Antitumor effect of tubeimoside-I on murine colorectal cancers through PKM2-dependent pyroptosis and immunomodulation.

Induction of cancer cell death is an established treatment strategy, but chemotherapy drug-mediated apoptosis can be evaded by many tumors. Pyroptosis is a type of inflammatory programmed cell death (PCD) that is important for organism immunity. Tubeimoside-I (TBMS1) is a plant-derived component that exhibits antitumor activity. However, it is unclear how TBMS1 induces pyroptosis to inhibit colorectal cancer (CRC). In this study, we demonstrated that TBMS1 is able to induce pyroptosis in murine CRC cells and releases pro-inflammatory cytokines. Mechanistically, we found that TBMS1 inhibits CRC cell proliferation and migration and induces pyroptosis by activating caspase-3 and cleaving gasdermin E (GSDME) through the inhibition of PKM2. In the animal experiments, TBMS1 attenuated the weight of solid tumors, increased the proportion of CD8+ cytotoxic T cells, and reduced the content of M2-type macrophages in the spleen of tumor-bearing mice. Furthermore, TBMS1 inhibited M2-type polarization by blocking STAT6 pathway activation in RAW 264.7 cells. To sum up, our findings suggest that TBMS1 triggers pyroptosis in CRC by acting on the PKM2/caspase-3/GSDME signaling pathway. Additionally, it modulates the antitumor immune response in CRC murine models. This study provides a promising basis for the potential use of TBMS1 in treating CRC.

[Revealing the role of gut microbiota in immune regulation and organ damage in sepsis using 16s rRNA and untargeted metabolomics].

OBJECTIVE: To analyze the composition and metabolites of gut microbiota in septic rats by fecal 16s rRNA sequencing and untargeted metabolomics, and to preliminarily explore the effect and potential mechanism of gut microbiota and its metabolites on inflammatory response and multiple organ damage in sepsis. METHODS: Ten males healthy male Wistar rats were randomly divided into a sham operated group (Sham group) and sepsis model group (CLP group) using a random number table method, with 5 rats in each group. A rat sepsis model was established by cecal ligation and perforation (CLP) method. The animals were sacrificed 24 hours after modeling, the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in peripheral blood were detected by enzyme-linked immunosorbent assay (ELISA). Hematoxylin-eosin (HE) staining was used to observe the pathological changes of lung and kidney tissues, and the pathological scores were evaluated. Fecal samples were collected, and 16s rRNA high-throughput sequencing and non-targeted metabolomics were used to screen microbiota, metabolites and potential signal pathways that may play an important role in disease outcomes. Spearman correlation analysis was conducted to jointly analyze the gut microbiota and non-targeted metabolism. RESULTS: Compared with the Sham group, the degree of pathological damage to lung and kidney tissues in the CLP group was significantly increased (lung tissue score: 3.60±0.80 vs. 0.00±0.00, kidney tissue score: 2.40±0.80 vs. 0.00±0.00, both P < 0.01), the level of IL-6 and TNF-α in peripheral blood significantly increased [TNF-α (ng/L): 248.12±55.98 vs. 143.28±36.57, IL-6 (ng/L): 260.26±39.47 vs. 116.01±26.43, both P < 0.05], the species diversity of intestinal flora of rats in the CLP group was significantly reduced, the relative abundance of Morganella, Bacteroides and Escherichia-Shigella were significantly increased, and the relative abundance of Lachnospiraceae NK4A136, Ruminococcus, Romboutsia and Roseburia were significantly reduced. In addition, the biosynthesis and bile secretion of phenylalanine, tyrosine, and tryptophan in the gut microbiota of the CLP group were significantly increased, while the biosynthesis of secondary bile acids was significantly reduced. There was a significant correlation between differential metabolites and differential microbiota. CONCLUSIONS: Sepsis can cause significant changes in the characteristics of gut microbiota and fecal metabolites in rats, which provides a basis for translational research to seek new targets for the treatment of sepsis.

Saikosaponin d modulates the polarization of tumor-associated macrophages by deactivating the PI3K/AKT/mTOR pathway in murine models of pancreatic cancer.

The tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) poses a major obstacle to traditional and immunomodulatory cancer therapies and is closely associated with macrophage polarization. Saikosaponin d (SSd), a major active component of triterpene saponins derived from Bupleurum falcatum, has anti-inflammatory and antitumor activities. However, whether SSd can regulate immune cells during the development of the TME in PDAC remains unknown. In the present study, we aimed to analyze the role of SSd in regulating immune cells in the PDAC TME, especially the polarization of macrophages, and examine the related mechanisms. An orthotopic PDAC cancer model was used to investigate the antitumor activities and the regulation of immune cells in vivo. In vitro, bone marrow mononuclear (BM-MNC) cells and RAW 264.7 cells were used to induce the M2 macrophage phenotype and examine the effects and molecular mechanism of SSd on M2 macrophage polarization. The results revealed that SSd could directly inhibit the apoptosis and invasion of pancreatic cancer cells, modulate the immunosuppressive microenvironment and reactivate the local immune response, especially by decreasing the shift toward M2 macrophage polarization by downregulating phosphorylated STAT6 levels and the PI3K/AKT/mTOR signaling pathway. Furthermore, 740-Y-P (PI3K activator) was used to verify that SSd inhibited M2 polarization in RAW264.7 cells via the PI3K/AKT/mTOR signaling pathway. In conclusion, this study provided experimental evidence of the antitumor effect of SSd, especially in the regulation of M2 macrophage polarization, and demonstrated that SSd may be a promising therapeutic agent in PDAC.

Protective effect of liriodendrin on IgG immune complex-induced acute lung injury via inhibiting SRC/STAT3/MAPK signaling pathway: a network pharmacology research.

The primary objectives of this research were to investigate the protective effects of liriodendrin against IgG immune complex (IgG-IC)-induced acute lung injury (ALI) and to elucidate the underlying mechanisms. This study employed a mouse and cell model of IgG-IC-induced acute lung injury. Lung tissue was stained with hematoxylin-eosin to observe pathological alterations and arterial blood gas analysis was tested. Inflammatory cytokines, including interleukin-6 (IL-6), interleukin-1ß (IL-1ß), and tumor necrosis factor-alpha (TNF-α), were measured using ELISA. The mRNA expression of inflammatory cytokines was assessed via RT-qPCR. Molecular docking and enrichment analysis were combined to identify the most potential signaling pathways modulated by liriodendrin, which were then verified using western blot analysis in IgG-IC-induced ALI models. We identified 253 shared targets between liriodendrin and IgG-IC-induced acute lung injury from the database. Through network pharmacology, enrichment analysis, and molecular docking, SRC was determined to be the most closely associated target of liriodendrin in IgG-IC-induced ALI. Pretreatment with liriodendrin notably reduced the increased cytokine secretion of IL-1ß, IL-6, and TNF-α. Histopathological analysis of lung tissue demonstrated a protective effect of liriodendrin on IgG-IC-induced acute lung injury in mice. Arterial blood gas analysis showed liriodendrin ameliorated acidosis and hypoxemia efficiently. Further studies revealed that liriodendrin pretreatment substantially attenuated the elevated phosphorylation levels of SRC's downstream components (JNK, P38, and STAT3), suggesting that liriodendrin may protect against IgG-IC-induced ALI via the SRC/STAT3/MAPK pathway. Our findings indicate that liriodendrin protects against IgG-IC-induced acute lung injury by inhibiting the SRC/STAT3/MAPK signaling pathway, suggesting that liriodendrin may serve as a potential treatment for acute lung injury caused by IgG-IC.

Isobavachalcone Activates Antitumor Immunity on Orthotopic Pancreatic Cancer Model: A Screening and Validation.

Pancreatic cancer is accompanied by poor prognosis and accounts for a significant number of deaths every year. Since Psoralea corylifolia L. (PCL) possesses a broad spectrum of bioactivities, it is commonly used in traditional Chinese medicine. The study explored potential antitumor agents of PCL and underlying mechanisms in vitro and vivo. Based on network pharmacology, bioinformatics, and molecular docking, we considered isobavachalcone (IBC) as a valuable compound. The activity and potential mechanisms of IBC were investigated by RT-qPCR, immunohistochemistry, immunofluorescence, and flow cytometry. It was confirmed that IBC could inhibit Panc 02 cell proliferation and induce apoptosis via increasing the production of reactive oxygen species. IBC could attenuate the weight of solid tumors, increase CD8+ T cells, and reduce M2 macrophages in the tumor tissue and spleen. Another promising finding was that IBC alleviated the proportion of myeloid-derived suppressor cells (MDSCs) in the tumor tissue but had no change in the spleen. The study of pharmacological effects of IBC was carried out and suggested IBC restrained M2-like polarization of RAW 264.7 cells by inhibiting the expression of ARG1 and MRC1 and suppressed the expression of ARG1 and TGF-ß in bone marrow-derived MDSC. In summary, this research screened IBC as an antineoplastic agent, which could attenuate the growth of pancreatic cancer via activating the immune activity and inducing cell apoptosis. It might be a reference for the antitumor ability of IBC and the treatment of the tumor microenvironment in pancreatic cancer.

