Synergistic and attenuated effects and molecular biological mechanisms of Shouhui Tongbian capsule in the treatment of slow transit constipation based on UPLC-MS/MS, network pharmacology and animal experimental validation.

BACKGROUND: Shouhui Tongbian capsule (SHTB) has been widely used for the treatment of constipation. There are few studies on SHTB at present. The current study aimed to explore the effects of multi-components compatibility of SHTB for efficacy enhancement and toxicity reduction and evaluate its molecular biological mechanisms in the treatment of slow transit constipation (STC). METHODS: Ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to quantify 17 anthraquinone components in different compatible systems of SHTB. Network pharmacological analysis was used to probe the potential mechanisms of SHTB in treating STC. In addition, an animal experiment combined with western blot analysis was performed to further validate the predicted results. RESULTS: After compatibility, the dissolution of 13 components with good effects in treating constipation increased, while the dissolution of 3 components with hepatotoxicity decreased. Overall, 145 common targets of 13 synergistic components and constipation were identified. A synergistic component-target-disease network showed that chrysoobtusin, obtusifolin, emodin, obtusin and 2-hydroxyl emodin-1-methyl ether were the potential key synergistic components. A protein-protein interaction network analysis identified 91 targets, and an analysis of topological characteristics was conducted to confirm the core targets. Gene Ontology function revealed that the 13 synergistic components for the treatment of STC mainly played roles via protein phosphorylation, positive regulation of phosphorylation, phosphotransferase activity, kinase activity and protein kinase activity, and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that these components were enriched in pathways in cancer, MAPK signaling pathway, IL-17 signaling pathway, NF-κB signaling pathway, etc. The results of animal experimental validation showed that SHTB significantly reduced the expression levels of p-p38 and p-ERK proteins in the colon tissue of the STC rats. CONCLUSION: This study preliminarily demonstrated that efficacy enhancement and toxicity reduction of SHTB could be achieved after compatibility, which expounded the connotation of compatibility theory of traditional Chinese medicine from the perspective of chemical composition, reflecting the rationality and scientificity of compatibility theory. Meanwhile, the study also revealed the core targets and potential molecular biological mechanisms of SHTB in the treatment of STC, which may serve as a reference for subsequent studies and clinical applications of SHTB.

Network pharmacology and experimental verification to explore the anti-superficial thrombophlebitis mechanism of Mailuo shutong pill.

ETHNOPHARMACOLOGICAL RELEVANCE: Mailuo shutong pill (MLST) has been widely used in clinical treatment of superficial thrombotic phlebitis (STP). Nevertheless, the major active components of MLST and the mechanism of synergistic action have not been reported. AIM OF THE STUDY: The present study aimed to evaluate the improving effects and the underlying mechanism of MLST on mannitol-induced STP in rabbits. MATERIAL AND METHODS: In this study, Ultrahigh-performance liquid chromatography electrospray ionization quadrupole-exactive orbitrap mass spectrometry (UHPLC-ESI-Q-Exactive-Orbitrap-MS) was used to analyze and identify the chemical composition of MLST and the prototype components absorbed into the blood. Then, according to the prototype components in serum, the targets and mechanisms of MLST were explored by applying network pharmacology. The rabbit model of STP was established by injecting 20% mannitol into bilateral auricular vein. The pathological changes of rabbit ear tissues, inflammatory factors, coagulation function and hemorheology were detected. In addition, molecular docking verified the interaction between the main active ingredient and the key target. Finally, the PI3K/AKT pathway and its regulated downstream pathways were verified by Western blot. RESULTS: A total of 96 MLST components and 53 prototypical components absorbed into the blood were successfully identified. Based on network pharmacology, PI3K/AKT pathway and 10 chemical components closely related to this pathway were obtained. Hematoxylin-eosin (HE) staining results indicated that MLST effectively improved of the pathological damage of ear tissues. MLST decreased levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6 and C-reactive protein (CRP). The expression of platelets (PLT) and fibrinogen concentration (FIB) was decreased, while prothrombin time (PT) and activated partial thromboplastin time (APTT) were prolonged. In addition, the plasma viscosity and whole blood viscosity in the MLST groups were significantly decreased. The more important discovery was that the expressions of P-PI3K, VEGF, P-AKT, P-IκB-α, P-NF-κB, NLRP3, ASC, Cleaved IL-1ß and Cleaved Caspase-1 were effectively reversed after treatment with MLST. CONCLUSIONS: This study comprehensively analyzed and characterized the chemical composition of MLST and the prototypical components absorbed into the blood. This study strongly confirmed the pharmacodynamic effect of MLST on STP. More importantly, this pharmacodynamic effect was achieved through inhibition of the PI3K/AKT pathway and its regulated NF-κB and NLRP3 pathways.

Investigation of anti-aging and anti-infection properties of Jingfang Granules using the Caenorhabditis elegans model.

Jingfang Granule (JFG), a traditional Chinese medicine, is frequently employed in clinical settings for the treatment of infectious diseases. Nevertheless, the anti-aging and anti-infection effects of JFG remain uncertain. In the present study, these effects were evaluated using the Caenorhabditis elegans (C. elegans) N2 as a model organism. The results demonstrated that JFG significantly increased the median lifespan of C. elegans by 31.2% at a dosage of 10 mg/mL, without any discernible adverse effects, such as alterations in the pharyngeal pumping rate or nematode motility. Moreover, JFG notably increased oviposition by 11.3%. Subsequent investigations revealed that JFG enhanced oxidative stress resistance in C. elegans by reducing reactive oxygen species levels and significantly improved survival rates in nematodes infected with Pseudomonas aeruginosa ATCC 9027. These findings suggest that JFG delays reproductive senescence in C. elegans and protects them from oxidative stress, thereby extending their lifespan. Additionally, JFG improves the survival of P. aeruginosa-infected nematodes. Consequently, JFG has potential as a candidate for the development of anti-aging and anti-infection functional medicines.

