sulfate sodium (DSS)-induced ulcerative colitis in mice. We found that atractylodin could significantly reverse the effects of DSS-induced ulcerative colitis, such as weight loss, disease activity index score; shorten the colon length, and reverse the pathological changes in the colon of mice. Atractylodin could inhibit the activation of colonic macrophages by inhibiting the MAPK pathway and alleviate intestinal inflammation in the mouse model of ulcerative colitis. Moreover, it could protect the intestinal barrier by inhibiting the decrease of the tight junction proteins, ZO-1, occludin, and MUC2. Additionally, atractylodin could decrease the abundance of harmful bacteria and increase that of beneficial bacteria in the intestinal tract of mice, effectively improving the intestinal microecology. In an LPS-induced macrophage model, atractylodin could inhibit the MAPK pathway and expression of the inflammatory factors of macrophages. Atractylodin could also inhibit the production of lactate, which is the end product of glycolysis; inhibit the activity of GAPDH, which is an important rate-limiting enzyme in glycolysis; inhibit the malonylation of GAPDH, and, thus, inhibit the translation of TNF-α. Therefore, ours is the first study to highlight the potential of atractylodin in the treatment of ulcerative colitis and reveal its possible mechanism.

Identification of different bran-fried Atractylodis Rhizoma and prediction of atractylodin content based on multivariate data mining combined with intelligent color recognition and near-infrared spectroscopy.

Unclear established standard of bran-fried Atractylodis Rhizoma (BFAR), a commonly used drug in Traditional Chinese Medicine (TCM), compromised its clinical efficacy. In this study, we explored the correlation between color and near-infrared spectroscopy (NIR) feature with content of atractylodin, then established a rapid recognition model for the optimal degree of processing for BFAR preparation. The results of the Pearson analysis indicated that the color values were significantly and positively correlated with atractylodin content. The back propagation artificial neural network algorithm and cluster analysis revealed the color of different BFAR could be accurately divided into three categories; subsequently, the color range for the optimal degrees of stir-frying was established as follows: R[red value (105.79-127.25)], G[green value(75.84-89.64)], B[blue value(33.33-42.73)], L[Lightness (81.26-95.09)].Using NIR, principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and cluster analysis, three types of BFAR were accurately identified. The prediction model of atractylodin content was established using partial least squares regression analysis. The R2 of the validation set was 0.9717 and the root mean square error was 0.026. In the color judgment model, the processing degree of 8 batches of BFAR from the market is inferior. According to the NIR judgment model, the processing degree of all samples from the market is inferior. In conclusion, the best fire degree of BFAR can be identified quickly and accurately based on our established model. It is a potential method for quality evaluation of Chinese Materia Medica processing.

Treatment of Spleen-Deficiency Syndrome With Atractyloside A From Bran-Processed Atractylodes lancea by Protection of the Intestinal Mucosal Barrier.

Atractylodes lancea (Thunb.) DC. (AL) is used in traditional Chinese medicine for the treatment of spleen-deficiency syndrome (SDS). Bran-processed Atractylodes lancea (BAL) has been found to be more effective than unprocessed AL. However, the compound in BAL active against SDS remains unclear. The pharmacological efficacy of BAL and its mechanism of action against SDS were investigated by HPLC-ELSD. Candidate compound AA (atractyloside A) in AL and BAL extracts was identified by HPLC-MS analysis. AA was tested in a rat model of SDS in which body weight, gastric residual rate, and intestinal propulsion were measured, and motilin (MTL), gastrin (GAS), and c-Kit were quantified by enzyme-linked immunosorbent assay. Potential targets and associated pathways were identified based on network pharmacology analysis. mRNA expression levels were measured by qRT-PCR and protein expression levels were measured by Western blot analysis and immunohistochemistry. AA increased body weight, intestinal propulsion, MTL, GAS, and c-Kit levels, while decreasing gastric residual volume and intestinal tissue damage, as same as Epidermal Growth Factor Receptor and Proliferating Cell Nuclear Antigen levels. Seventy-one potential pharmacologic targets were identified. Analysis of protein interaction, Gene Ontology (GO) functional analysis, pathway enrichment analysis, and docking and molecular interactions highlighted MAPK signaling as the potential signal transduction pathway. Validation experiments indicated that treatment with AA increased MTL, GAS, ZO-1, and OCLN levels, while reducing AQP1, AQP3, and FGF2 levels. In addition, phosphorylation of p38 and myosin light-chain kinase (MLCK) expression were inhibited. AA improved gastrointestinal function by protecting the intestinal mucosal barrier via inhibition of the p38 MAPK pathway. The results have clinical implications for the therapy of SDS.