Dihydroartemisinin inhibits the growth of pancreatic cells by inducing ferroptosis and activating antitumor immunity.

Dihydroartemisinin (DHA) exhibits a direct antitumor effect in various tumor models. However, the mechanism of DHA inducing ferroptosis and activating antitumor immunity remains obscure. Therefore, our study was dedicated to investigate the effect of DHA on ferroptosis and tumor microenvironment and elucidate the underlying molecular mechanism. PDAC orthotopic tumor model was used to investigate tumor proliferation and the population of immune cell in vivo, including M2-type macrophages (M2), myeloid-derived suppressor cells (MDSCs), CD4+T cells, CD8+T cells, NK cells and NKT cells. Levels of GPX4, SLC7A11, P53 and ALOX12 were determined by Real-time PCR and Western blot. CCK8 assay was performed to detect cell viability, and the ferroptosis was distinguished by flow cytometry. Our results showed that DHA inhibited pancreatic cancer cell proliferation. In addition, DHA induced cell ferroptosis by up-regulating the expression of P53 and ALOX12, which was blocked by baicalein (a selective ALOX12 inhibitor). However, DHA also up-regulated the expression of GPX4 and SLC7A11. On the other hand, DHA significantly decreased the suppressive expansion of M2 and MDSCs. Moreover, DHA increased the immune cell population of CD8+T cells, NK cells and NKT cells in the tumor tissues of the tumor-bearing mice. Whereas, the DHA treatment did not affect the frequencies of M2, MDSCs, CD4+T, CD8+T, NK and NKT cells in the spleen. Our research provided experimental evidences on the activity and mechanism of ferroptosis induced by DHA and revealed that DHA regulated tumor local immunosuppressive microenvironment.

3,4-dihydroxyphenylethyl alcohol glycoside reduces acetaminophen-induced acute liver failure in mice by inhibiting hepatocyte ferroptosis and pyroptosis.

APAP is one of the most commonly used antipyretic and pain medications, but excessive use can cause liver toxicity and damage. 3,4-dihydroxyphenylethyl alcohol glycoside (DAG) is a component isolated from Sargentodoxa cuneata known to have anti-apoptotic, anti-oxidation and anti-inflammatory effects. However, the effects of DAG on acute liver failure (ALF) are largely unknown. The purpose of this study is to study the protective effects and mechanism of DAG on APAP-induced ALF in mice. We established an ALF model in adult male pathogen-free C57BL/6 mice treated with APAP (300 mg/kg) by intraperitoneal injection and resolved by 24 h. Hematoxylin and eosin (HE) staining was used to evaluate the pathological changes in mouse liver tissue. The infiltration of neutrophils in liver tissue and reactive oxygen species (ROS) in AML12 cells were analyzed by flow cytometry. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutathione (GSH), malondialdehyde (MDA), catalase (CAT), and superoxide dismutase (SOD) were analyzed using relevant kits. Our results show that DAG reduced APAP-induced serum ALT and AST levels, histopathological changes, liver neutrophil infiltration and proinflammatory cytokines production, also attenuated the accumulation of MDA and the exhaustion of GSH, CAT and SOD. In vitro experiment indicated that DAG dose-dependently inhibited APAP-induced the levels of pro-inflammatory factors (IL-1ß and IL18), and reactive oxygen species (ROS) and preventing GSH depletion in mouse AML12 hepatocytes. More interestingly, DAG inhibited the expression of ERK, HO-1, NLRP3, Caspase1 (p20) and Gasdermin-D and upregulated the expression of GPX4 in liver tissues and AML12hepatocytes. Therefore, our results indicate that DAG may act as a potential agent to treat ALF induced by APAP by inhibiting hepatocyte ferroptosis and pyroptosis.

Syringaresinol Resisted Sepsis-Induced Acute Lung Injury by Suppressing Pyroptosis Via the Oestrogen Receptor-ß Signalling Pathway.