[Antidepressant mechanism of Shenling Kaixin Granules based on BDNF/TrkB/CREB pathway].

To investigate the antidepressant mechanism of Shenling Kaixin Granules(SLKX) in treating chronic unpredictable mild stress(CUMS) model rats. Ninety male SD rats were randomly divided into control group, model group, Shugan Jieyu Capsules(110 mg·kg~(-1)) group and SLKX low-(90 mg·kg~(-1)), medium-(180 mg·kg~(-1)), and high-dose(360 mg·kg~(-1)) groups. Depression rat model was replicated by CUMS method. After treatment, the behavioral changes of rats were evaluated by sugar preference, open field, elevated cross maze and forced swimming experiments. The contents of interleukin 1 beta(IL-1ß), tumor necrosis factor α(TNF-α), brain-derived neurotrophic factor(BDNF) and 5-hydroxytryptamine(5-HT) in serum were determined by enzyme linked immunosorbent assay(ELISA), and the activities of superoxide dismutase(SOD) and catalase(CAT) in hippocampal CA1 region were also detected. Pathological changes in hippocampal CA1 region were detected by hematoxylin-eosin(HE) staining, and Western blot was used to determine the expression of nerve growth factor(NGF), BDNF, phospho-tyrosine kinase receptor(p-TrkB)/TrkB, phospho-cAMP-response element binding protein(p-CREB)/CREB, nuclear factor E2 related factor 2(Nrf2), heme oxygenase 1(HO-1), B-cell lymphoma-2(Bcl-2)/Bcl-2 associated X protein(Bax) and caspase-3 in hippocampal CA1 region. RESULTS:: showed that compared with the control group, the model group had decreased sugar preference, reduced number of entries and time spent in the center of open field and shortened total distance of movement, reduced number of entries and proportion of time spent in open arm, and increased number and time of immobility in forced swimming experiment. Additionally, the serum contents of IL-1ß and TNF-α and the expression of caspase-3 were higher, while the contents of BDNF and 5-HT, the activities of SOD and CAT in hippocampal CA1 region, the expressions of NGF, BDNF, p-TrkB/TrkB, p-CREB/CREB, HO-1 and Bcl-2/Bax, and the Nrf2 nuclear translocation were lower in model group than in control group. Compared with the conditions in model group, the sugar preference, the number of entries and time spent in the center of open, total distance of movement, and the number of entries and proportion of time spent in open arm in treatment groups were increased while the number and time of immobility in forced swimming experiment were decreased; the serum contents of IL-1ß and TNF-α and the expression of caspase-3 were down regulated, while the contents of BDNF and 5-HT, the activities of SOD and CAT in hippocampal CA1 region, the expressions of NGF, BDNF, p-TrkB/TrkB, p-CREB/CREB, HO-1, Bcl-2/Bax, and Nrf2 nuclear translocation were enhanced. In conclusion, SLKX might regulate the Nrf2 nucleus translocation by activating BDNF/TrkB/CREB pathway, lower oxidative stress damage in hippocampus, inhibit caspase-3 activity, and reduce apoptosis of hippocampal nerve cells, thereby playing an antidepressant role.

[Potentiating effect and mechanism of extract of Jingfang Granules on activation of macrophages].

This study aimed to explore the potentiating effect and mechanism of the extract of Jingfang Granules(JFG) on the activation of macrophages. The RAW264.7 cells were treated with JFG extract and then stimulated by multiple agents. Subsequently, mRNA was extracted, and reverse transcription-polymerase chain reaction(RT-PCR) was used to measure the mRNA transcription of multiple cytokines in RAW264.7 cells. The levels of cytokines in the cell supernatant were detected by enzyme-linked immunosorbent assay(ELISA). In addition, the intracellular proteins were extracted and the activation of signaling pathways was determined by Western blot. The results showed that JFG extract alone could not promote or slightly promote the mRNA transcription of TNF-α, IL-6, IL-1ß, MIP-1α, MCP-1, CCL5, IP-10, and IFN-ß, and significantly enhance the mRNA transcription of these cytokines in RAW264.7 cells induced by R848 and CpG in a dose-dependent manner. Furthermore, JFG extract also potentiated the secretion of TNF-α, IL-6, MCP-1, and IFN-ß by RAW264.7 cells stimulated with R848 and CpG. As revealed by mechanism analysis, JFG extract enhanced the phosphorylation of p38, ERK1/2, IRF3, STAT1, and STAT3 in RAW264.7 cells induced by CpG. The findings of this study indicate that JFG extract can selectively potentiate the activation of macrophages induced by R848 and CpG, which may be attributed to the promotion of the activation of MAPKs, IRF3, and STAT1/3 signaling pathways.

[Neuroprotective effect of ginsenoside Re on drosophila model of Parkinson's disease].