Deep-Fried Atractylodis Rhizoma Protects against Spleen Deficiency-Induced Diarrhea through Regulating Intestinal Inflammatory Response and Gut Microbiota.

According to the theories of traditional Chinese medicine, spleen deficiency often leads to diarrhea, and deep-fried Atractylodis Rhizoma (DAR) is commonly used for the treatment. However, the association between spleen deficiency and diarrhea remains unclear. The present study aimed to investigate the therapeutic effect of DAR for the treatment of diarrhea caused by spleen deficiency and analyze the related mechanisms. It was found that a high dose group of an ethanolic extract of deep-fried Atractylodis Rhizoma (EEDAR-H) significantly inhibited weight loss, diarrhea, and pathological changes in colon tissue induced by rhubarb. EEDAR-H was found to significantly reduce the level of intestinal inflammatory cytokines and increase the expression of gastrointestinal motility hormones. In addition, EEDAR-H significantly increased the expression of aquaporin 3 (AQP3) and aquaporin 8 (AQP8) and restored abnormal water metabolism; Shen-Ling-Bai-Zhu-San (SLBZS) induced the same effect as EEDAR-H. Additional tests on the mechanism found that EEDAR-H and SLBZS promoted the integrity of the intestinal barrier. Both significantly increased the expression of the tight junction protein ZO-1 and Occludin, inhibited the phosphorylation of p38MAPK and MLC, and significantly reduced the expression levels of PAR-2. Analysis of the gut microbiota indicated that overall changes in its structure were reversed after treatment with EEDAR-H or SLBZS, in addition to significant modulation of the abundance of different phyla. At the genus level, EEDAR-H or SLBZS significantly reduced the levels of potential pathogens and increased those of beneficial bacteria.

[Study on dryness effect of Atractylodis Rhizoma based on theory of dry-dry and dryness-induced Yin deficiency].

In order to establish a more perfect evaluation system for dryness effect of Atractylodis Rhizoma, determine the main dry parts of Atractylodis Rhizoma，and further define the mechanism of stir-baked Atractylodis Rhizoma in reducing the dryness. The healthy rats were given with different doses of water extract and volatile oil of raw Atractylodis Rhizoma and stir-baked Atractylodis Rhizoma for 21 days. Based on the theory of the dry-dry and dryness-induced Yin deficiency, the amount of drinking water, tissue morphology of submandibular glands, urine volume and the expression of aquaporin 2 (AQP2) in the kidneys, as well as blood rheology, ratio of cAMP/cGMP in serum and the content of Na⁺-K⁺-ATP enzyme were selected as the evaluation indexes. The results indicated that the rats with high dose volatile oil from raw Atractylodis Rhizoma had a significant increase in the amount of drinking water, urine volume, blood viscosity, ratio of cAMP/cGMP and content of Na⁺-K⁺-ATP enzyme in the serum（P<0.05）as compared with the soybean oil group; meanwhile, atrophy of submandibular acinar gland was obvious，and the expression of aquaporin 2 was reduced significantly（P<0.05）. There were significant differences between volatile oil high dose group of raw Atractylodis Rhizoma and volatile oil high dose group of stir-baked Atractylodis Rhizoma. There was no significant difference between the water extract groups of raw and stir-baked Atractylodis Rhizoma and the saline group. A comprehensive evaluation system for the dryness effect of Atractylodis Rhizoma was established. It was confirmed that the volatile oil part was the main dry part of Atractylodis Rhizoma. It revealed that the mechanism of dryness effect of Atractylodis Rhizoma was not only related to the decrease of the total content of the volatile oil, but also may be related to the transformation of dryness components in the volatile oil. It provides references for the study of material basis of Atractylodis Rhizoma dryness, provides an experimental basis for the clinical application of Atractylodis Rhizoma, further clarifies the mechanism of stir-baked Atractylodis Rhizoma in reducing the dryness, and provides thoughts for the evaluation of other dry traditional Chinese medicines.