Inhibited Pancreatic Stellate Cells Activation Research Articles

A Nanodrug Simultaneously Inhibits Pancreatic Stellate Cell Activation and Regulatory T cell Infiltration to Promote the Immunotherapy of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense extracellular matrix flooded with immune suppressive cells, resulting in extremely poor clinical response to immunotherapy. It has been revealed that the activation of pancreatic stellate cells (PSCs) makes considerable contributions to the immunological "cold" tumor microenvironment (TME). Herein, we developed a polyamino acid-based nanodrug incorporating the PSC activation inhibitor calcipotriol and anti-CXCL12 siRNA. The nanodrug was easily prepared with a small particle size and is capable of penetrating pancreatic tumors to inactivate PSCs and downregulate CXCL12. The in vivo results of orthotopic pancreatic tumor treatment demonstrated that codelivery of calcipotriol and anti-CXCL12 siRNA remodeled the PDAC TME with reduced extracellular matrix and decreased immunosuppressive T cells. Eventually, the infiltration of cytotoxic T cells was increased, thereby acting with immune checkpoint blockade (ICB) therapy for immunologically "cold" pancreatic tumors. In the present study, we propose a promising paradigm to improve the immunotherapy outcome of PDAC using nanodrugs that synchronously inhibit PSC activation and regulatory T-cell infiltration. STATEMENT OF SIGNIFICANCE: Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense extracellular matrix (ECM) that impedes the tumor infiltration of therapeutic agents and cytotoxic T lymphocytes, resulting in a poor clinical response to immunotherapy. In the present study, we proposed a promising approach for enhanced immunotherapy of pancreatic cancer. Specifically, a nanodrug incorporating calcipotriol and anti-CXCL12 siRNA was synthesized to synchronously inactivate matrix-producing pancreatic stellate cells and suppress the infiltration of regulatory T cells. The reduced ECM removed the pathological barrier, preventing nanodrug penetration and effector T-cell infiltration, leading to a conversion of the immunosuppressive "cold" microenvironment to a "hot" microenvironment, which eventually boosted the immunotherapy of anti-PD-1 antibodies in pancreatic cancer.

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.

Saikosaponin A inhibits the activation of pancreatic stellate cells by suppressing autophagy and the NLRP3 inflammasome via the AMPK/mTOR pathway

Pancreatic stellate cells (PSCs) are the main effector cells in the development of pancreatic fibrosis. Finding substances that inhibit PSC activation is an important approach to inhibiting pancreatic fibrosis. Saikosaponin A (SSa) has numerous pharmacological activities, but its effect on PSCs remains unknown. This study was conducted to explore the effects of SSa on PSC activation in cultured rat PSCs. Cell viability, proliferation, migration and apoptosis were evaluated by MTT assays, the iCELLigence System, Transwell assays and flow cytometry. Markers of PSC activation, autophagy and the NLRP3 inflammasome were measured by real-time PCR, immunofluorescence and western blotting. Rapamycin and phenformin hydrochloride were used to determine the effect of SSa via the AMPK/mTOR pathway. The results showed that SSa suppressed PSC viability, proliferation, and migration and promoted apoptosis. SSa inhibited PSC activation, restrained PSC autophagy and suppressed the NLRP3 inflammasome. In addition, there was interaction between autophagy and the NLRP3 inflammasome during SSa inhibition of PSCs. Moreover, promotion of p-AMPK increased autophagy and the NLRP3 inflammasome. Inhibition of p-mTOR increased autophagy and decreased the NLRP3 inflammasome. Our results indicated that SSa inhibited PSC activation by inhibiting PSC autophagy and the NLRP3 inflammasome via the AMPK/mTOR pathway. These findings provide a theoretical basis for the use of SSa to treat pancreatic fibrosis and further suggest that targeting autophagy and the NLRP3 inflammasome may provide new strategies for the treatment of pancreatic fibrosis.

Inhibition of PAK1 suppresses pancreatic cancer by stimulation of anti-tumour immunity through down-regulation of PD-L1.