This study aims to explore the neuroprotective mechanism of ginsenoside Re(GS-Re) on drosophila model of Parkinson's disease(PD) induced by rotenone(Rot). To be specific, Rot was used to induce PD in drosophilas. Then the drosophilas were grouped and respectively treated(GS-Re: 0.1, 0.4, 1.6 mmol·L~(-1); L-dopa: 80 µmol·L~(-1)). Life span and crawling ability of drosophilas were determined. The brain antioxidant activity [content of catalase(CAT), malondialdehyde(MDA), reactive oxygen species(ROS), superoxide dismutase(SOD)], dopamine(DA) content, and mitochondrial function [content of adenosine triphosphate(ATP), NADH:ubiquinone oxidoreductase subunit B8(NDUFB8) Ⅰ activity, succinate dehydrogenase complex, subunit B(SDHB) Ⅱ activity] were detected by enzyme-linked immunosorbent assay(ELISA). The number of DA neurons in the brains of drosophilas was measured with the immunofluorescence method. The levels of NDUFB8 Ⅰ, SDHB Ⅱ, cytochrome C(Cyt C), nuclear factor-E2-related factor 2(Nrf2), heme oxygenase-1(HO-1), B-cell lymphoma/leukemia 2(Bcl-2)/Bcl-2-assaciated X protein(Bax), and cleaved caspase-3/caspase-3 in the brain were detected by Western blot. The results showed that model group [475 µmol·L~(-1) Rot(IC_(50))] demonstrated significantly low survival rate, obvious dyskinesia, small number of neurons and low DA content in the brain, high ROS level and MDA content, low content of SOD and CAT, significantly low ATP content, NDUFB8 Ⅰ activity, and SDHB Ⅱ activity, significantly low expression of NDUFB8 Ⅰ, SDHB Ⅱ, and Bcl-2/Bax, large amount of Cyt C released from mitochondria to cytoplasm, low nuclear transfer of Nrf2, and significantly high expression of cleaved caspase-3/caspase-3 compared with the control group. GS-Re(0.1, 0.4, and 1.6 mmol·L~(-1)) significantly improved the survival rate of PD drosophilas, alleviated the dyskinesia, increased DA content, reduced the loss of DA neurons, ROS level, and MDA content in brain, improved content of SOD and CAT and antioxidant activity in brain, maintained mitochondrial homeostasis(significantly increased ATP content and activity of NDUFB8 Ⅰ and SDHB Ⅱ, significantly up-regulated expression of NDUFB8 Ⅰ, SDHB Ⅱ, and Bcl-2/Bax), significantly reduced the expression of Cyt C, increased the nuclear transfer of Nrf2, and down-regulated the expression of cleaved caspase-3/caspase-3. In conclusion, GS-Re can significantly relieve the Rot-induced cerebral neurotoxicity in drosophilas. The mechanism may be that GS-Re activates Keap1-Nrf2-ARE signaling pathway by maintaining mitochondrial homeostasis, improves antioxidant capacity of brain neurons, then inhibits mitochondria-mediated caspase-3 signaling pathway, and the apoptosis of neuronal cells, thereby exerting the neuroprotective effect.

Integrated fecal microbiota and metabolomics analysis of the orlistat intervention effect on polycystic ovary syndrome rats induced by letrozole combined with a high-fat diet.

BACKGROUND: This study aimed to compare the characteristics of the gut microbiota and their metabolite profiles between polycystic ovary syndrome (PCOS) and orlistat-treated PCOS rats (ORL-PCOS), which could help to better understand the underlying mechanism of the effect of orlistat on PCOS. METHODS: PCOS rat models were established using letrozole combined with a high-fat diet. Ten rats were randomly selected as a PCOS control group (PCOS). The other three groups (n = 10/group) were additionally supplemented with different doses of orlistat (low, medium, high). Then, fecal samples of the PCOS and ORL-PCOS groups were analysed by 16S rRNA gene sequencing and untargeted metabolomics. Blood samples were collected to detect serum sex hormones and lipids. RESULTS: The results showed that orlistat attenuated the body weight gain, decreased the levels of T, LH, the LH/FSH ratio, TC, TG and LDL-C; increased the level of E2; and improved estrous cycle disorder in PCOS rats. The bacterial richness and diversity of the gut microbiota in the ORL-PCOS group were higher than those in the PCOS group. The ratio of Firmicutes to Bacteroidetes was decreased with orlistat treatment. Moreover, orlistat treatment led to a significant decrease in the relative abundance of Ruminococcaceae and Lactobacillaceae, and increases in the abundances of Muribaculaceae and Bacteroidaceae. Metabolic analysis identified 216 differential fecal metabolites in total and 6 enriched KEGG pathways between the two groups, including steroid hormone biosynthesis, neuroactive ligand-receptor interaction and vitamin digestion and absorption. Steroid hormone biosynthesis was the pathway with the most significant enrichment. The correlations between the gut microbiota and differential metabolites were calculated, which may provide a basis for understanding the composition and function of microbial communities. CONCLUSIONS: Our data suggested that orlistat exerts a PCOS treatment effect, which may be mediated by modifying the structure and composition of the gut microbiota, as well as the metabolite profiles of PCOS rats.

Jingfang granules exert anti-psoriasis effect by targeting MAPK-mediated dendritic cell maturation and PPARγ-mediated keratinocytes cell cycle progression in vitro and in vivo.

