In vitro anti-Helicobacter pylori activity and the underlining mechanism of an empirical herbal formula - Hezi Qingyou.

Background: Helicobacter pylori (H. pylori) is thought to primarily colonize the human stomach and lead to various gastrointestinal disorders, such as gastritis and gastric cancer. Currently, main eradication treatment is triple or quadruple therapy centered on antibiotics. Due to antibiotic resistance, the eradication rate of H. pylori is decreasing gradually. Therefore, searching for anti-H. pylori drugs from herbal sources has become a strategy for the treatment. Our team proposed a Hezi Qingyou Formula (HZQYF), composed of Chebulae Fructus, Ficus hirta Vahl and Cloves, and studied its anti-H. pylori activity and mechanism. Methods: Chemical components of HZQYF were studied using UHPLC-MS/MS and HPLC. Broth microdilution method and agar dilution method were used to evaluate HZQYF's antibacterial activity. The effects of HZQYF on expression of adhesion genes (alpA, alpB, babA), urease genes (ureE, ureF), and flagellar genes (flaA, flaB) were explored using Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) technology. Effects on morphology and permeability of the extracellular membrane were studied using scanning electron microscopy (SEM) and N-phenylnaphthalen-1-amine (NPN) uptake. Effect on urease activity was studied using a urease kinetics analysis in vitro. Immunofluorescence staining method was used to examine the effect on adhesion. Western blot was used to examine the effect on cagA protein. Results: Minimum inhibitory concentration (MIC) values of the formula against H. pylori clinical strains and standard strains were 80-160 µg/mL, and minimum bactericidal concentration (MBC) values were 160-320 µg/mL. The formula could down-regulate the expression of adhesion genes (alpA, alpB, babA), urease genes (ureE, ureF) and flagellar genes (flaA, flaB), change the morphology of H. pylori, increase its extracellular membrane permeability, and decrease its urease activity. Conclusion: Present studies confirmed that HZQYF had promising in vitro anti-H. pylori activities and demonstrated its possible mechanism of action by down-regulating the bacterial adhesion, urease, and flagellar gene expression, which provided scientific bases for further clinical investigations.

1,3,6-Trigalloylglucose: A Novel Potent Anti-Helicobacter pylori Adhesion Agent Derived from Aqueous Extracts of Terminalia chebula Retz.

1,3,6-Trigalloylglucose is a natural compound that can be extracted from the aqueous extracts of ripe fruit of Terminalia chebula Retz, commonly known as "Haritaki". The potential anti-Helicobacter pylori (HP) activity of this compound has not been extensively studied or confirmed in scientific research. This compound was isolated using a semi-preparative liquid chromatography (LC) system and identified through Ultra-high-performance liquid chromatography-MS/MS (UPLC-MS/MS) and Nuclear Magnetic Resonance (NMR). Its role was evaluated using Minimum inhibitory concentration (MIC) assay and minimum bactericidal concentration (MBC) assay, scanning electron microscope (SEM), inhibiting kinetics curves, urea fast test, Cell Counting Kit-8 (CCK-8) assay, Western blot, and Griess Reagent System. Results showed that this compound effectively inhibits the growth of HP strain ATCC 700392, damages the HP structure, and suppresses the Cytotoxin-associated gene A (Cag A) protein, a crucial factor in HP infection. Importantly, it exhibits selective antimicrobial activity without impacting normal epithelial cells GES-1. In vitro studies have revealed that 1,3,6-Trigalloylglucose acts as an anti-adhesive agent, disrupting the adhesion of HP to host cells, a critical step in HP infection. These findings underscore the potential of 1,3,6-Trigalloylglucose as a targeted therapeutic agent against HP infections.

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.

Sanguisorba officinalis L. enhances the 5-fluorouracil sensitivity and overcomes chemoresistance in 5-fluorouracil-resistant colorectal cancer cells via Ras/MEK/ERK and PI3K/Akt pathways.

