Dachaihu decoction ameliorates pancreatic fibrosis by inhibiting macrophage infiltration in chronic pancreatitis

AIM To explore the role of macrophages in chronic pancreatitis(CP) and the effect of Dachaihu decoction(DCHD) on pancreatic fibrosis in mice.METHODS Kun Ming mice were randomly divided into a control group, CP group, and DCHD group. In the CP and DCHD groups, mice were intraperitoneally injected with 20% L-arginine(3 g/kg twice 1 d/wk for 6 wk). Mice in the DCHD group were administered DCHD intragastrically at a dose of 14 g/kg/d 1 wk after CP induction. At 2 wk, 4 wk and 6 wk post-modeling, the morphology of the pancreas was observed using hematoxylin and eosin, and Masson staining. Interleukin-6(IL-6) serum levels were assayed using an enzyme-linked immunosorbent assay. Double immunofluorescence staining was performed to observe the co-expression of F4/80 and IL-6 in the pancreas. Inflammatory factors including monocyte chemoattractant protein-1(MCP-1), macrophage inflammatory protein-1α(MIP-1α) and IL-6 were determined using real time-polymerase chain reaction. Western blot analysis was used to detect fibronectin levels in the pancreas. RESULTS Compared with the control group, mice with 20% L-arginine-induced CP had obvious macrophage infiltration and a higher level of fibrosis. IL-6 serum concentrations were significantly increased. Double immunofluorescence staining showed that IL-6 and F4/80 were co-expressed in the pancreas. With the administration of DCHD, the infiltration of macrophages and degree of fibrosis in the pancreas were significantly attenuated; IL-6, MCP-1 and MIP-1α m RNA, and fibronectin levels were reduced. CONCLUSION The dominant role of macrophages in the development of CP was mainly related to IL-6 production. DCHD was effective in ameliorating pancreatic fibrosis by inhibiting macrophage infiltration and inflammatory factor secretion in the pancreas.

An immortalized rat pancreatic stellate cell line RP-2 as a new cell model for evaluating pancreatic fibrosis,inflammation and immunity

BACKGROUND： Pancreatic stellate cells （PSCs） play a critical role in the pathogenesis of pancreatic fibrosis and have emerging functions as progenitor cells, immune cells or intermediaries in pancreatic exocrine secretion. Increasing evidence has shown that desmin as an exclusive cytoskeleton marker of PSC is only expressed in part of these cells. This study was to establish a desmin-positive PSC cell line and evaluate its actions on pancreatic fibrosis, inflammation and immunity.

Pancreatic parenchymal injection of ethanol and octreotide to induce focal pancreatic fibrosis in rats: Strategies to eliminate postoperative pancreatic fistula

Background: Postoperative pancreatic fistula(POPF) is more likely to occur in a soft pancreas compared to a hard pancreas in which fibrosis has progressed. There is almost no leakage at the anastomosis site or cut surface of a hard pancreas. The aim of this study was to induce localized fibrosis at the cut surface of the pancreas in a rat model.Methods: Thirty-six rats were divided into three groups(group S: normal saline group; group E: ethanol group; and group O: octreotide group). Each rat was directly injected with a particular compound at the duodenal lobe of the pancreatic parenchyma. Each group was divided into three subgroups according to the time of post-injection sacrifice(1, 2, or 4 weeks). The hardness, suture holding capacity(SHC), and histological fibrosis grade of each pancreas were measured.Results: The hardness, SHC, and fibrosis grade of groups E and O were increased at week 1, with greater increases in group E(all P < 0.001). In a subgroup comparison, the hardness, SHC, and fibrosis grade of group E tended to decrease gradually over time, with no regular pattern evident in group O. A comparison between the injected site(duodenal lobe) and non-injected site(splenic lobe) of the pancreas revealed increases in the three parameters of group E only in the duodenal lobe, with increases in group O at both the duodenal and splenic lobes.Conclusions: Parenchymal injection of ethanol and octreotide increased pancreatic fibrosis. Unlike octreotide, ethanol provoked localized fibrosis that was maintained over time. It is expected that ethanol injection could eliminate POPF during pancreatic surgery.