Acute lung injury (ALI) is a common lung disease characterized by severe acute inflammatory lung injury in patients with sepsis. Syringaresinol (SYR) has been reported to have anti-apoptotic and anti-inflammatory effects, but whether it could prevent pyroptosis to improve sepsis-induced ALI remains unclear. The purpose of this work was to examine the impact of SYR on sepsis-induced ALI and investigate the underlying mechanisms. The ALI model was induced by caecal ligation and puncture (CLP) in C57BL/6 mice, structural damage in the lung tissues was determined using haematoxylin and eosin (HE) staining, and the levels of related inflammatory cytokines and macrophage polarization were examined by enzyme-linked immunosorbent assays (ELISAs) and flow cytometry, respectively. The activation of the NLRP3 inflammasome and the protein levels of TLR4, NF-κB and MAPKs was measured by western blotting. The results demonstrated that SYR pretreatment significantly reduced lung tissue histological damage, inhibited the production of proinflammatory cytokines and albumin in bronchoalveolar lavage fluid (BALF), and decreased myeloperoxidase (MPO) levels, thereby alleviating lung tissue injury. Meanwhile, septic mice treated with SYR displayed a higher survival rate and lower percentage of M1 macrophages in the BALF and spleen than septic mice. In addition, lung tissues from the CLP + SYR group exhibited downregulated protein expression of NLRP3, ASC, GSDMD caspase-1 p20 and TLR4, along with decreased phosphorylated levels of NF-κB, ERK, JNK and P38, indicating that SYR administration effectively prevented CLP-induced pyroptosis in the lung. SYR also suppressed LPS-induced pyroptosis in RAW 264.7 cells by inhibiting the activation of the NLRP3 inflammasome, which was abolished by an oestrogen receptor-ß (ERß) antagonist (PHTPP). In conclusion, SYR exerted protective effects on CLP-induced ALI via the oestrogen receptor-ß signalling pathway.

Hydroxytyrosol Inhibits MDSCs and Promotes M1 Macrophages in Mice With Orthotopic Pancreatic Tumor.

The poor immunotherapy of pancreatic cancer is mainly due to its complex immunosuppressive microenvironment. The Mediterranean diet contributes to low cancer incidence. Hydroxytyrosol (HT) derived from olive oil has multiple health-promoting effects, but its therapeutic effect on pancreatic cancer remains controversial. Here, we evaluated the inhibitory effect of HT on mouse pancreatic cancer, and the effect of HT on the immune microenvironment. We found that HT can inhibit the proliferation of Panc 02 cells through signal transducer and activator of transcription (STAT) 3/Cyclin D1 signaling pathway. In the tumor-bearing mice treated with HT, the orthotopic pancreatic tumors were suppressed, accompanied by a decrease in the proportion of myeloid-derived suppressor cells (MDSCs) and an increase in the proportion of M1 macrophages. In addition, we found that HT inhibited the expression of immunosuppressive molecules in bone marrow (BM)-derived MDSCs, as well as down-regulated CCAAT/enhancer-binding protein beta (C/EBPß) and phosphorylation of STAT3. Moreover, HT enhanced the anti-tumor effect of anti-CD47 antibody in vivo. HT combined with plumbagin (PLB) induced more Panc 02 cells death than HT or PLB alone. This combination therapy not only inhibited the accumulation of MDSCs, but also promoted the infiltration of CD4+ and CD8+ T cells in the tumors. In summary, HT is a potential immunomodulatory drug for the treatment of pancreatic cancer.

Syringaresinol attenuates sepsis-induced cardiac dysfunction by inhibiting inflammation and pyroptosis in mice.

The mortality of sepsis-induced cardiac dysfunction (SICD) is very high due to the complex pathophysiological mechanism. Syringaresinol (SYR) is a natural abstract which possesses anti-inflammatory property. The present study aims was to identify the protective impact of SYR on sepsis-induced cardiac dysfunction and investigate the specific mechanisms. We found that SYR improved the cardiac function and alleviated myocardial injury in mice that subjected to cecal ligation and puncture, in addition, SIRT1 expression was significantly elevated after SYR treatment compared to sepsis group both in vivo and in vitro, along with suppression of NLRP3 activation and proinflammatory cytokines release. However, SIRT1 inhibitor EX427 abolished the impact of SYR on LPS-induced pyroptosis in cardiomyocytes. Furthermore, molecular docking analysis predicted that there is high affinity between SYR and estrogen receptor (ER), ER inhibitor ICI182780, the specific ERß inhibitor PHTP and the specific ERαinhibitor AZD9496 were used to examine the role of ER in the protective effect of SYR against SICD, and the results suggested that ER activation was essential for the cardioprotective function of SYR. In conclusion, SYR ameliorates SICD via the ER/SIRT1/NLRP3/GSDMD pathway.