BACKGROUND: Jingfang granules (JFG), derived from JingFangBaiDu San (JFBDS), are a traditional herbal formulas used for the treatment of respiratory tract infections. They were initially prescribed to treat skin disease, such as psoriasis in Chinese Taiwan, but are not widely used for psoriasis treatment in mainland China because of the lack of anti-psoriasis mechanism research. PURPOSES: The present study was designed to evaluate the anti-psoriasis effect of JFG and reveal the correlated mechanisms of JFG in vivo and in vitro using network pharmacology, UPLC-Q-TOF-MS technology and molecular biotechnology methods. RESULTS: An imiquimod-induced psoriasis-like murine model was used to verify the anti-psoriasis effect in vivo, with inhibition of lymphocytosis and CD3+CD19+B cell proliferation in the peripheral blood and prevention of the activation of CD4+IL17+T cells and CD11c+ MHC Ⅱ+ dendritic cells (DCs) in the spleen. Network pharmacology analysis demonstrated that the targets of the active components were significantly enriched in pathways involved in cancer, inflammatory bowel disease and rheumatoid arthritis, which were closely related to cell proliferation and immune regulation. The drug-component-target networks and molecular docking analysis demonstrated the active ingredients to be luteolin, naringin and 6'-feruloylnodakenin, which had a good binding affinity to PPARγ, p38a MAPK and TNF-a. Finally, UPLC-Q-TOF-MS analysis to validate the active ingredients in drug-containing serum and in vitro experiments showed that JFG inhibited the maturation and activation of BMDCs via the p38a MAPK signaling pathway and translocation of the agonist PPARγ into the nuclei to reduce the activity of NF-κB/STAT3 inflammatory signaling pathway in keratinocytes. CONCLUSIONS: Our study demonstrated that JFG improved psoriasis by inhibiting the maturation and activation of BMDCs and proliferation and inflammation of keratinocytes, which may facilitate the applications of JFG in anti-psoriasis therapy in clinical settings.

Jingfang Granule alleviates bleomycin-induced acute lung injury via CD200-CD200R immunoregulatory pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Jingfang granules (JF), one famous traditional Chinese formula in "She Sheng Zhong Miao Fang" written by Shi-Che Zhang during the Ming Dynasty era, has been widely used to prevent epidemic diseases in history and now was recommended for the treatment of coronavirus disease 2019 (COVID-19) in China. However, the roles of JF against acute lung injury and its mechanisms remain unclear. AIM OF THE STUDY: Acute lung injury (ALI) and its progressive acute respiratory distress syndrome (ARDS) are a continuum of lung inflammatory disease with high morbidity and mortality in clinic, especially in COVID-19 patients. The present study aims to investigate the effect of JF on ALI and clarify its underlying mechanisms for clinical application in COVID-19 control. METHODS: Bleomycin-induced ALI mice were given oral gavage daily for seven days with or without Jingfang granules (2, 4 g/kg). The body weight, lung wet/dry weight ratios, lung appearance and tissue histopathology were evaluated. Quantitative real-time PCR, biochemical bronchoalveolar lavage fluids analysis was used to determine the gene expression of proinflammation factor and infiltrated inflammatory cells in lung. Immunofluorescence image and western blot were used to detect the markers of alveolar macrophages (AMs), endothelial cell apoptosis and changes of CD200-CD200R pathway. RESULTS: Firstly, histopathological analysis showed that JF significantly attenuated pulmonary injury and inflammatory response in ALI mice. Then, cytokine detection, inflammatory cells assay, and JNKs and p38 pathway analysis indicated that the recruitment and activation of alveolar macrophages was the main reason to cause ALI and JF could reverse this variation. Next, immunofluorescence staining and TUNEL assay showed that JF upregulated the expression of CD200 and suppressed the apoptosis of alveolar endothelial cells. Finally, double immunofluorescence staining of CD200 and CD11c indicated that the seriously damaged tissue had the lower CD200 while more AMs infiltration, which was confirmed by RT-PCR analysis of CD200/CD200R. CONCLUSIONS: Jingfang granules can protect lung from acu te injury and mitigate the recruitment and overactive AMs-induced inflammation via CD200-CD200R immunoregulatory signal axis, which will provide an experimental basis for Jingfang granules clinical applications in COVID-19.

In vitro outcomes of quercetin on Candida albicans planktonic and biofilm cells and in vivo effects on vulvovaginal candidiasis. Evidences of its mechanisms of action.

BACKGROUND AND PURPOSE: Candida albicans is a fungus that produces common fungal infection in humans, including vulvovaginal candidiasis (VVC). While quercetin (QC) has potential antifungal activities against C. albicans, studies on the in vivo anti-VVC activity of QC are limited. This study evaluated the antifungal capacity of QC against cultured C. albicans strain SC5314 or in C. albicans-infected mice. METHODS: Microdilution and XTT reduction assay were used to determine the minimum inhibitory concentration (MIC) and biofilm formation of QC on C. albicans, respectively. Immunofluorescence was performed to detect the anti-invasive capacity of QC upon co-culturing C. albicans with VK2/E6E7 cells. The potential anti-VVC effects of QC were assessed in C. albicans-infected mice with VVC. Further, inflammatory cytokine levels were determined using ELISA. PAS and Papanicolaou staining were used to detect C. albicans cells and polymorphonuclear leukocytes (PMNs) in vaginal tissues. Western blotting and immunohistochemistry were performed to measure the expression of MAPK, ERK, JUN, and P38. RESULTS: MIC and minimal fungicidal concentration (MFC) of QC for C. albicans were 128 µM and > 512 µM, respectively. QC concentration lower than 128 µM (32-128 µM) could not inhibit C. albicans. QC (16 µM) notably inhibited C. albicans biofilm formation and suppressed the adhesion and invasion of C. albicans to VK2/E6E7 cells. In addition, the pharmacokinetic parameters of orally administered QC in mice showed rapid absorption (approximately 1 h) and slow elimination (approximately 6 h). Oral QC showed an effective protective function against C. albicans infection with no toxic effects a in mouse VVC model. QC significantly reduced IL-1α, TNF-α, IL-22 and IL-23 levels in vaginal lavage solution, inhibited invasive C. albicans and PMN infiltration in vaginal tissue, and effectively protected the integrity of vaginal mucosa. CONCLUSIONS: The present study showed that QC has rapid oral absorption, slow elimination, good viral distribution, and a lack of toxicity. QC not only inhibited biofilm formation, adhesion, and invasion of C. albicans in vitro, but also ameliorated C. albicans-induced inflammation and protected the integrity of the vaginal mucosa in vivo, suggesting that QC has the potential for the treatment of candidiasis.

