Matrine inhibits invasion and migration of gallbladder cancer via regulating the PI3K/AKT signaling pathway.

Gallbladder cancer (GBC) is a common malignant cancer in the biliary system, which poses a serious threat to human health. It is urgent to explore ideal drugs for the treatment of GBC. Matrine is the main active ingredient of Sophora flavescentis, with a wide range of biological activities encompassing anti-inflammatory, antiviral, immunomodulatory, and anti-tumor. However, the underlying mechanism by which Matrine treats GBC is still unclear. The purpose of this study is to investigate the anti-tumor effects of Matrine on GBC in vivo and in vitro and to clarify the potential regulatory mechanisms. Here, we found that Matrine had a significant killing effect on GBC through CCK8 and flow cytometry, including arrest of cell cycle, inhibition of GBC cell, and induction of apoptosis. Further in vivo studies confirmed the inhibitory effect of Matrine on tumor growth in NOZ xenografted nude mouse. At the same time, Matrine also significantly suppressed the migration and invasion of GBC cells through scratch and Transwell experiments. In addition, by detecting the mRNA and protein levels of epithelial-mesenchymal transition (EMT) and matrix metalloproteinases, Matrine furtherly substantiated the inhibitory role on invasion and migration of GBC. From a mechanistic perspective, network pharmacology analysis suggests that the potential targets of Matrine in the treatment of GBC are enriched in the PI3K/AKT signaling pathway. Subsequently, Matrine effectively decreased the abundance of p-PI3K and p-AKT protein in vivo and in vitro. More importantly, PI3K activator (740 Y-P) antagonized the anti-tumor effect of Matrine, while PI3K inhibitor (LY294002) increased the sensitivity of Matrine for GBC. Based on the above findings, we conclude that Matrine inhibits the invasion and migration of GBC by regulating PI3K/AKT signaling pathway. Our results indicate the crucial role and regulatory mechanism of Matrine in suppressing the growth of GBC, which provides a theoretical basis for Matrine to be a candidate drug for the treatment and research of GBC.

Dihydrotanshinone I inhibits gallbladder cancer growth by targeting the Keap1-Nrf2 signaling pathway and Nrf2 phosphorylation.

BACKGROUND: Gallbladder cancer (GBC) poses a significant risk to human health. Its development is influenced by numerous factors, particularly the homeostasis of reactive oxygen species (ROS) within cells. This homeostasis is crucial for tumor cell survival, and abnormal regulation of ROS is associated with the occurrence and progression of many cancers. Dihydrotanshinone I (DHT I), a biologically effective ingredient isolated from Salvia miltiorrhiza, has exhibited cytotoxic properties against various tumor cells by inducing apoptosis. However, the precise molecular mechanisms by which dht I exerts its cytotoxic effects remain unclear. PURPOSE: To explore the anti-tumor impact of dht I on GBC and elucidate the potential molecular mechanisms. METHODS: The proliferation of GBC cells, NOZ and SGC-996, was assessed using various assays, including CCK-8 assay, colony formation assay and EdU staining. We also examined cell apoptosis, cell cycle progression, ROS levels, and alterations in mitochondrial membrane potential to delve into the intricate molecular mechanism. Quantitative PCR (qPCR), immunofluorescence staining, and Western blotting were performed to evaluate target gene expression at both the mRNA and protein levels. The correlation between nuclear factor erythroid 2-related factor 2 (Nrf2) and kelch-like ECH-associated protein 1 (Keap1) were examined using co-immunoprecipitation. Finally, the in vivo effect of dht I was investigated using a xenograft model of gallbladder cancer in mice. RESULTS: Our research findings indicated that dht I exerted cytotoxic effects on GBC cells, including inhibiting proliferation, disrupting mitochondrial membrane potential, inducing oxidative stress and apoptosis. Our in vivo studies substantiated the inhibition of dht I on tumor growth in xenograft nude mice. Mechanistically, dht I primarily targeted Nrf2 by promoting Keap1 mediated Nrf2 degradation and inhibiting protein kinase C (PKC) induced Nrf2 phosphorylation. This leads to the suppression of Nrf2 nuclear translocation and reduction of its target gene expression. Moreover, Nrf2 overexpression effectively counteracted the anti-tumor effects of dht I, while Nrf2 knockdown significantly enhanced the inhibitory effect of dht I on GBC. Meanwhile, PKC inhibitors and nuclear import inhibitors increased the sensitivity of GBC cells to dht I treatment. Conversely, Nrf2 activators, proteasome inhibitors, antioxidants and PKC activators all antagonized dht I induced apoptosis and ROS generation in NOZ and SGC-996 cells. CONCLUSION: Our findings indicated that dht I inhibited the growth of GBC cells by regulating the Keap1-Nrf2 signaling pathway and Nrf2 phosphorylation. These insights provide a strong rationale for further investigation of dht I as a potential therapeutic agent for GBC treatment.

[Study on efficacy of liujunzi decoction combined with zuojin pills in treating acute radioactive duodenitis and their mechanism].

