Protease-activated Receptor-2 Protein Expression Research Articles

Evaluation of Cisplatin-Induced Acute Renal Failure Amelioration Using Fondaparinux and Alteplase.

Acute renal failure (ARF) is a deleterious condition with increased mortality or healthcare costs or dialysis-dependent end-stage renal disease. The study aims to compare prophylaxis with fondaparinux (Fund) vs. treatment with alteplase (Alt) in ameliorating cisplatin (Cis)-induced ARF. Sixty male mice were equally divided randomly into six groups of control, Cis, Alt, and Cis + Alt groups receiving normal saline for 10 days. All four groups except for the control received Cis (30 mg/kg, i.p.) on day 7, and 6 h later, both the Alt groups received Alt (0.9 mg/kg, i.v.). The animal groups Fund and Fund + Cis received Fund (5 mg/kg, i.p.) for 10 days, and the Fund + Cis group on day 7 received Cis. All the animal groups were euthanized 72 h after the Cis dose. The Fund + Cis group showed significantly increased expression levels of platelet count, retinoid X receptor alpha (RXR-α) and phosphorylated Akt (p-Akt) in addition to decreased levels of urea, blood urea nitrogen (BUN), uric acid, white blood cells (WBCs), red blood cells (RBCs), relative kidney body weight, kidney injury score, glucose, prothrombin (PT), A Disintegrin And Metalloproteinases-10 (ADAM10), extracellular matrix deposition, protease-activated receptor 2 (PAR-2), and fibrinogen expression when compared to the Cis-only group. Meanwhile, the Cis + Alt group showed increased caspase-3 expression in addition to decreased levels of urea, BUN, uric acid, WBCs, RBCs, glucose, platelet count and PT expression with a marked decrease in PAR-2 protein expression compared to the Cis group. The creatinine levels for both the Fund + Cis and Cis + Alt groups were found to be comparable to those of the Cis-only group. The results demonstrate that the coagulation system's activation through the stimulation of PAR-2 and fibrinogen due to Cis-induced ADAM10 protein expression mediated the apoptotic pathway, as indicated by caspase-3 expression through the p-Akt pathway. This is normally accompanied by the loss of RXR-α distal and proximal tubules as lipid droplets. When the animals were pre-treated with the anticoagulant, Fund, the previous deleterious effect was halted while the fibrinolytic agent, Alt, most of the time failed to treat Cis-induced toxicity.

PAR2 promotes tumor-associated angiogenesis in lung adenocarcinoma through activating EGFR pathway

BackgroundMetastasis of advanced lung adenocarcinoma (LUAD) is a key cause of cancer-related death, and angiogenesis is the main feature of tumor growth and metastasis. MethodsThe expression level of F2R like trypsin receptor 1(F2RL1) which encodes protease-activated receptor 2 (PAR2) protein in LUAD tissues was analyzed by bioinformatics. The effects of F2RL1 overexpression/silencing on cell proliferation and sphere-forming were analyzed by Cell Counting Kit-8 and colony formation assays, stem cell sphere-forming assay, and angiogenesis assay, respectively. The F2RL1 mRNA expression level and the PAR2 protein expression level, vascular endothelial growth factor A (VEGFA), and epidermal growth factor receptor (EGFR) in lung cancer cell lines were evaluated by real-time quantitative polymerase chain reaction and western blot. The level of VEGFA secreted by lung cancer cells was analyzed by Enzyme-linked immunosorbent assay (ELISA). The effect of F2RL1-mediated EGFR signaling on angiogenesis was further explored by EGFR inhibitor AG1478. ResultsF2RL1 was substantially up-regulated in LUAD tissues and cells, and overexpression of F2RL1 could promote proliferation and stem cell sphere-forming of lung cancer cell lines, as well as formation of blood vessels and branch points of human umbilical vein endothelial cells (HUVECs). Meanwhile, overexpression of F2RL1 significantly upregulated VEGFA expression and promoted EGFR phosphorylation. EGFR inhibitor AG1478 treatment significantly down-regulated pEGFR, and AG1478 treatment reversed the promoting effect of cancer cell cultured medium (oe-F2RL1) on HUVEC angiogenesis. SignificanceIn summary, this study revealed the molecular mechanism of PAR2 promoting LUAD angiogenesis by activating EGFR signaling pathway, which further improves our understanding of LUAD angiogenesis, and provides a potential therapeutic strategy for LUAD treatment.

