Exploring the mechanism of JiGuCao capsule formula on treating hepatitis B virus infection via network pharmacology analysis and in vivo/vitro experiment verification.

The JiGuCao capsule formula (JCF) has demonstrated promising curative effects in treating chronic hepatitis B (CHB) in clinical trials. Here, we aimed to investigate JCF's function and mechanism in diseases related to the hepatitis B virus (HBV). We used mass spectrometry (MS) to identify the active metabolites of JCF and established the HBV replication mouse model by hydrodynamically injecting HBV replication plasmids into the mice's tail vein. Liposomes were used to transfect the plasmids into the cells. The CCK-8 kit identified cell viability. We detected the levels of HBV s antigen (HBsAg) and HBV e antigen (HBeAg) by the quantitative determination kits. qRT-PCR and Western blot were used to detect the genes' expression. The key pathways and key genes related to JCF on CHB treatment were obtained by network pharmacological analysis. Our results showed that JCF accelerated the elimination of HBsAg in mice. JCF and its medicated serum inhibited HBV replication and proliferation of HBV-replicating hepatoma cells in vitro. And the key targets of JCF in treating CHB were CASP3, CXCL8, EGFR, HSPA8, IL6, MDM2, MMP9, NR3C1, PTGS2, and VEGFA. Furthermore, these key targets were related to pathways in cancer, hepatitis B, microRNAs in cancer, PI3K-Akt signaling, and proteoglycans in cancer pathways. Finally, Cholic Acid, Deoxycholic Acid, and 3', 4', 7-Trihydroxyflavone were the main active metabolites of JCF that we obtained. JCF employed its active metabolites to perform an anti-HBV effect and prevent the development of HBV-related diseases.

Mucus adhesion vs. mucus penetration? Screening nanomaterials for nasal inhalation by MD simulation.

Nanocarrier-aided drug delivery techniques have improved the absorption and permeability of drugs in nose-to-brain delivery. However, the molecular properties of nanocarriers during the delivery process are of great interest; in particular, the characteristics when penetrating barriers in vivo are crucial for the screening and optimization of materials for nasal inhalation. In this study, we have focused on two types of delivery systems: mucoadhesive nanoparticles (MAPs) and mucopenetrating nanoparticles (MPPs); both have been widely used for mucosal delivery, although a method for selecting the more effective type of drug carriers for mucosal delivery has not been established. Molecular dynamics (MD) simulations were used to reveal the all-atom dynamic characteristics of the interaction between different delivery systems and the nasal mucus protein MUC5AC. Among the systems tested, hydroxypropyltrimethyl ammonium chloride chitosan (HTCC) had the strongest interaction with mucin, suggesting it had better mucoadhesive performance, and that it interacted with MUC5AC more strongly than unmodified chitosan. In contrast, the mucus-penetrating material polyethylene glycol-poly lactic acid-co-glycolic acid (PEG-PLGA), had almost no interaction with MUC5AC. The results of the MD simulations were verified by in vitro experiments on nanoparticles (NPs) and mucin binding. The drug delivery performance of the four types of NPs, analyzed by in vitro and ex vivo mucosal penetration, were all generally consistent with the properties of the material predicted from the MD simulation. These clues to the molecular mechanism of MAPs and MPPs may provide useful insight into the screening and optimization of nanomaterials suitable for nasal inhalation.

Network pharmacology and in vitro experiments-based strategy to investigate the mechanisms of KangXianYiAi formula for hepatitis B virus-related hepatocellular carcinoma.