OBJECTIVE: To evaluate the therapeutic effect of Liujunzi decoction combined with Zuojin pills in treating the radioactive duodenitis and their mechanism, and compare with clinical routine acid suppressants combined with mucous membrane protective preparations to study the mechanism of their efficacy. METHOD: According to the study of Williams J P and characteristics of duodenitis, and by reference to the radiation enteritis modeling standard, we took the lead in establishing the mouse radioactive duodenal injury model. The model mice were randomly divided into the control group (n = 26), traditional Chinese medicine (TCM) group (n = 16) and the western medicine (oral administration with famotidine 0.5 mL + almagate suspension 0.3 mL per mouse, once a day) group (n = 16). After the standard administrating, such objective indexes as general condition, weight, changes in health score, pathology and expression of inflammatory factors were observed to evaluate the efficacy. RESULT: The radioactive duodenitis model of mice was successfully established with 12 Gy. Mice in the control group suffered from weight loss, anorexia, low fluid intake, loose stools, and occasionally mucous bloody stool, poor spirit, dim fur, lack of exercise and arch back. Mice in drug intervention groups were generally better than those in the pure irradiation group. The IL-6, IL-1beta, TNF-alpha mRNA expressions in spleen and mesenteric lymph node tissues in TCM and western medicine groups showed a declining trend compared with the control group. Their concentrations in peripheral blood serum also slightly changed. The TCM group revealed notable advantage in reducing inflammatory factors. The microscopic observation showed that a better mucosa repair in intervention groups than the pure irradiation group. The improved Chiu's scoring method showed a statistical significance in the difference between TCM and western medicine groups (P < 0.05). CONCLUSION: Liujunzi decoction combined with Zuojin pills could treat acute radiation enteritis, regulate organic immunity, and inhibit acute injury, promote local tissue repair, with the potential to resist such adverse effects as radiation intestinal fibrosis. The regulation of inflammatory factor release is one of efficacy generation mechanisms.

[Establishment and evaluation of experimental sepsis mouse model].

After treating with chemotherapy or immunosuppressant, malignant diseases of hematopoietic system such as leukemia, malignant lymphoma and aplastic anemia usually induced severe infection such as sepsis. Sepsis which is hard to be diagnosed causes high death rate. This study was purposed to establish an experimental sepsis mouse model so as to provide a basis for pathogenesis and intervention study. A classic caecal ligation and puncture (CLP) was used to establish experimental sepsis model. ELISA was used to detect levels of C5a, IL-6, TNFalpha, and IFN-gamma. Flow Cytometry was applied to measure apoptosis of lymphocytes in thymus and mesentery. The pathologic changes of thymus and spleen were confirmed by HE staining. The results showed that almost 70%-80% mice died at 72 hours after CLP. Only approximate 20% animal survived during finite time, mice in CLP group had significant weight lose. Meanwhile large release of different inflammatory mediators which are related with sepsis (C5a, IL-6, TNF-alpha, and IFN-gamma) was observed after CLP. Apoptosis of lymphocytes in thymus and mesentery lymphonodus was enhanced markedly after CLP. Significantly pathologic injury was also observed in thymus and spleen. It is concluded that a mouse model of experimental sepsis was successfully established by caecal ligation and puncture which can well mimic the clinical symptom of sepsis. The experimental sepsis mouse model provides an excellent tool for exploring the pathogenesis and intervention ways for sepsis accompanied with complicated malignant hematological diseases in vivo.

[Development of rAAV2-GAD-Ig for IDDM gene therapy].

AIM: To develop rAAV2 containing cDNA of GAD-Ig fusion gene [GAD-Ig fusion gene is constructed by inserting glutamic acid decarboxylase(GAD) into N-terminal of murine IgG3 heavy chain(Ig)], and to study whether rAAV2 mediated GAD-Ig fusion gene expression could induce specific tolerance in IDDM animal model-nonobese diabetic (NOD) mice. METHODS: GAD-Ig cDNA was amplified by PCR from the plasmid BSSK-GAD-Ig and inserted into an eukaryotic expression plasmid pSNAV to construct a recombinant expression plasmid pSNAV/GAD-Ig. Plasmid pSNAV-GAD-Ig was then transfected into the rAAV2 packaging cell-BHK-21 cells using LipofectAMINE TM 2000 to produce rAAV-GAD-Ig. Target gene expression in BHK-21 cells infected by rAAV-GAD-Ig was confirmed by RT-PCR, Western blot and ELISA respectively. Then rAAV-GAD-Ig was injected into intramuscularly NOD mice. Antigen-specific tolerance in NOD mice was detected by specific lymphocytic proliferation reaction to GAD antigen. RESULTS: GAD-Ig gene was inserted into the genome of target cells. Target cells transfected by rAAV2-GAD-Ig could secret GAD-Ig. rAAV2-GAD-Ig induced specific tolerance in NOD mice. CONCLUSION: rAAV2-GAD-Ig was obtained, which can be used in IDDM gene therapy in NOD mice.