Chaihu Guizhi Ganjiang Decoction Ameliorates Pancreatic Fibrosis via JNK/mTOR Signaling Pathway.

Pancreatic fibrosis is a pathological characteristic of chronic pancreatitis (CP) and pancreatic cancer. Chaihu Guizhi Ganjiang Decoction (CGGD) is a traditional Chinese medicine, which is widely used in the clinical treatment of digestive diseases. However, the potential anti-fibrosis mechanism of CGGD in treating CP remains unclear. Here, we conducted a series of experiments to examine the effect of CGGD on the CP rat model and primary isolated pancreatic stellate cells (PSCs). The results revealed that CGGD attenuated pancreatic damage, decreased collagen deposition, and inhibited PSC activation in the pancreas of CP rats. However, compared with the CP group, CGGD had no effect on body weight and serum amylase and lipase. In addition, CGGD suppressed autophagy by downregulating Atg5, Beclin-1, and LC3B and facilitated phosphorylation of mTOR and JNK in pancreatic tissues and PSCs. Moreover, the CGGD-containing serum also decreased LC3B or collagen I expression after rapamycin (mTOR inhibitor) or SP600125 (JNK inhibitor) treatment in PSCs. In conclusion, CGGD attenuated pancreatic fibrosis and PSC activation, possibly by suppressing autophagy of PSCs through the JNK/mTOR signaling pathway.

Pancreatic Stellate Cells Promote Tumor Progression by Promoting an Immunosuppressive Microenvironment in Murine Models of Pancreatic Cancer.

OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer with poor prognosis. Pancreatic stellate cells (PSCs) play a vital role in PDAC development. The aim of this study was to explore tumor microenvironment response to PSCs in an orthotopic pancreatic cancer mouse model and to assess if PSCs secreted factors that can facilitate an immunosuppressive microenvironment. METHODS: Pancreatic ductal adenocarcinoma orthotopic tumor model, derived from coinjection of Panc02 cells plus PSCs, was used to investigate tumor proliferation, metastasis, and the population of immune cells in vivo, including regulatory T cells, M2-type macrophages, myeloid-derived suppressor cells, CD8 T cells, CD4 T cells, M1-type macrophages, natural killer (NK), and NK T cells. RESULTS: Pancreatic stellate cells promoted PDAC growth not only induced cell proliferation and metastasis, but also significantly increased the suppressive immune cell population of regulatory T cells, M2-type macrophages, and myeloid-derived suppressor cells. In addition, PSCs decreased the immune cell population of CD8 T, CD4 T cells, and M1-type macrophages in the spleen and tumor tissues of the tumor-bearing mice. Moreover, PSCs decreased the population of NK and NK T cells in the tumor tissues. CONCLUSIONS: Our findings support PSCs playing multiple roles in PDAC development via promoting immunosuppressive microenvironment.

Modified Da-chai-hu Decoction regulates the expression of occludin and NF-κB to alleviate organ injury in severe acute pancreatitis rats.

Modified Da-chai-hu Decoction (MDD), a traditional Chinese medicinal formulation, which was empirically generated from Da-chai-hu decoction, has been utilized to treat severe acute pancreatitis (SAP) for decades. The aim of the present study was to explore its potential organprotective mechanism in SAP. In the present study, rat SAP model was induced by retrograde injection of 3.5% sodium taurocholate into the biliopancreatic duct, MDD (23.35 g/kg body weight, twelve times the clinical dose) were orally given at 2 h before and 10 h after injection. At 12 h after model induction, blood was taken from vena cava for analysis of amylase, diamine oxidase (DAO), pulmonary surfactant protein-A (SP-A), and C-reactive protein (CRP). Histopathological change of pancreas, ileum and lung was assayed by H&E staining, myeloperoxidase (MPO) activity were determinated using colorimetric assay, and the expressions of occludin and nuclear factor-κB (NF-κB) were detected by real-time RT-PCR and western blot, respectively. In addition, the tissue concentrations of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and monocyte chemoattractant protein-1 (MCP-1) were measured by enzyme-linked immunosorbent assay (ELISA). The results showed that in SAP rats, MDD significantly alleviated histopathological damage, depressed the MPO activity and the concentrations of TNF-α, IL-1ß, and MCP-1 of pancreas, ileum and lung, and reduced the serum levels of amylase [(3283.4 ± 585.5) U·L-1vs (5626.4 ± 795.1)U·L-1], DAO [(1100.1 ± 334.3) U·L-1vs (1666.4 ± 525.3) U·L-1] and CRP [(7.6 ± 1.2) µg·mL-1vs (17.8 ± 3.8) µg·mL-1]. However, the serum SP-A concentration [(106.1 ± 16.6) pg·mL-1vs (90.1 ± 14.9) pg·mL-1] was elevated when treated SAP rats with MDD. Furthermore, MDD increased the occludin expression and reduced the NF-κB expression in pancreas, ileum and lung of SAP rats. Our findings suggested that MDD administration was an effective therapeutic approach for SAP treatment. It could up-regulate occludin expression to protect intercellular tight junction and down-regulate NF-κB expression to inhibit inflammatory reaction of pancreas, ileum and lung.