The Chinese traditional medicine KangXianYiAi formula (KXYA) is used to treat hepatic disease in the clinic. Here we aim to confirm the therapeutic effects and explore the pharmacological mechanisms of KXYA on hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). We first collected and analyzed clinical data of 40 chronic hepatitis B (CHB) patients with precancerous liver lesions under KXYA treatment. Then, the cell viability, migration, cell cycle, and apoptosis of HepAD38 cells with KXYA treatment were examined. Next, we performed network pharmacological analysis based on database mining to obtain the key target pathways and genes of KXYA treatment on HBV-related HCC. We finally analyzed the expression of the key genes between normal and HBV-related HCC tissues in databases and measured the mRNA expression of the key genes in HepAD38 cells after KXYA treatment. The KXYA treatment could reduce the liver nodule size of CHB patients, suppress the proliferation and migration capabilities, and promote apoptosis of HepAD38 cells. The key pathways of KXYA on HBV-related HCC were Cancer, Hepatitis B, Viral carcinogenesis, Focal adhesion, and PI3K-Akt signaling, and KXYA treatment could regulate the expression of the key genes including HNF4A, MAPK8, NR3C1, PTEN, EGFR, and HDAC1. The KXYA exhibited a curative effect via inhibiting proliferation, migration, and promoting apoptosis of HBV-related HCC and the pharmacological mechanism was related to the regulation of the expression of HNF4A, MAPK8, NR3C1, PTEN, EGFR, and HDAC1.

Uncovering the Pharmacological Mechanisms of Gexia-Zhuyu Formula (GXZY) in Treating Liver Cirrhosis by an Integrative Pharmacology Strategy.

Liver cirrhosis (LC) is a fibrotic lesion of liver tissue caused by the repeated progression of chronic hepatitis. The traditional Chinese medicine Gexia-Zhuyu formula (GXZY) has a therapeutic effect on LC. However, its pharmacological mechanisms on LC remain elucidated. Here, we used the network pharmacology approach to explore the action mechanisms of GXZY on LC. The compounds of GXZY were from the traditional Chinese medicine systems pharmacology (TCMSP) database, and their potential targets were from SwissTargetPrediction and STITCH databases. The disease targets of LC came from GeneCards, DisGeNET, NCBI gene, and OMIM databases. Then we constructed the protein-protein interaction (PPI) network to obtain the key target genes. And the gene ontology (GO), pathway enrichment, and expression analysis of the key genes were also performed. Subsequently, the potential action mechanisms of GXZY on LC predicted by the network pharmacology analyses were experimentally validated in LC rats and LX2 cells. A total of 150 components in GXZY were obtained, among which 111 were chosen as key compounds. The PPI network included 525 targets, and the key targets were obtained by network topological parameters analysis, whereas the predicted key genes of GXZY on LC were AR, JUN, MYC, CASP3, MMP9, GAPDH, and RELA. Furthermore, these key genes were related to pathways in cancer, hepatitis B, TNF signaling pathway, and MAPK signaling pathway. The in vitro and in vivo experiments validated that GXZY inhibited the process of LC mainly via the regulation of cells proliferation and migration through reducing the expression of MMP9. In conclusion, through the combination of network pharmacology and experimental verification, this study offered more insight molecular mechanisms of GXZY on LC.

The host immune response of a discharged COVID-19 patient with twice reemergence of SARS-CoV-2: a case report.

BACKGROUND: The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. There have been reports that long-term SARS-CoV-2 RNA shedding and re-infection of COVID-19 patients existed. However, the specific mechanism, diagnosis, and treatment of COVID-19 are still unclarified. CASE PRESENTATION: In this case, we reported a 64-year-old patient who had a long-term course of COVID-19 for 174 days with two retests of SARS-CoV-2 RNA positive after discharging from the hospital. The patient's serum immunoglobulin G (IgG) of SARS-CoV-2 tested positive after the initial infection. And during treatment, the CD4 + T cell count and ratio to peripheral blood mononuclear cell (PBMC) were in dynamic change. CONCLUSIONS: Our results suggested that the host immune system responded with IgG production after SARS-CoV-2 infection, but was not protective enough for the patient. The reemergence of SARS-CoV-2 could be related to the cell count and proportion of CD4 + T cells in PBMC. And the increase of CD4 + T cells after treatment may help to clear the virus.

Versatile modification of the CRISPR/Cas9 ribonucleoprotein system to facilitate in vivo application.