Date palm seed extract and herbal mixture mitigate gentamicin-induced renal injury in mice: Role of Protease-activated receptors (PARs) and Retinoid X receptor alpha (RXR-α)

Introduction: Gentamicin (Gen) causes renal toxicity by inhibiting protein synthesis in kidney cells, causing proximal tubule cell necrosis and renal failure. Herein, we examined the nephroprotective effect of date palm seed extract (DPSE) and one herbal mixture (HM; composed of Tribulus terrestris, Aerva lanata, Andrographis paniculata, and Raphanus sativus) against Gen-induced renal toxicity in mice with special reference to the possible role of retinoid X receptor alpha (RXR-α) and protease-activated receptor 2 (PAR-2) in this effect. Methods: Thirty-two male Balb/c mice divided randomly into four groups were either treated with saline, Gen (225 mg/kg/i.p., daily from day 3 to day 10), Gen (225 mg/kg i.p.) daily from day 3 to day 10 and DPSE (100 mg/kg/p.o.) daily for 10 days, or Gen (225 mg/kg i.p.) daily from day 3 to day 10 and HM (100 mg/kg/p.o., daily for 10 days). Mice were sacrificed 24 hours after the last dose administration, and kidney tissues were dissected out, weighed, and subjected to histological, immunofluorescence, and biochemical assays. Results: The Gen-induced renal toxicity group demonstrated a significant decrease in RXR-α and a significant increase in PAR-2 protein expression. Treatment with DPSE or HM significantly improved Gen-induced effects on serum creatinine, blood urea nitrogen (BUN), white blood cells (WBCs), platelets, RXR-α extracellular matrix deposition, and PAR-2. Conclusion: The present study stated the nephroprotective effects of DPSE and HM and revealed, for the first time, the involvement of retinoid receptors and PAR-2 in Gen-induced renal toxicity as well as in the protective effects of the two tested natural products.

High glucose upregulates PAR-1 in SH-SY5Y cells via deficiency of miR-20a and miR-190a.

Thrombin activity enhancement and its receptor protease-activated receptor 1 (PAR-1) activation play vital roles in neurologic deficits in the central nervous system. Our recent study showed that PAR-1 upregulation stimulated by chronic high glucose (HG) caused central neuron injury through neuroinflammation; however, the molecular mechanisms are far from clear. In the present study, we found that HG resulted in neuronal injury of SH-SY5Y cells as evidenced by decreased cell viability and increased lactate dehydrogenase release and elevated the mRNA level of PAR-1. Moreover, we predicted and determined several potential microRNAs (miRs) combining with the 3'-UTR of PAR-1 mRNA, finding that miR-20a-5p, miR-93-5p, and miR-190a-5p were significantly decreased in HG-cultured SH-SY5Y cells compared with control. Further, SH-SY5Y cells stably transfected with miR-20a-5p or miR-190a-5p mimic were established, and overexpression efficiency were confirmed. It was found that miR-20a-5p or miR-190a-5p overexpression markedly decreased PAR-1 mRNA level and protein expression in SH-SY5Y cells cultured with HG and normal glucose, indicating that miR-20a or miR-19a deficiency contributed to HG-induced PAR-1 upregulation. Together, our findings demonstrated that PAR-1 upregulation mediated HG-induced neuronal damage in central neurons, which was achieved through miR-20a or miR-190a deficiency.