[Anti-infectious pneumonia target discovery and molecular mechanism study of Jingfang Granules].

This study aimed to identify the direct pharmacological targets of Jingfang Granules in treating infectious pneumonia via "target fishing" strategy. Moreover, the molecular mechanism of Jingfang Granules in treating infectious pneumonia was also investigated based on target-related pharmacological signaling pathways. First, the Jingfang Granules extract-bound magnetic nanoparticles were prepared, which were incubated with lipopolysaccharide(LPS)-induced mouse pneumonia tissue lysates. The captured proteins were analyzed by high-resolution mass spectrometry(HRMS), and the target groups with specific binding to the Jingfang Granules extract were screened out. Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis was used to identify the target protein-associated signaling pathways. On this basis, the LPS-induced mouse model of infectious pneumonia was established. The possible biological functions of target proteins were verified by hematoxylin-eosin(HE) staining and immunohistochemical assay. A total of 186 Jingfang Granules-specific binding proteins were identified from lung tissues. KEGG pathway enrichment analysis showed that the target protein-associated signaling pathways mainly included Salmonella infection, vascular and pulmonary epithelial adherens junction, ribosomal viral replication, viral endocytosis, and fatty acid degradation. The target functions of Jingfang Granules were related to pulmonary inflammation and immunity, pulmonary energy metabolism, pulmonary microcirculation, and viral infection. Based on the in vivo inflammation model, Jingfang Granules significantly improved the alveolar structure of the LPS-induced mouse model of infectious pneumonia and down-regulated the expressions of tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6). Meanwhile, Jingfang Gra-nules significantly up-regulated the expressions of key proteins of mitochondrial function COX Ⅳ and ATP, microcirculation-related proteins CD31 and Occludin, and proteins associated with viral infection DDX21 and DDX3. These results suggest that Jingfang Gra-nules can inhibit lung inflammation, improve lung energy metabolism and pulmonary microcirculation, resist virus infection, thus playing a protective role in the lung. This study systematically explains the molecular mechanism of Jingfang Granules in the treatment of respiratory inflammation from the perspective of target-signaling pathway-pharmacological efficacy, thereby providing key information for clinical rational use of Jingfang Granules and expanding potential pharmacological application.

Shouhui Tongbian Capsules induce regression of inflammation to improve intestinal barrier in mice with constipation by targeted binding to Prkaa1: With no obvious toxicity.

Constipation arising from the poor bowel movement is a rife enteric health problem. Shouhui Tongbian Capsule (SHTB) is a traditional Chinese medicine (TCM) which effectively improve the symptoms of constipation. However, the mechanism has not been fully evaluated. The purpose of this study was to evaluate the effect of SHTB on the symptoms and intestinal barrier of mice with constipation. Our data showed that SHTB effectively improved the constipation induced by diphenoxylate, which was confirmed by shorter first defecation time, higher internal propulsion rate and fecal water content. Additionally, SHTB improved the intestinal barrier function, which was manifested by inhibiting the leakage of Evans blue in intestinal tissues and increasing the expression of occludin and ZO-1. SHTB inhibited NLRP3 inflammasome signaling pathway and TLR4/NF-κB signaling pathway, reduced the number of proinflammatory cell subsets and increased the number of immunosuppressive cell subsets to relieve inflammation. The photochemically induced reaction coupling system combined with cellular thermal shift assay and central carbon metabolomics technology confirmed that SHTB activated AMPKα through targeted binding to Prkaa1 to regulate Glycolysis/Gluconeogenesis and Pentose Phosphate Pathway, and finally inhibited intestinal inflammation. Finally, no obvious toxicity related to SHTB was found in a repeated drug administration toxicity test for consecutive 13 weeks. Collectively, we reported SHTB as a TCM targeting Prkaa1 for anti-inflammation to improve intestinal barrier in mice with constipation. These findings broaden our knowledge of Prkaa1 as a druggable target protein for inflammation inhibition, and open a new avenue to novel therapy strategy for constipation injury.

Dynamic changes in marker components during the stir-frying of Pharbitidis Semen, and network analysis of its potential effects on nephritis.