A PDGFRβ-targeting nanodrill system for pancreatic fibrosis therapy

Activated pancreatic stellate cells(PSCs)are the main source of collagen layer deposition and the key target in pancreatic fibrosis.However,no effective treatment specific to pancreatic fibrosis clinically,owing to the drug accumulation blocked by the collagen barrier and thus it is difficult to inhibit activated PSCs precisely.Herein,a PSCs-targeting nano-system based on“nanodrill”strategy(LA-PC)was designed to enhance the accumulation of all-trans retinoic acid(ATRA)in PSCs,relying on the platelet-derived growth factor receptor beta(PDGFRβ)-targeting peptide(p PB:C*SRNLIDC*)and collagenase(Col).After being injected into fibrotic mice via tail vein,the Col modified on LA-PC can remove the excess collagen layer,and the drug delivery efficiency through pPB targeting peptide was more than 5 times higher than that of free ATRA,as well as the degree of fibrosis significantly reduced.Notably,this nano-system effectively inhibited platelet-derived growth factor subunit B(PDGF-BB)/PDGFRβaxis on PSCs via a down-regulated extracellular signal-regulated protein kinase(ERK)pathway,and accordingly reduced the level of PDGFBB.Thus,the smart platform provided a promising strategy for the treatment of pancreatic fibrosis to achieve the precise regulation of PSCs.

Activation of JAK-STAT pathway is required for platelet-derived growth factor-induced proliferation of pancreatic stellate cells

AM: To clarify the role of Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway in platelet-derived growth factor (PDGF) induced proliferation in activated pancreatic stellate cells (PSCs). METHODS: PSCs were isolated from rat pancreas tissue, and used in their culture-activated, myofibroblast-like phenotype. STAT-specific binding activity was assessed by electrophoretic mobility shift assay. Activation of Src, JAK2, STAT1, STAT3, and ERK was determined by Western blotting using anti-phosphospecific antibodies. Cell proliferation was assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine. RESULTS: PDGF-BB induced STAT-specific binding activity, and activation of Src, JAK2, STAT1, STAB, and ERK. Ethanol and acetaldehyde at clinically relevant concentrations decreased basal activation of JAK2 and STAT3. PDGF-induced activation of STAT1 and STAT3 was inhibited by a Src inhibitor PP1 and a JAK2 inhibitor AG490, whereas PDGF-induced activation of ERK was inhibited by PP1, and not by AG490. PDGF-induced proliferation was inhibited by PP1 and AG490 as well as by STAT3 antisense oligonucleotide. CONCLUSION: PDGF-BB activated JAK2-STAT pathway via Src-dependent mechanism. Activation of 3AK2-STAT3 pathway, in addition to ERK, may play a role in PDGF-induced proliferation of PSCs.

Endothelin-1 stimulates contraction and migration of rat pancreatic stellate cells

AIM： To examine the ability of FT-1 to affect the cell functions of PSCs and the underlying molecular mechanisms. METHODS： PSCs were isolated from the pancreas of male Wistar rats after perfusion with collagenase, and cells between passages two and five were used. Expression of ET-1 and FT receptors was assessed by reverse transcription-PCR and immunostaining. Phosphorylation of myosin regulatory light chain （MLC）, extracellular-signal regulated kinase （FRK）, and Akt was examined by Western blotting. Contraction of PSCs was assessed on hydrated collagen lattices. Cell migration was examined using modified Boyden chambers. Ceil proliferation was assessed by measuring the incorporation of 5-bromo-2＇- deoxyuridine. RESULTS： Culture-activated PSCs expressed ETA and ETB receptors, and ET-1. ET-1 induced phosphorylation of NLC and FRK, but not Akt. ET-1 induced contraction and migration, but did not alter proliferation of PSCs. FT-1-induced contraction was inhibited by an ETA receptor antagonist BQ-123 and an ETB receptor antagonist BQ-788, whereas migration was inhibited by BQ-788 but not by BQ-123. A Rho kinase inhibitor Y-27632 abolished both contraction and migration. CONCLUSION： ET-1 induced contraction and migration of PSCs through El receptors and activation of Rho-Rho kinase. ETA and FTB receptors play different roles in the regulation of these cellular functions in response to ET-1.