The development of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems has created a tremendous wave that is sweeping the world of genome editing. The ribonucleoprotein (RNP) method has evolved to be the most advantageous form for in vivo application. Modification of the CRISPR/Cas9 RNP method to adapt delivery through a variety of carriers can either directly improve the stability and specificity of the gene-editing tool in vivo or indirectly endow the system with high gene-editing efficiency that induces few off-target mutations through different delivery methods. The exploration of in vivo applications mediated by various delivery methods lays the foundation for genome research and variety improvements, which is especially promising for better in vivo research in the field of translational biomedicine. In this review, we illustrate the modifiable structures of the Cas9 nuclease and single guide RNA (sgRNA), summarize the latest research progress and discuss the feasibility and advantages of various methods. The highlighted results will enhance our knowledge, stimulate extensive research and application of Cas9 and provide alternatives for the development of rational delivery carriers in multiple fields.

The mechanism of TiaoGanYiPi formula for treating chronic hepatitis B by network pharmacology and molecular docking verification.

The Chinese herbal formula TiaoGanYiPi (TGYP) showed effective against chronic hepatitis B (CHB) caused by hepatitis B virus (HBV) infection. Hence, we aimed to clarify the mechanisms and potential targets between TGYP and CHB. The active compounds and related putative targets of TGYP, and disease targets of CHB were obtained from the public databases. The key targets between TGYP and CHB were identified through the network construction and module analysis. The expression of the key targets was detected in Gene Expression Omnibus (GEO) dataset and normal hepatocyte cell line LO2. We first obtained 11 key targets which were predominantly enriched in the Cancer, Cell cycle and HBV-related pathways. And the expression of the key targets was related to HBV infection and liver inflammation verified in GSE83148 database. Furthermore, the results of real-time quantitative PCR and CCK-8 assay indicated that TGYP could regulate the expression of key targets including CCNA2, ABL1, CDK4, CDKN1A, IGFR and MAP2K1, and promote proliferation of LO2 cells. In coclusion, we identified the active compounds and key targets btween TGYP and CHB, and found that the TGYP might exhibite curative effect on CHB via promoting hepatocyte proliferation and inhibiting the liver inflammatory processes.

Coptidis alkaloids extracted from Coptis chinensis Franch attenuate IFN-γ-induced destruction of bone marrow cells.

Coptidis alkaloids are the primary active components of Coptis chinensis Franch. Clinical and pharmacodynamic studies have confirmed that Coptidis alkaloids have multiple therapeutic effects including anti-inflammatory, antioxidant and antitumor effects, and they are usually used to treat various inflammatory disorders and related diseases. Mouse bone marrow cells (BMCs) were isolated from BALB/c mice. Immune-mediated destruction of BMCs was induced by interferon (IFN) -γ. High-performance liquid chromatography-electrospray ionization/ mass spectrometry was used to analyze the ingredients of the aqueous extract from Coptis chinensis Franch. The results confirmed that Coptidis alkaloids were the predominant ingredients in the aqueous extract from Coptis chinensis. The functional mechanism of Coptidis alkaloids in inhibiting immune-mediated destruction of BMCs was studied in vitro. After Coptidis alkaloid treatment, the percentages of apoptotic BMCs and the proliferation and differentiation of helper T (Th) cells and regulatory T (Treg) cells were measured by flow cytometry. The expression and distribution of T-bet in BMCs were observed by immunofluorescence. Western blotting analysis was used to assay the expression of key molecules in the Fas apoptosis and Jak/Stats signaling pathways in BMCs. We identified five alkaloids in the aqueous extract of Coptis chinensis. The apoptotic ratios of BMCs induced by IFN-γ were decreased significantly after Coptidis alkaloid treatment. The levels of key molecules (Fas, Caspase-3, cleaved Caspase-3, Caspase-8 and Caspase-8) in Fas apoptosis signaling pathways also decreased significantly after treatment with low concentrations of Coptidis alkaloids. Coptidis alkaloids were also found to inhibit the proliferation of Th1 and Th17 cells and induce the differentiation of Th2 and Treg cells; further, the distribution of T-bet in BMCs was decreased significantly. In addition, the levels of Stat-1, phospho-Stat-1 and phospho-Stat-3 were also reduced after Coptidis alkaloid treatment. These results indicate that Coptidis alkaloids extracted by water decoction from Coptis chinensis Franch could inhibit the proliferation and differentiation of T lymphocytes, attenuate the apoptosis of BMCs, and suppress the immune-mediated destruction of the BMCs induced by pro-inflammatory cytokines.