The role of 2-arachidonoylglycerol in the regulation of the tumor-immune microenvironment in murine models of pancreatic cancer.

The pancreatic ductal adenocarcinoma (PDAC) microenvironment plays a critical role in the antitumor immune response. 2-arachidonoylglycerol (2-AG) exhibits a direct antitumor effect in various tumor models. However, the immunomodulatory effect of 2-AG on PDAC remains obscure. The aim of this study was to explore the tumor microenvironment response to 2-AG in pancreatic cancer. A PDAC orthotopic tumor model was used to investigate tumor proliferation and the population of immune cells in vivo, including dendritic cells (DCs), myeloid-derived suppressor cells (MDSCs), CD8+ T cells and CD4+ T cells. The effect of 2-AG on panc02 cell proliferation and DC2.4 cell maturation in vitro by mediating activation the typical cannabinoid receptors (CB1, CB2) was evaluated by flow cytometry and CCK8. The protein levels of P-STAT6, STAT6 and GADPH were measured by Western blotting.2-AG inhibited pancreatic cancer cell proliferation in tumor bearing mice and panc02 cell. Inhibition of proliferation was blocked by the CB1receptor antagonist (AM251) but not the CB2 receptor antagonist (AM630). In addition, 2-AG promoted DC phenotypic maturation and the production of proinflammatory cytokines by up-regulating p-STAT6. These effects were also blocked by AM251 but not AM630. Moreover, we also provide evidence that 2-AG administration induced the expansion of MDSCs in tumor bearing mice. However, no effect on the population of CD8+ T cells and CD4+ T cells was observed. Our findings support 2-AG exhibited direct antitumor effects via inhibiting pancreatic cancer proliferation and inducing DC phenotypic maturation, but also significantly promoted an immunosuppressive microenvironment via increasing the suppressive immune cell population of MDSCs.

Treatment with 3,4-dihydroxyphenylethyl alcohol glycoside ameliorates sepsis-induced ALI in mice by reducing inflammation and regulating M1 polarization.

The bioactive phenylethanoid 3,4-dihydroxyphenylethyl alcohol glycoside (DAG) is a component isolated from Sargentodoxa cuneata. The effects of DAG on acute lung injury (ALI) are largely unknown. Here, the effects of DAG on sepsis-induced ALI were investigated, and the related mechanisms were explored. Male C57BL/6 mice were used to establish a sepsis-induced ALI model. Levels of inflammatory cytokines were determined using real-time quantitative reverse transcription PCRs (qRT-PCR) and enzyme-linked immunosorbent assays (ELISAs). Pathological changes in the lung tissues were evaluated using haematoxylin and eosin (HE) staining. Mouse survival was quantified, and macrophage polarization was analyzed using flow cytometry. Our results showed that, in septic mice, pretreatment with DAG significantly improved survival, reduced histological damage in the lung, and suppressed the inflammatory response by inhibiting the activation of the NF-κB, STAT3, and p38 MAPK signaling pathways. Moreover, DAG treatment reduced the percentage of M1 macrophages in the bronchoalveolar lavage fluid (BALF) and spleen. In addition, DAG treatment decreased the production of pro-inflammatory cytokines and suppressed the activation of the NF-κB, STAT3, and p38 MAPK signaling pathways in LPS-induced MH-S cells. DAG treatment also reduced the relative abundances of M1 macrophages and M1 macrophage markers by suppressing the activation of the Notch1 signaling pathway. Thus, our results provided new insights for the development of drugs to treat ALI.