Immunotherapies have not yielded significant clinical benefits for pancreatic ductal adenocarcinoma (PDA) because of the existence of an immunosuppressive tumour microenvironment (TME) characterized by a desmoplastic stroma containing infiltrated immune cells and activated pancreatic stellate cells (PSCs). This study aims to investigate the involvement of PAK1 in anti-tumour immunity. In PDA patients, low PAK1 expression, low activation of PSC and high CD8+ T cell/PAK1 ratios correlated with longer overall survival. In a murine PDA model, PAK1 knockout increased intra-tumoral CD4+ and CD8+ T cells, inhibited PSCs activation and extended survival. Inhibition of PAK1 reduced PSC-stimulated PDA cell proliferation and migration, blocked PSC-mediated protection of PDA cells from killing by cytotoxic lymphocytes and decreased intrinsic and PSC-stimulated PD-L1 expression in PDA cells, which further sensitized PDA cells to cytotoxic lymphocytes. Inhibition of PAK1 stimulates anti-tumour immunity by increasing intra-tumoral CD4+ and CD8+ T cells, and by sensitizing PDA cells to killing by cytotoxic lymphocytes via down-regulation of intrinsic and PSC-stimulated PD-L1 expression. PAK1 inhibitors, especially in combination with immune checkpoint inhibitors may result in improved efficacy of immunotherapy of PDA.

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-β1 (TGF-β1). 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-β1/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-β1/Smads pathway.

Suppression of ROS Production by Exendin-4 in PSC Attenuates the High Glucose-Induced Islet Fibrosis.

Pancreatic stellate cells (PSCs) play a major role to fibrotic islet destruction observed in diabetic patients and animal model of diabetes. Exendin-4 (Ex-4) is a potent insulinotropic agent and has been approved for the treatment of type 2 diabetes. However, there have been no reports demonstrating the effects of Ex-4 on pancreatic islet fibrosis. In this study, Ex-4 treatment clearly attenuated fibrotic islet destruction and improved glucose tolerance and islet survival. GLP-1 receptor expression was upregulated during activation and proliferation of PSCs by hyperglycemia. The activation of PKA pathway by Ex-4 plays a role in ROS production and angiotensin II (Ang II) production. Exposure to high glucose stimulated ERK activation and Ang II-TGF- β1 production in PSCs. Interestingly, Ex-4 significantly reduced Ang II and TGF-β1 production by inhibition of ROS production but not ERK phosphorylation. Ex-4 may be useful not only as an anti-diabetic agent but also as an anti-fibrotic agent in type 2 diabetes due to its ability to inhibit PSC activation and proliferation and improve islet fibrosis in OLETF rats.

Generation of an in vitro 3D PDAC stroma rich spheroid model

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prominent desmoplastic/stromal reaction, which contributes to the poor clinical outcome of this disease. Therefore, greater understanding of the stroma development and tumor-stroma interactions is highly required. Pancreatic stellate cells (PSC) are myofibroblast-like cells located in exocrine areas of the pancreas, which as a result of inflammation produced by PDAC migrate and accumulate in the tumor mass, secreting extracellular matrix components and producing the dense PDAC stroma. Currently, only a few orthotopic or ectopic animal tumor models, where PDAC cells are injected into the pancreas or subcutaneous tissue layer, or genetically engineered animals offer tumors that encompass some stromal component. Herein, we report generation of a simple 3D PDAC invitro micro-tumor model without an addition of external extracellular matrix, which encompasses a rich, dense and active stromal compartment. We have achieved this invitro model by incorporating PSCs into 3D PDAC cell culture using a modified hanging drop method. It is now known that PSCs are the principal source of fibrosis in the stroma and interact closely with cancer cells to create a tumor facilitatory environment that stimulates local and distant tumor growth. The 3D micro-stroma models are highly reproducible with excellent uniformity, which can be used for PDAC-stroma interaction analysis and high throughput automated drug-screening assays. Additionally, the increased expression of collagenous regions means that molecular based perfusion and cytostaticity of gemcitabine is decreased in our Pancreatic adenocarcinoma stroma spheroids (PDAC-SS) model when compared to spheroids grown without PSCs. We believe this model will allow an improved knowledge of PDAC biology and has the potential to provide an insight into pathways that may be therapeutically targeted to inhibit PSC activation, thereby inhibiting the development of fibrosis in PDAC and interrupting PSC-PDAC cell interactions so as to inhibit cancer progression.