Introduction: Pharbitidis Semen (PS) has been widely used in traditional Chinese medicine to treat several diseases such as nephritis. PS is usually stir-fried to enhance its therapeutic efficacy before use in clinical practice. However, the changes in phenolic acids during stir-frying and the mechanisms of their therapeutic effects on nephritis are still unclear. Methods: Here, we studied the processing-induced chemical changes and elucidated the mechanism of PS in the treatment of nephritis. We determined the levels of the 7 phenolic acids in raw PS (RPS) and stir-fried PS (SPS) using high-performance liquid chromatography, analyzed the dynamic compositional changes during stir-frying, and used network analysis and molecular docking to predict and verify compound targets and pathways corresponding to nephritis. Results: The dynamic changes in the 7 phenolic acids in PS during stir-frying are suggestive of a transesterification reaction. Pathway analysis revealed that the targets of nephritis were mainly enriched in the AGE-RAGE, hypoxia-inducible factor-1, interleukin-17, and tumor necrosis factor signaling pathways among others. Molecular docking results showed that the 7 phenolic acids had good binding ability with the key nephritic targets. Discussion: The potential pharmaceutical basis, targets, and mechanisms of PS in treating nephritis were explored. Our findings provide a scientific basis for the clinical use of PS in treating nephritis.

[Anti-depression targets and mechanism study of Kaixin San].

This study identified the anti-depression targets of Kaixin San(KXS) in the brain tissue with "target fishing" strategy, and explored the target-associated pharmacological signaling pathways to reveal the anti-depression molecular mechanism of KXS. The Balb/c mouse model of depression was established by chronic unpredictable mild stress(CUMS) and the anti-depression effect of KXS was evaluated by forced swimming test and sucrose preference test. KXS active components were bonded to the benzophenone-modified magnetic nanoparticles by photocrosslinking reaction for capturing target proteins from cortex, thalamus and hippocampus of depressive mice. The target proteins were identified by liquid chromatography-mass spectrometry/mass spectrometry(LC-MS/MS). The enrichment analysis on signaling pathways was performed by Cytoscape. The potential biological functions of targets were verified by immunohistochemistry and Western blot assay. The results showed that KXS significantly improved the behavioral indexes. There were 64, 91, and 44 potential targets of KXS identified in cortex, thalamus, and hippocampus, respectively, according to the target identification experiment. The functions of these targets were mainly associated with vasopressin-regulated water reabsorption, salmonella infection, thyroid hormone synthesis, and other signaling pathways. Besides, the results of immunohistochemistry and Western blot showed that KXS up-regulated the expressions of argipressine(AVP) in the cortex, heat shock protein 60(HSP60), cytochrome C oxidase 4(COX4), and thyrotropin-releasing hormone(TRH) in the thalamus, and down-regulated the expressions of tumor necrosis factor-α(TNF-α) and nuclear factor kappa B(NF-κB) p65 in the thalamus. Therefore, KXS may exert anti-depression effect through regulating vasopressin signaling pathway in the cortex and inflammation, energy metabolism, and thyroid hormone signaling pathways in the thalamus, and the effect of KXS on hippocampus is not significant.

Jingfang Granules improve glucose metabolism disturbance and inflammation in mice with urticaria by up-regulating LKB1/AMPK/SIRT1 axis.

ETHNOPHARMACOLOGICAL RELEVANCE: Jingfang Granule (JFG) is a Traditional Chinese Medicine prescription to empirically treat skin disease such as urticaria in clinical practice. However, the potential mechanisms of JFG on urticaria are not fully defined. AIM OF STUDY: The aim of this study is to investigate the mechanisms of JFG in treating urticaria through an OVA/aluminum hydroxide induced urticaria mice model. MATERIALS AND METHODS: KM mice were injected intraperitoneally (i.p.) with OVA/aluminium hydroxide to establish the model with urticaria. After the mice were administered JFG, itching degree and hematoxylin and eosin (H&E) staining were used to assess the protective effect of JFG on mice with urticaria. The regulatory networks were investigated by proteomics and central carbon metabolomics. Spleen T lymphocyte subsets were detected by flow cytometry. Peripheral blood cytokines were detected using ELISA kits or Cytometric Bead Array (CBA) kits. The protein expression of skin tissue was detected by western blot or immunohistochemical staining. RESULTS: JFG significantly relived skin tissue lesions and skin pruritus in mice with urticaria. Meanwhile, JFG significantly decreased IgE, IL-1ß, IL-6, IL-4, TNF-α and IL-17A levels and increased IFN-γ levels in the serum of urticaria mice by inhibiting the expression of inflammation associated proteins including TLR4 and p-NF-κB p65, p-ERK1/2, p-JNK and p-p38, NLRP3, ASC and cleaved caspase-1. The results of proteomics, central carbon metabolomics, western blot and immunohistochemical staining confirmed that JFG inhibited Glycolysis/Gluconeogenesis and Pentose phosphate pathway in the skin tissue of urticaria mice by activating the LKB1/AMPK/SIRT1 axis and then downregulating the protein expressions of Glut1, TORC2, p-CREB, PEPCK, HNF4α and G6Pase. CONCLUSION: The current study demonstrates that JFG is effective in treating OVA/aluminum hydroxide-induced skin lesions and inflammation in mice, and JFG exhibits the clinical benefits via modulating LKB1/AMPK/SIRT1 axis, which in turn inhibits Glycolysis/Gluconeogenesis and Pentose phosphate pathway.

[Therapeutic effect of Jingfang Granules on CCl_4-induced liver fibrosis in mice and its mechanism].