Green tea polyphenol epigallocatechin-3-gallate blocks PDGF-induced proliferation and migration of rat pancreatic stellate cells

AIM: To clarify the effects of epigallocatechin-3-gallate (EGCG) on the platelet-derived growth factor (PDGF)-BB-induced proliferation and migration of pancreatic stellate cells (PSCs). METHODS: PSCs were isolated from rat pancreas tissue and used in their culture-activated, myofibroblast-like phenotype. Cell proliferation was assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine. Cell migration was assessed using modified Boyden chambers. Cyclin D1, p21waf1, and p27kip1 expression and phosphorylation of PDGF β-receptor, extracellular signal-regulated kinase, and Akt were examined by Western blotting. Activation of phospha-tidylinositol 3-kinase was examined by kinase assay using phosphatidylinositol as a substrate. Cell cycle was assessed by flow cytometry after staining with propidium iodide. RESULTS: EGCG at non-cytotoxic concentrations inhibited PDGF-induced proliferation and migration. This effect was associated with the inhibition of cell cycle progression beyond the G1 phase, decreased cyclin Dl and increased p27kip1 expression. EGCG inhibited tyrosine phosphorylation of PDGF p-receptor and downstream activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/ Akt pathways. CONCLUSION: EGCG inhibited PDGFBB-induced proliferation and migration of PSCs through the inhibition of PDGFmediated signaling pathways.

Inhibition of pancreatic stellate cell activity by adipose-derived stem cells

BACKGROUND：Pancreatic stellate cells（PSCs）play a critical role in the development of pancreatic fibrosis.In this study we used a novel method to isolate and culture rat PSCs and then investigated the inhibitory effects of adipose-derived stem cells（ADSCs）on activation and proliferation of PSCs.METHODS：Pancreatic tissue was obtained from SpragueDawley rats for PSCs isolation.Transwell cell cultures were adopted for co-culture of ADSCs and PSCs. PSCs prolifera- tion and apoptosis were determined using CCK-8 and flow cytometry, respectively, a-SMA expressions were analyzed using Western blotting. The levels of cytokines [nerve growth factor （NGF）, interleukin-10 （IL-10） and transforming growth factor-ill （TGF-[31）] in conditioned medium were detected by ELISA. Gene expression （MMP-2, MMP-9 and TIMP-1） was analyzed using qRT-PCR. RESULTS： This method produced 17.6_＋6.5 ~ 103 ceils per gram of the body weight with a purity of 90%-95% and a viability of 92%-97%. Co-culture of PSCs with ADSCs significantly inhib- ited PSCs proliferation and induced PSCs apoptosis. Moreover, a-SMA expression was significantly reduced in PSCs＋ADSCs compared with that in PSC-only cultures, while expression of fibrinolytic proteins （e.g., MMP-2 and MMP-9） was upregulated and anti-fibrinolytic protein （TIMP-1） was downregulated. In addition, NGF expression was up-regulated, but IL-10 and TGF-β1 expressions were down-regulated in the coculture conditioned medium compared with those in the PSC- only culture medium. CONCLUSIONS： This study provided an easy and reliable technique to isolate PSCs. The data demonstrated the inhibitory effects of ADSCs on the activation and proliferation of PSCs in vitro.