Purification of 3, 4-dihydroxyphenylethyl alcohol glycoside from Sargentodoxa cuneata (Oliv.) Rehd. et Wils. and its protective effects against DSS-induced colitis.

Sargentodoxa cuneata is a tropical plant used in traditional Chinese medicine to treat intestinal inflammation. In this study, 3, 4-dihydroxyphenylethyl alcohol glycoside (DAG) was purified from the stem of S. cuneata using macroporous resins and its bioactivity was also investigated. The adsorption/desorption of DAG on macroporous resins was investigated systematically. HPD300 resin was selected as the most suitable medium for DAG purification. Further dynamic absorption/desorption experiments on the HPD300 column were conducted to obtain the optimal parameters. To obtain more than 95% DAG, a second stage procedure was developed to purify the DAG using SiliaSphere C18 with 8% v/v acetonitrile through elution at low pressure. Further investigation showed that DAG pretreatment significantly reversed the shortening of colon length, the increase in the disease activity index (DAI) scores and histological damage in the colon. Moreover, DAG greatly increased SOD and GPx activities, significantly decreased MPO and MDA activities and reduced the levels of pro-inflammatory cytokines in the colon. Free radical scavenging activities of DAG were assessed using DPPH, with an IC50 value of 17.03 ug/mL. Additionally, DAG suppressed ROS and proinflammatory cytokine production in LPS-stimulated RAW 264.7 macrophages by suppressing activation of the ERK1/2 and NF-κB pathways. The results were indicative of the antioxidant and anti-inflammatory properties of DAG. When viewed together, these findings indicated that DAG can be used to expand future pharmacological research and to potentially treat colitis.

Saikosaponin d ameliorates pancreatic fibrosis by inhibiting autophagy of pancreatic stellate cells via PI3K/Akt/mTOR pathway.

Chronic pancreatitis is characterized by pancreatic fibrosis, associated with excessive activation of pancreatic stellate cells (PSCs) and increased expression of transforming growth factor-ß1 (TGF-ß1). Recently, our studies have shown that autophagy inhibitor could inhibit PSCs activation and reduce collagen secretion. Saikosaponin d (SSd), the major active component of bupleurum falcatum (a medicinal plant), has anti-fibrosis effects in liver. However, it is unclear whether SSd has a role in pancreatic fibrosis. This study aimed to investigate the effect of SSd on the autophagy and activation of PSCs in vivo and in vitro. In vivo, a rat chronic pancreatitis model was induced by intravenous injection of dibutyltin dichloride. SSd was administered at a dose of 2.0 mg/kg body weight per day by gavage. After 4 weeks, the pancreas was collected for histological and molecular analysis. In vitro, PSCs were isolated and cultured for treatment with different dosages of SSd. The results showed that SSd inhibited PSCs autophagy and activation while also reducing extracellular matrix (ECM) formation and pancreatic damage. SSd inhibited autophagy through activating the PI3K/Akt/mTOR pathway. SSd also promoted degradation of ECM with an increasing ratio of MMPs/TIMPs and suppressed the TGF-ß1/Smads pathway. From these results, we concluded that SSd prevents pancreatic fibrosis by reducing autophagy of PSCs through PI3K/Akt/mTOR pathway, which has crosstalk with the TGF-ß1/Smads pathway.

Resolvin D1 Promotes SIRT1 Expression to Counteract the Activation of STAT3 and NF-κB in Mice with Septic-Associated Lung Injury.

Resolvin D1 (RvD1) is a novel endogenous docosahexaenoic acid (DHA)-derived lipid mediators, which possesses a dual role of anti-inflammation and promotes inflammation resolution. The aim of the present study was to assess the effects of RvD1 on cecal ligation and puncture (CLP) model of sepsis and explore the underlying mechanism. Six-to-eight-week-old male C57BL/6 mice were randomly divided into following three groups: sham-operated group (SO), CLP model group (CLP), and CLP+RvD1 group (RvD1). The SO group underwent the sham operation. The RvD1 groups were administered RvD1 (10-ng/g body weight) by penile vein injection, but the CLP groups were administered the same volume of vehicle (PBS) after CLP. We assessed the survival benefit of RvD1 in CLP-induced septic mice for 7 days. After 24 h, mice were sacrificed, bronchoalveolar lavage fluids (BALF) was collected for proinflammatory cytokines assay, and albumin assay and the lung tissues were harvested for histologic analysis, myeloperoxidase (MPO) activity and the expression of Sirtuin 1 (SIRT1), signal transducers, and activators of transcription 3 (STAT3), nuclear factor-κB (NF-κB), and mitogen-activated protein kinases (MAPKs). RvD1 treatment increased the survival time in mice with sepsis induced by CLP, reducing the MPO activity and albumin level at 24 h. The levels of inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and interleukin-6 (IL-6) in BALF were significantly decreased by RvD1. RvD1 promoted SIRT1 expression and suppressed the activation of NF-κB, STAT3, ERK, and p38 in lung tissues of septic mice. These results suggest that RvD1 may improve survival and attenuate the degree of lung inflammation reaction in mice with CLP by suppressing STAT3, NF-κB, ERK, and p38 expressions through a mechanism partly dependent on SIRT1.