Sanguisorba officinalis L., a traditional Chinese medicine (TCM) called DiYu (DY) in China, has a strong tradition of utilization as a scorching, blood-cooling, and hemostatic medication, and was used for cancer prevention and treatment due to its potential immune-enhancing and hematological toxicity-reducing effects. Previous studies have reported significant effects of DY on cancers including colorectal cancer (CRC), which is one of the most common malignancies worldwide. The first-line cure 5-fluorouracil (5-FU) plays decisive commerce in the sedative of CRC as a clinically available chemotherapeutic agent. One of the primary causes of cancer treatment failure is the acquisition of chemotherapy drug resistance. In order to successfully combat the emergence of chemoresistance, it is essential to identify herbs or traditional Chinese medicine that have adjuvant therapeutic effects on CRC. Therefore, this study aimed to determine whether DY could improve the sensitivity, conquer the chemoresistance of 5-FU-resistant CRC cells, and investigate its intrinsic mechanism. Materials and methods: MTT, Hoechst 33258 staining, and flow cytometry assays were used to determine the anticancer activity of DY alone or in combination with 5-FU against 5-FU-resistant CRC cells (RKO-R and HCT15-R) and wound healing assays were conducted to detect cell migration. Transcriptomic techniques were carried out to explore the effect and mechanism of DY on drug-resistant CRC cells. Western Blot and RT q-PCR assays were performed to validate the mechanism by which DY overcomes drug-resistant CRC cells. Results: These results indicated that DY alone or in combination with 5-FU significantly inhibited the proliferation and the migration of resistant CRC cells, and potentiated the susceptibility of 5-FU to drug-resistant CRC cells. GO and KEGG enrichment analysis showed that the mechanisms of drug resistance in CRC cells and DY against drug-resistant CRC cells highly overlapped, involved in the modulation of biological processes such as cell migration, positive regulation of protein binding and cytoskeleton, and MAPK (Ras-ERK-MEK), PI3K/Akt, and other signaling pathways. Moreover, DY can mediate the expression of p-R-Ras, p-ERK1/2, p-MEK1/2, p-PI3K, p-AKT, HIF-1A and VEGFA proteins. In addition, DY significantly suppressed the expression of AKT3, NEDD9, BMI-1, and CXCL1 genes in resistant CRC cells. Conclusion: In conclusion, DY could inhibit the proliferation and migration of 5-FU-resistant cells and strengthen the sensitivity of 5-FU to CRC-resistant cells. Furthermore, DY may prevail over chemoresistance through the Ras/MEK/ERK and PI3K/Akt pathways. These findings imply that DY may be a potential drug for clinical treatment or adjuvant treatment of drug-resistant CRC.

[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.

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.

Integrating network pharmacology and experimental validation to decipher the mechanism of action of Jingfang Granule in the treatment of viral myocarditis.

This study investigated the mechanisms of Jingfang Granule (JFG) in viral myocarditis (VMC) treatment via network pharmacology-based approach combined with molecular docking and validated the results through in vitro and in vivo experiments. The chemical composition of JFG and its therapeutic targets was queried in Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. The targets related to VMC were retrieved from the disease database, and the overlapping targets were screened. Based on the STRING database, a protein-protein interaction network was constructed. Cytoscape software was used to construct the "component-target-disease" interaction network for visualization. GO and KEGG pathway enrichment analyses were performed using Metascape data. Molecular docking was performed using PyMoL2.3.0 and AutoDock Vina software programs. The target genes were further verified in vitro and in vivo. JFG contains 88 active components. The main biological targets of JFG in VMC include quercetin, luteolin, and kaempferol. The molecular docking results showed that the three key targets showed strong binding properties with both the height components of the molecular docking interaction energies. The results of experimental verification results showed that JFG may be used to treat VMC mainly by down-regulating inflammatory factors TNF-α and NF-κB and inhibiting myocardial apoptosis. The results support the network pharmacological data. JFG reduces myocardial inflammation and myocardial cell apoptosis in VMC and protects myocardial tissue.