Effects of Octreotide on Activated Pancreatic Stellate Cell-Induced Pancreas Graft Fibrosis in Rats

Of solid organ transplantations, pancreas transplantation is associated with the highest incidence of pancreatic fibrosis in the early post-transplantation period. Activated pancreatic stellate cells (PSCs) are the main source of pancreatic fibrosis. Octreotide is widely used as a prophylactic for postoperative complications in pancreas transplant recipients. Recent studies have shown that it can inhibit liver fibrosis. This study investigated the effect of octreotide in pancreas graft fibrosis in rats. Isolated PSCs from Sprague Dawley rats were co-cultured with different doses of octreotide (1.25, 2.5, 5, 10, 20, and 40 ng/mL). PSC proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide at 48, 72, and 96 h. The α-smooth muscle actin (α-SMA) and collagen I expressions of PSCs were detected by immunohistochemistry and reverse-transcriptase polymerase chain reaction. Rat heterotopic pancreaticoduodenal transplantation was performed with and without octreotide treatment (0.01 mg/kg). Pancreas grafts were harvested at postoperative d 1, 3, 5, and 7. Hematoxylin-eosin staining, Masson's trichrome staining, and immunohistochemical staining for α-SMA, collagen I, and tumor growth factor-β1 (TGF-β1) were performed. Octreotide at a concentration of >20 ng/mL significantly inhibited PSC activation and proliferation in vitro. Inflammatory infiltration was reduced in the octreotide group in vivo, and the expression levels of α-SMA, collagen I, and TGF-β1 were also lower, with statistic significant difference or not. Masson's trichrome staining showed a decrease in collagen deposition with octreotide treatment. Octreotide effectively inhibits PSC activation and proliferation in vitro, but has a limited inhibitory effect on the development of pancreas graft fibrosis.

New insights into alcoholic pancreatitis and pancreatic cancer

Pancreatitis and pancreatic cancer represent two major diseases of the exocrine pancreas. Pancreatitis exhibits both acute and chronic manifestations. The commonest causes of acute pancreatitis are gallstones and alcohol abuse; the latter is also the predominant cause of chronic pancreatitis. Recent evidence indicates that endotoxinemia, which occurs in alcoholics due to increased gut permeability, may trigger overt necroinflammation of the pancreas in alcoholics and one that may also play a critical role in progression to chronic pancreatitis (acinar atrophy and fibrosis) via activation of pancreatic stellate cells (PSCs). Chronic pancreatitis is a major risk factor for the development of pancreatic cancer, which is the fourth leading cause of cancer-related deaths in humans. Increasing attention has been paid in recent years to the role of the stroma in pancreatic cancer progression. It is now well established that PSCs play a key role in the production of cancer stroma and that they interact closely with cancer cells to create a tumor facilitatory environment that stimulates local tumor growth and distant metastasis. This review summarizes recent advances in our understanding of the pathogenesis of alcoholic pancreatitis and pancreatic cancer, with particular reference to the central role played by PSCs in both diseases. An improved knowledge of PSC biology has the potential to provide an insight into pathways that may be therapeutically targeted to inhibit PSC activation, thereby inhibiting the development of fibrosis in chronic pancreatitis and interrupting stellate cell-cancer cell interactions so as to retard cancer progression.