To investigate the therapeutic effect of Jingfang Granules on carbon tetrachloride(CCl_4)-induced liver fibrosis in mice and its mechanism. Forty-nine 8-week-old male C57 BL/6 J mice were randomly divided into a blank group, a CCl_4 group, a silybin group(positive control, 100 mg·kg~(-1))+CCl_4, a Jingfang high-dose(16 g·kg~(-1)) group, a Jingfang high-dose(16 g·kg~(-1))+CCl_4 group, a Jingfang medium-dose(8 g·kg~(-1))+CCl_4 group, and a Jingfang low-dose(4 g·kg~(-1))+CCl_4 group, with 7 mice in each group. The mice in the blank group and Jingfang high-dose group were intraperitoneally injected olive oil solution, and mice in other groups were intraperitoneally injected with 10% CCl_4 olive oil solution(5 mL·kg~(-1)) to induce liver fibrosis, twice a week with an interval of 3 d, for 8 weeks. At the same time, except for the blank group and CCl_4 group, which were given deionized water, the mice in other groups were given the corresponding dose of drugs by gavage once daily for 8 weeks with the gavage volume of 10 mL·kg~(-1). All mice were fasted and freely drank for 12 h after the last administration, and then the eyeballs were removed for blood collection. The liver and spleen were collected, and the organ index was calculated. The levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), total bile acid(TBA), and triglyceride(TG) in the serum of mice were detected by an automated analyzer. Tumor necrosis factor-α(TNF-α), interleukin-6(IL-6) and interleukin-1ß(IL-1ß) levels were detected by enzyme-linked immunosorbent assay(ELISA). Kits were used to detect the contents of superoxide dismutase(SOD), malondialdehyde(MDA), and glutathione(GSH) in the liver tissue. Pathological changes in the liver tissue were observed by hematoxylin-eosin(HE), Masson, and Sirius red staining. Western blot was used to detect protein expressions of transforming growth factor-ß(TGF-ß), α-smooth muscle actin(α-SMA) and Smad4 in the liver tissue. The results indicated that Jingfang Granules significantly reduced the organ index, levels of ALT, AST, TBA,TG, TNF-α, IL-6, and IL-1ß in the serum, and the content of MDA in the liver tissue of mice with CCl_4-induced liver fibrosis. Jingfang Granules also significantly increased the content of SOD and GSH in the liver tissue. Meanwhile, Jingfang Granules down-regulated the protein levels of TGF-ß, α-SMA, and Smad4. Furthermore, Jingfang Granules had no significant effect on the liver tissue morphology and the above indexes in the normal mice. In conclusion, Jingfang Granules has obvious therapeutic effect on CCl_4-induced liver fibrosis, and its mechanism may be related to reducing the expression of pro-inflammatory factors, anti-oxidation, and regulating TGF-ß/Smad4 signaling pathway.

[Intervention effect of Jingfang Mixture on urticaria mice based on NF-κB/NLRP3/IL-1ß signaling pathway].

This study explored the curative effect of Jingfang Mixture on urticaria mice induced by aluminum hydroxide/ovalbumin, and discussed its mechanism. Sixty male Kunming mice were randomly divided into a normal group, a model group, three Jingfang Mixture(low-dose, medium-dose, and high-dose) groups, and a positive drug(cetirizine hydrochloride) group. The urticarial model in mice was induced by the intraperitoneal injection of the mixed solution of ovalbumin and aluminum hydroxide. The degrees of pruritus were observed after the second immunization. Pathological changes were detected by hematoxylin and eosin(HE) staining. Levels of interleukin 1ß(IL-1ß) and tumor necrosis factor α(TNF-α) in the serum were detected by enzyme linked immunosorbent assay(ELISA). Expressions of NOD-like receptor protein 3(NLRP3) and IL-1ß were detected by immunohistochemistry(IHC). Expressions of nuclear factor kappa-B(NF-κB p65), NLRP3, apoptosis-associated speck-like protein containing a CARD(ASC), cysteinyl aspartate-specific proteases 1(caspase-1), and IL-1ß proteins were detected by Western blot. The results showed that, except for the normal group, the mice in all groups had different degrees of pruritus. Compared with the model group, the Jingfang Mixture groups and the positive drug group prolonged the scratching latency of mice(P<0.05), and significantly reduced the number of scratching(P<0.05). In addition, the Jingfang Mixture groups and the positive drug group improved the pathological morphology of skin tissue. The expression levels of IL-1ß and TNF-α in serum were significantly reduced(P<0.05), and the number of NLRP3 and IL-1ß positive cells was decreased(P<0.01). The expressions of p-NF-κB p65, NLRP3, ASC, cleaved caspase-1, and IL-1ß protein were significantly down-regulated(P<0.05). The results of the above study indicate that Jingfang Mixture inhibit the inflammatory response in urticaria mice, and the mechanism may be related to the inhibition of activating NF-κB/NLRP3/IL-1ß signaling pathway.

[Jingfang Mixture regulates balance of spleen T lymphocyte subsets in urticaria mice by inhibiting JAK2-STAT3 signaling pathway].