Enhanced expression of interleukin-18 in serum and pancreas of patients with chronic pancreatitis

AIM： To investigate interleukin-18 （IL-18） in patients with chronic panreatitis （CP）. METHODS： We studied 29 patients with CP and 30 healthy controls. Peripheral blood mononuclear cells （PBMC） were isolated and incubated with 50 mmol/L ethanol, lipopolysaccharide （LPS） （doses 25 g/L, 250 g/L, 2500 g/L） and both agents for 24 h. Levels of IL-18 in the supernatants, and levels of IL-18, IL-12, interferon （IFN）-T and soluble CD14 in the serum were analysed by EI_ISA technique. Expression of IL-18 in PBMC was investigated by reverse-transcription （RT）-PCR. IL-18 protein levels in CP tissue and in normal pancreas were studied by ELISA technique. IL-18 levels in PBMC and pancreatic tissue were determined by Westernblot. Immunohistochemistry for pancreatic IL-18 expression was performed.RESULTS： In patients, IL-18 serum levels were significantly enhanced by 76% （mean： 289.9 ± 167.7 ng/L） compared with controls （mean： 165.2 ± 43.6 ng/L; P 〈 0.0005）. IL-12 levels were enhanced by 25% in patients （18.3 ± 7.3 ng/L） compared with controls （14.7 ± 6.8 ng/L, P = 0.0576） although not reaching the statistical significance. IFN-γ, and soluble CD14 levels were not increased. In vitro, LPS stimulated significantly and dosedependently IL-18 secretion from PBMC. Incubation with ethanol reduced LPS-stimulated IL-18 secretion by about 50%. The mRNA expression of IL-18 in PBMC and the response of PBMC to ethanol and LPS was similar in CP patients and controls. In PBMC, no significant differences in IL-18 protein levels were detected between patients and controls. IL-18 protein levels were increased in CP tissues compared to normal pancreatic tissues. IL-18 was expressed by pancreatic acinar cells and by infiltrating inflammatory cells within the pancreas. CONCLUSION： IL-18 originates from the chronically inflammed pancreas and appears to be involved in the fibrotic destruction of the organ.

Comprehensive review of diagnostic modalities for early chronic pancreatitis

Chronic pancreatitis(CP)is a progressive condition caused by several factors and characterised by pancreatic fibrosis and dysfunction.However,CP is difficult to diagnose at an early stage.Various advanced methods including endoscopic ultrasound based elastography and confocal laser endomicroscopy have been used to diagnose early CP,although no unified diagnostic standards have been established.In the past,the diagnosis was mainly based on imaging,and no comprehensive evaluations were performed.This review describes and compares the advantages and limitations of the traditional and latest diagnostic modalities and suggests guidelines for the standardisation of the methods used to diagnose early CP.

Nanomedicine regulating PSC-mediated intercellular crosstalk:Mechanisms and therapeutic strategies

Pancreatic fibrosis(PF)is primarily distinguished by the stimulation of pancreatic stellate cells(PSCs)and excessive extracellular matrix deposition,which is the main barrier impeding drug delivery and distribution.Recently,nanomedicine,with efficient,targeted,and controllable drug release characteristics,has demonstrated enormous advantages in the regression of pancreas fibrotic diseases.Notably,paracrine signals from parenchymal and immune cells such as pancreatic acinar cells,islet cells,pancreatic cancer cells,and immune cells can directly or indirectly modulate PSC differentiation and activation.The intercellular crosstalk between PSCs and these cells has been a critical event involved in fibrogenesis.However,the connections between PSCs and other pancreatic cells during the progression of diseases have yet to be discussed.Herein,we summarize intercellular crosstalk in the activation of PSCs and its contribution to the development of common pancreatic diseases,including pancreatitis,pancreatic cancer,and diabetes.Then,we also examine the latest treatment strategies of nanomedicine and potential targets for PSCs crosstalk in fibrosis,thereby offering innovative insights for the design of antifibrotic nanomedicine.Ultimately,the enhanced understanding of PF will facilitate the development of more precise intervention strategies and foster individually tailored therapeutic approaches for pancreatic diseases.