Pairing with Enriched Sound Exposure Restores Auditory Processing Degraded by an Antidepressant.

Antidepressants, while effective in treating depression and anxiety disorders, also induce deficits in sensory (particularly auditory) processing, which in turn may exacerbate psychiatric symptoms. How antidepressants cause auditory signature deficits remains largely unknown. Here, we found that fluoxetine-treated adult female rats were significantly less accurate when performing a tone-frequency discrimination task compared with age-matched control rats. Their cortical neurons also responded less selectively to sound frequencies. The degraded behavioral and cortical processing was accompanied by decreased cortical perineuronal nets, particularly those wrapped around parvalbumin-expressing inhibitory interneurons. Furthermore, fluoxetine induced critical period-like plasticity in their already mature auditory cortices; therefore, a brief rearing of these drug-treated rats under an enriched acoustic environment renormalized auditory processing degraded by fluoxetine. The altered cortical expression of perineuronal nets was also reversed as a result of enriched sound exposure. These findings suggest that the adverse effects of antidepressants on auditory processing, possibly because of a reduction in intracortical inhibition, can be substantially alleviated by simply pairing drug treatment with passive, enriched sound exposure. They have important implications for understanding the neurobiological basis of antidepressant effects on hearing and for designing novel pharmacological treatment strategies for psychiatric disorders.SIGNIFICANCE STATEMENT Clinical experience suggests that antidepressants adversely affect sensory (particularly auditory) processing, which can exacerbate patients' psychiatric symptoms. Here, we show that the antidepressant fluoxetine reduces cortical inhibition in adult rats, leading to degraded behavioral and cortical spectral processing of sound. Importantly, fluoxetine induces a critical period-like state of plasticity in the mature cortex; therefore, a brief rearing under an enriched acoustic environment is sufficient to reverse the changes in auditory processing caused by the administration of fluoxetine. These results provide a putative neurobiological basis for the effects of antidepressants on hearing and indicate that antidepressant treatment combined with enriched sensory experiences could optimize clinical outcomes.

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.

[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.

[Anti-inflammatory mechanism of active ingredients in Jingfang Mixture based on network pharmacology and experimental verification].

The present study aimed to explore the regulatory targets and anti-inflammatory mechanism of Jingfang Mixture based on network pharmacology and animal tests. The active ingredients of Jingfang Mixture and the corresponding targets were screened out by the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). Inflammation-related targets were searched from GeneCards and DisGeNET, and the targets of active ingredients of Jingfang Mixture against inflammation were obtained. The protein-protein interaction(PPI) network was analyzed by STRING and plotted. Gene ontology(GO) function and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses were carried out based on DAVID. The results of network pharmacology showed 159 active ingredients and 276 targets of Jingfang Mixture and 664 inflammation-related targets were screened out, and 90 targets of active ingredients of Jingfang Mixture against inflammation were obtained. As revealed by the PPI network, protein kinase B1(AKT1), caspase-3(CASP3), interleukin-1ß(IL1 B), prostaglandin-endoperoxide synthase 2(PTGS2), and tumor necrosis factor(TNF) might be the key proteins for the anti-inflammatory effect of Jingfang Mixture. KEGG enrichment analysis demonstrated the pathways involved TNF, nuclear factor-kappa B(NF-κB), and mitogen-activated protein kinase(MAPK). The anti-inflammatory effect of Jingfang Mixture was explored through the mouse model of urticaria. The results indicated that Jingfang Mixture could down-regulate the phosphorylation levels of p38 MAPK, extracellular regulated protein kinases(ERK1/2), and NF-κB. The present study revealed the anti-inflammatory effect of Jingfang Mixture with multi-component and multi-target characteristics, which is expected to provide a scientific basis and important support for further research, development, and application.