Xuebijing Injection () and Resolvin D1 Synergize Regulate Leukocyte Adhesion and Improve Survival Rate in Mice with Sepsis-Induced Lung Injury.

OBJECTIVE: To investigate the effect of combined application of Xuebijing Injection ( , XBJ) and resolvin D1 (RvD1) on survival rate and the underlying mechanisms in mice with sepsisinduced lung injury. METHODS: The cecal ligation and puncture (CLP) method was used to develop a mouse sepsis model. Specific pathogen free male C57BL/6 mice were randomly divided into 5 groups (n=20 each): sham, CLP, CLP+XBJ, CLP+RvD1 and CLP+XBJ+RvD1. After surgery, mice in the CLP+XBJ, CLP+RvD1 and CLP+XBJ+RvD1 groups were given XBJ (25 µL/g body weight), RvD1 (10 ng/g body weight), and their combination (the same dose of XBJ and RvD1), respectively. In each group, 12 mice were used to observe 1-week survival rate, while the rest were executed at 12 h. Whole blood was collected for flow cytometric analysis of leukocyte adhesion molecules CD18, lung tissues were harvested for observing pathological changes, and testing the activity of myeloperoxidase (MPO) and the expression of intercellular cell adhesion molecule 1 (ICAM-1). RESULTS: Compared with the CLP group, the histopathological damage of the lung tissues was mitigated, MPO activity was decreased in the CLP+XBJ and CLP+RvD1 groups (P<0.05). In addition, the 1-week survival rate was improved, proportion of CD18-expressing cells in whole blood and ICAM-1 protein expression in lung tissue were decreased in the CLP+XBJ+RvD1 group (P<0.05 or P<0.01). CONCLUSIONS: XBJ together with RvD1 could effectively inhibit leukocyte adhesion, reduce lung injury, and improve the survival rate of mice with sepsis.

Protective Role of Liriodendrin in Sepsis-Induced Acute Lung Injury.

In current study, we investigated the role of liriodendrin, a constituent isolated from Sargentodoxa cuneata (Oliv.) Rehd. Et Wils (Sargentodoxaceae), in cecal ligation and puncture (CLP)-induced acute lung inflammatory response and injury (ALI). The inflammatory mediator levels in bronchoalveolar lavage fluid (BALF) were determined by enzyme-linked immunosorbent assay (ELISA). Pathologic changes in lung tissues were evaluated via pathological section with hematoxylin and eosin (H&E) staining. To investigate the mechanism whereby liriodendrin regulates lung inflammation, the phosphorylation of the NF-kB (p65) and expression of vascular endothelial growth factor (VEGF) were determined by western blot assay. We show that liriodendrin treatment significantly improved the survival rate of mice with CLP-induced sepsis. Pulmonary histopathologic changes, alveolar hemorrhage, and neutrophil infiltration were markedly decreased by liriodendrin. In addition, liriodendrin decreased the production of the proinflammatory mediators including (TNF-α, IL-1ß, MCP-1, and IL-6) in lung tissues. Vascular permeability and lung myeloperoxidase (MPO) accumulation in the liriodendrin-treated mice were substantially reduced. Moreover, liriodendrin treatment significantly suppressed the expression of VEGF and activation of NF-kB in the lung. We further show that liriodendrin significantly reduced the production of proinflammatory mediators and downregulated NF-kB signaling in LPS-stimulated RAW 264.7 macrophage cells. Moreover, liriodendrin prevented the generation of reactive oxygen species (ROS) by upregulating the expression of SIRT1 in RAW 264.7 cells. These findings provide a novel theoretical basis for the possible application of liriodendrin in clinic.