Pancreatic stellate cells: Molecular mechanism of pancreatic fibrosis

Pancreatic stellate cells (PSC) are known to play a crucial role in pancreatic fibrogenesis in chronic pancreatitis and in the desmoplastic reaction of pancreatic cancer. When PSC are stimulated by oxidative stress, the phenotype of quiescent fat-storing cells converts to myofibroblast-like activated PSC that then produce extracellular matrix (ECM), adhesion molecules, and various chemokines in response to inflammatory cytokines, chemokines, and growth factors. Platelet-derived growth factor (PDGF) is a potent stimulator of PSC proliferation, and transforming growth factor-beta, PDGF, and basic fibroblast growth factor stimulate ECM synthesis by PSC. As induction of acinar cell apoptosis and necrosis is a major mechanism in the pathogenesis of mild to severe acute pancreatitis, the rapid removal of apoptotic acinar cells may be helpful in preventing the release of toxic intracellular materials, preventing further progression of the inflammation, and restoring normal tissue homeostasis. Although PSC are not professional phagocytes, we have demonstrated that PSC engulf apoptotic polymorphonuclear neutrophils (PMN) and necrotic acinar cells. Apoptotic PMN are ingested into the cytoplasm of the PSC, and troglitazone induces a dose-dependent increase in both phagocytic activity and expression of CD36, which is a representative scavenger receptor for phagocytic function. Engulfment of necrotic acinar cells by PSC induced PSC death and inhibited PSC activation in an in vitro study, suggesting that engulfment of necrotic acinar cells by PSC may inhibit the progression of fibrogenesis. These findings suggest that engulfment of damaged cells by PSC may be one of the mechanisms that prevent the progression of pancreatitis and restore tissue homeostasis.

PD98059 inhibited the activation of pancreatic stellate cells mediated by platelet-derived growth factor BB in rats.

To determine the biological effects of extracelluar signal regulated kinase (ERK) specific inhibitor PD98059 on pancreatic stellate cells (PSCs) activated by platelet-derived factor-BB (PDGF-BB), cultured rat PSCs were co-incubated at 37 DC for 24 h with 25 ng/ml PDGF-BB and different doses of PD98059 (ranging from 5 ng/ml to 40 ng/ml). Expression of pERK1 protein was detected by Western blot and collagen alpha1 ( I ) mRNA was tested by reverse transcription-polymerase chain reaction (RT-PCR). Our results showed that there were statistical differences in the expression of pERK1 protein in all groups. Expression of pERK1 protein was up-regulated in the group treated by PDGF-BB, and gradually down-regulated in the other groups treated by PD98059 of different doses. An excellent positive correlation was revealed between the inhibitory effect and PD98059 concentrations. It was also observed that the expression of collagen alpha1 ( I ) mRNA had similar response to pERK1. The level of collagen alpha1 ( I ) mRNA was the highest in the PDGF-BB group, and gradually reduced in the other groups treated by PD98059 of different doses. It is concluded that expression of pERK1 protein and collagen alpha1 ( I ) mRNA was up-regulated in rat PSCs activated by PDGF-BB. Meanwhile, PD98059 could inhibit PSCs activation mediated by PDGF. It is suggested that ERK1 protein plays an important role on PSCs activation mediated by PDGF signal pathway.

Distinct Roles of Smad2-, Smad3-, and ERK-dependent Pathways in Transforming Growth Factor-β1 Regulation of Pancreatic Stellate Cellular Functions

Pancreatic stellate cells (PSCs) play a major role in promoting pancreatic fibrosis. Transforming growth factor-beta(1) (TGF-beta(1)) regulates PSC activation and proliferation in an autocrine manner. The intracellular signaling pathways of the regulation were examined in this study. Immunoprecipitation and immunocytochemistry revealed that Smad2, Smad3, and Smad4 were functionally expressed in PSCs. Adenovirus-mediated expression of Smad2, Smad3, or dominant-negative Smad2/3 did not alter TGF-beta(1) mRNA expression level or the amount of autocrine TGF-beta(1) peptide. However, expression of dominant-negative Smad2/3 inhibited PSC activation and enhanced their proliferation. Co-expression of Smad2 with dominant-negative Smad2/3 restored PSC activation inhibited by dominant-negative Smad2/3 expression without changing their proliferation. By contrast, co-expression of Smad3 with dominant-negative Smad2/3 attenuated PSC proliferation enhanced by dominant-negative Smad2/3 expression without altering their activation. Exogenous TGF-beta(1) increased TGFbeta(1) mRNA expression in PSCs. However, PD98059, a specific inhibitor of mitogen-activated protein kinase kinase (MEK1), inhibited ERK activation by TGF-beta(1), and consequently attenuated TGF-beta(1) enhancement of its own mRNA expression in PSCs. We propose that TGF-beta(1) differentially regulates PSC activation, proliferation, and TGF-beta(1) mRNA expression through Smad2-, Smad3-, and ERK-dependent pathways, respectively.