[Mechanism of pathogenesis of Jingfang Mixture in intervention of chronic spontaneous urticaria based on serum metabolomics].

This study aims to clarify the effect of Jingfang Mixture on the treatment of chronic urticarial and its mechanism, and investigate the regulatory effect of chronic urticaria on the metabolic disorder of endogenous metabolites in the blood. The mice were randomly divided into normal group, model group, and Jingfang Mixture group, and modeling and administration continued for 21 d. The changes in endogenous small molecules in rat serum were determined by ultra-high performance liquid chromatography-electrospray ionization-Q Exactive-Orbitrap-mass spectrometry(UHPLC-ESI-QE-Orbitrap-MS) metabolomics technology. The change trend of endogenous metabolites in rat serum was analyzed to find potential biomarkers. The results showed that Jingfang Mixture regulate 16 biomarkers, mainly including taurine, glutamate, succinic acid, docosahexaenoic acid, and arachidonic acid. Metabolic pathway analysis was carried out by MetaboAnalyst, and P<0.01 was taken as the potential key metabolic pathway. Ten metabolic pathways were closely related to the treatment of chronic urticarial by Jingfang Mixture, mainly involved in the glutamate metabolism, taurine and hypotaurine metabolism, arginine and proline metabolism, arachidonic acid metabolism, tricarboxylic acid cycle, unsaturated fatty acid biosynthesis, glutathione metabolism, phenylalanine metabolism, alanine, aspartic acid, and glutamate metabolism, and butyric acid metabolism. Glutamate metabolism and butyric acid metabolism involved more metabolic pathways than others. Therefore, it was speculated that Jingfang Mixture had a balanced regulating effect on the related metabolic pathways which caused the serum disorder in the rats with urticaria, and tended to regulate the metabolic differential to the normal level in the rats with urticaria. This paper provides references for studying the mechanism of Jingfang Mixture from the perspective of endogenous metabolites and metabolic pathways in vivo. At the same time, the endogenous substances explored in this paper can be used as important biomarkers for the prevention of urticaria.

The anti-aging effects of Renshen Guben on thyrotoxicosis mice: Improving immunosenescence, hypoproteinemia, lipotoxicity, and intestinal flora.

With the rapid aging of the population, the control of age-related disease susceptibility and prognosis faces greater challenges. There is an urgent need for a strategy to maintain the vitality of elderly people. In this study, the effect of Renshen Guben (RSGB) oral liquid was investigated on an accelerated aging mice model of thyrotoxicosis by conventional detection methods combined with multiomics technology. The results showed that RSGB increased the number of neutrophils and lymphocytes, enhanced the function of lymphocytes, and increased the levels of complement and antimicrobial peptides, which indicated that RSGB improved the immunity of thyrotoxicosis mice at the cellular and molecular levels. RSGB corrected malnutrition in thyrotoxicosis mice by improving anemia, hypoalbuminemia, ion transporters, and vitamin-binding proteins. RSGB significantly reduced the lipotoxicity by reducing the level of fatty acids, triglyceride, sphingolipids, and glucocorticoids, thus increasing the level of docosapentaenoic acid (DPA) and bile acids, which contributed to improve immunosenescence. The intestinal defense ability of thyrotoxicosis mice was enhanced with the increase of bile acids and lactic acid bacteria by the RSGB treatment. The plant metabolomics analysis showed that there were various active components in RSGB oral liquid and medicated serum, including terpenoids, phenolic acids, flavonoids, tannin, alkaloids, organic acids, phenolamines, amino acids, and others. They have antioxidant, immune regulation, and anti-aging effects, which was the material basis of RSGB. Totally, RSGB protected the thyrotoxicosis mice against aging by improving immunosenescence, hypoproteinemia, lipotoxicity, and the intestinal flora. It will be beneficial for improving the disease susceptibility and prognosis of the elderly.

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.