Urticaria is an immune-mediated allergic disease. This study explored the effect of Jingfang Mixture on spleen T lymphocyte subsets of urticaria mice. A total of 50 Kunming mice were randomized into normal group(C), model group(V), and low-(JF-L, 0.5 g·kg~(-1)), medium-(JF-M, 1 g·kg~(-1)) and high-dose(JF-H, 2 g·kg~(-1)) Jingfang Mixture groups, with 10 mice in each group. The mixture of ovalbumin and aluminum hydroxide(0.1 mg + 0.1 mL) was used(intraperitoneal injection) to induce urticaria in mice. The administration began 6 days after the first immunization, and the second immunization was carried out 10 days after the first immunization. The pruritus index was detected within 30 min after the second immunization. The administration lasted 21 days. After 21 days, the serum was taken to detect the total IgE level. Based on hematoxylin and eosin(HE) staining, the pathological changes of skin tissue were observed, and Western blot was used to detect the levels of p-Janus kinase 2(JAK2)/JAK2 and p-signal transducer and activator of transcription 3(STAT3)/STAT3 in skin tissue. The spleen was taken to detect the spleen index, and flow cytometry was employed to determine the expression of lymphocyte subsets. The results showed that group V had obvious pathological changes in skin tissue compared with group C. Moreover, group V showed more scratches, higher spleen index, and higher level of total serum IgE than group C. In addition, higher levels of p-JAK2 and p-STAT3, lower proportions of CD4~+T, Th1, and Treg, higher proportions of CD8~+T, Th2, and Th17, and lower ratios of CD4~+/CD8~+, Th1/Th2, and Terg/Th17 were observed in group V than in group C. Compared with group V, each administration group showed alleviation of the pathological morphology of skin tissue, obvious epidermal thickening, relatively intact collagen fiber structure of dermal reticular layer, alleviated edema, and relief of vasodilation and peripheral inflammatory cell infiltration. Moreover, less scratching, lower spleen index, lower p-JAK2/JAK2 and p-STAT3/STAT3 were observed in the administration groups than in group V. JF-M group and JF-H group demonstrated lower levels of total IgE, larger proportions of CD4~+T, Th1, and Treg, smaller proportions of CD8~+ T, Th2, and Th17, and higher ratios of CD4~+/CD8~+, Th1/Th2, and Terg/Th17. In conclusion, Jingfang Mixture may improve the symptoms of urticaria mice by regulating the balance of spleen T lymphocyte subsets through JAK2-STAT3 signaling pathway.

Study on Integrated Pharmacokinetics of the Component-Based Chinese Medicine of Ginkgo biloba Leaves Based on Nanocrystalline Solid Dispersion Technology.

Background: To improve the dissolution and bioavailability of the component-based Chinese medicine of Ginkgo biloba leaves (GBCCM), a novel nanocrystalline solid dispersion of GBCCM (GBCCM NC-SD) was first prepared. Methods: GBCCM mainly containing high pure flavonoid aglycones (FAs) and terpenoid lactones (TLs) was used as the model drug. PVP K30 and SDS were used as solubilizers, combined stabilizers and carriers, and GBCCM NC-SD was prepared by high-pressure homogenization combined with freeze-dryer. Morphology and crystal characteristic of GBCCM NC-SD were analyzed. The dissolution and bioavailability evaluation were performed to investigate the feasibility of GBCCM NC-SD by in vitro dissolution and in vivo integrated pharmacokinetic models. Results: After homogenizing for 30 cycles under the pressure of 650 bar and freeze-drying, GBCCM NC-SD with uniform quality would be obtained. The particle size, PDI and zeta potential were found to be 335.9 ± 32.8 nm, 0.29 ± 0.02 and -28.4 ± 0.7 mV respectively. Based on charged aerosol detector (CAD) technology, a new chromatographic method for simultaneous detection of eight components in GBCCM was developed. In vitro drug release study showed that the cumulative dissolution of FAs and TLs in GBCCM NC-SD increased from 12.77% to 52.92% (P < 0.01) and 90.91% to 99.21% (P < 0.05) respectively. In comparison with physical mixture of GBCCM and stabilizer (PM), the integrated pharmacokinetics AUC0-t of FAs and TLs in GBCCM NC-SD were significantly increased (P < 0.05), and the T1/2 of TLs was also significantly prolonged (P < 0.05). Conclusion: This study demonstrated that novel GBCCM NC-SD was prepared using Polyvinylpyrrolidone K30 (PVP K30) and Sodium dodecyl sulfate (SDS) as a synergetic stabilizer and also provided a feasible way to improve the dissolution and oral bioavailability of poorly soluble candidate antihypertensive drugs.

[Anti-pneumonia targets and mechanism of Xiaoer Xiaoji Zhike Oral Liquid].

This study aims to identify the anti-pneumonia targets of Xiaoer Xiaoji Zhike Oral Liquid(XXZL) with "target fishing" strategy and investigate the related signaling pathways, thereby clarifying the anti-pneumonia mechanism of XXZL. To be specific, the magnetic nanoparticles cross-linked with XXZL extract were prepared based on the photochemical activity of benzophenone, which were then used to capture the target proteins from the lysate of tissue with lipopolysaccharide(LPS)-induced pneumonia in mice. Then, the target proteins were identified by liquid chromatography-tandem mass spectrometry(LC-MS/MS). The signaling pathways and interactions of target proteins were explored with KEGG and STRING analysis on Cytoscape, and the possible biological functions of the target proteins were verified by immunohistochemistry(IHC) and RT-PCR. The result showed that LC-MS/MS identified 62 potential anti-pneumonia targets of XXZL in the lungs. The targets were involved in Ras signaling pathway, mitophagy, leukocyte transendothelial migration, mitogen-activated protein kinase(MAPK) signaling pathway, platelet activation, and actomyosin structure organization, which were closely related to inflammation, pulmonary microcirculation, pulmonary fibrosis, and energy metabolism. XXZL up-regulated the content of CD31, and heat shock protein 60(HSP60) and ATP5 b mRNA expression, down-regulated interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), COL1 A1 content, and alleviated fibrosis, which suggested the obvious effects of XXZL such as anti-inflammation, pulmonary microcirculation improvement, pulmonary fibrosis inhibition, and energy metabolism regulation. This study explained the anti-pneumonia mechanism of XXZL from targets, which can serve as a reference for the clinical application of the prescription.