Chinese Herbal Formula, Huayu Tongbi Fang, Attenuates Inflammatory Proliferation of Rat Synoviocytes Induced by IL-1ß by Regulating Proliferation and Differentiation of T Lymphocytes.

The inflammatory proliferation of fibroblast-like synoviocytes (FLSs) and functional imbalances in T lymphocytes play critical roles in the pathogenesis of rheumatoid arthritis (RA). The clinical efficacy of Huayu Tongbi Fang (HYTB, a traditional herbal formula) in RA treatment has been validated. In this study, we aimed to explore the regulatory mechanisms of HYTB on the proliferation and differentiation of T lymphocytes, and the inhibitory effect of HYTB on inflammatory proliferation of FLSs. The RCS-364 (Rat FLSs) cells were cocultured with rat splenic lymphocytes that were induced by interleukin-1ß in Transwell chambers. After freeze-dried HYTB powder treatment, the percentage of T-cell subset and apoptosis rates of FLSs were measured using flow cytometry. Furthermore, protein expression of key molecules of NF-κB and JAK/STAT signaling pathways was quantified using Western blot. The granulocyte-macrophage colony-stimulating factor (GM-CSF) was measured using enzyme-linked immunosorbent assay. The results showed that HYTB could inhibit the inflammatory proliferation of FLSs through inducing cell apoptosis. Additionally, HYTB treatment could intervene in the proliferation and differentiation of T lymphocytes and regulate protein expression of key molecules in NF-κB and JAK/STAT cell signaling pathways. Moreover, it could inhibit FLS activation by suppressing GM-CSF production by T cells and FLSs. Therefore, the HYTB formula should be used as a traditional medicine against RA in modern complementary and alternative therapies.

The combination of Radix Astragali and Radix Angelicae Sinensis attenuates the IFN-γ-induced immune destruction of hematopoiesis in bone marrow cells.

BACKGROUND: Radix Astragali and Radix Angelicae Sinensis are two herbs that compose Danggui Buxue Tang (an herbal formula for treatment of anemia diseases). In this study, we explored the molecular mechanism and effective targets to immune destruction of bone marrow (BM) cells treated with Radix Astragali, Radix Angelicae Sinensis or a combination of two agents. The potential synergic advantages of two herbs should also be explored. METHODS: The constituents of Radix Astragali and Radix Angelicae Sinensis were analyzed by high performance liquid chromatography-electrospray ionization/mass spectrometer system BM cells were separated from limbs of BALB/c mice, and immune destruction was induced with IFN-γ. The percentages of hematopoietic stem cells (HSCs) and CD3+ T cells were detected by flow cytometry. The distribution of T-bet and changes in the combination of SAP and SLAM in BM cells were observed by immunofluorescence. Western blotting was used to assay the expression of key molecules of the eIF2 signaling pathway in BM cells. RESULTS: Seven constituents of Radix Astragali and six constituents of Radix Angelicae Sinensis were identified. The percentages of HSCs increased significantly after treatment with Radix Angelicae Sinensis, especially at high concentrations. The percentages of CD3+ T cells were significantly decreased after Radix Astragali and Radix Angelicae Sinensis treatment. However, the synergistic function of two-herb combinations was superior to that of the individual herbs alone. The distribution of T-bet in BM cells was decreased significantly after Radix Angelicae Sinensis treatment. The number of SLAM/SAP double-stained cells was increased significantly after Radix Astragali treatment at low concentrations. The phosphorylation levels of eIF2α were also reduced after Radix Astragali and Radix Angelicae Sinensis treatment. CONCLUSIONS: Radix Astragali and Radix Angelicae Sinensis could intervene in the immunologic balance of T lymphocytes, inhibit the apoptosis of BM cells induced by immune attack, restore the balance of the T cell immune response network and recover the hematopoietic function of HSCs. The synergistic effects of Radix Astragali and Radix Angelicae Sinensis were superior to those of each herb alone.