Electrospun tube reduces adhesion in rabbit Achilles tendon 12\xa0weeks post-surgery without PAR-2 overexpression

One great challenge in surgical tendon repair is the minimization of peritendinous adhesions. An electrospun tube can serve as a physical barrier around a conventionally sutured tendon. Six New Zealand White rabbits had one Achilles tendon fully transsected and sutured by a 4-strand suture. Another six rabbits had the same treatment, but with the additional electrospun DegraPol tube set around the sutured tendon. The adhesion formation to the surrounding tissue was investigated 12 weeks post-operation. Moreover, inflammation-related protease-activated receptor-2 (PAR-2) protein expression was assessed. Finally, rabbit Achilles tenocyte cultures were exposed to platelet-derived growth factor-BB (PDGF-BB), which mimicks the tendon healing environment, where PAR-2 gene expression was assessed as well as immunofluorescent staining intensity for F-actin and α-tubulin, respectively. At 12 weeks post-operation, the partially degraded DegraPol tube exhibited significantly lower adhesion formation (− 20%). PAR-2 protein expression was similar for time points 3 and 6 weeks, but increased at 12 weeks post-operation. In vitro cell culture experiments showed a significantly higher PAR-2 gene expression on day 3 after exposure to PDGF-BB, but not on day 7. The cytoskeleton of the tenocytes changed upon PDGF-BB stimulation, with signs of reorganization, and significantly decreased F-actin intensity. An electrospun DegraPol tube significantly reduces adhesion up to twelve weeks post-operation. At this time point, the tube is partially degraded, and a slight PAR-2 increase was detected in the DP treated tendons, which might however arise from particles of degrading DegraPol that were stained dark brown. PAR-2 gene expression in rabbit tenocytes reveals sensitivity at around day 10 after injury.

Protease activated receptor 2 signaling promotes self-renewal and metastasis in colorectal cancer through β-catenin and periostin.

The maintenance and expansion of cancer stem-like cells (CSCs) is necessary for metastasis. Although protease-activated receptor 2 (PAR2) is strongly associated with colorectal cancer (CRC) progression, it is unclear how it regulates distal metastasis, and no studies have shown the involvement of CSCs. In this study, we demonstrated that high PAR2 protein expression was correlated with metastatic CRC and poor prognosis in patients with stage III-IV CRC. CSCs from cell lines and patients showed higher levels of PAR2 than that of corresponding non-CSCs, and PAR2 inhibition reduced the CSC properties of the cell lines. Mechanistically, PAR2 inhibition switched the division mode of CSCs from symmetrical to asymmetrical via the ERK/GSK-3β/β-catenin pathway. We also identified periostin as a direct transcriptional target of β-catenin that mediates CSC self-renewal via PAR2 signaling. In a mouse xenograft model, PAR2 knockdown significantly attenuated liver metastasis. Finally, PAR2 expression was positively correlated with β-catenin and periostin in the primary sites of CRC with distant metastasis. Overall, our results indicate that PAR2 activation enhances CSC self-renewal and promotes metastasis through β-catenin and its target gene, periostin, in CRC.

Preservation of renal endothelial integrity and reduction of renal edema by aprotinin does not preserve renal perfusion and function following experimental cardiopulmonary bypass

BackgroundAcute kidney injury is a severe complication following cardiopulmonary bypass (CPB) and is associated with capillary leakage and microcirculatory perfusion disturbances. CPB-induced thrombin release results in capillary hyperpermeability via activation of protease-activated receptor 1 (PAR1). We investigated whether aprotinin, which is thought to prevent thrombin from activating PAR1, preserves renal endothelial structure, reduces renal edema and preserves renal perfusion and reduces renal injury following CPB.MethodsRats were subjected to CPB after treatment with 33.000 KIU/kg aprotinin (n = 15) or PBS (n = 15) as control. A secondary dose of 33.000 KIU/kg aprotinin was given 60 min after initiation of CPB. Cremaster and renal microcirculatory perfusion were assessed using intravital microscopy and contrast echography before CPB and 10 and 60 min after weaning from CPB. Renal edema was determined by wet/dry weight ratio and renal endothelial structure by electron microscopy. Renal PAR1 gene and protein expression and markers of renal injury were determined.ResultsCPB reduced cremaster microcirculatory perfusion by 2.5-fold (15 (10–16) to 6 (2–10) perfused microvessels, p < 0.0001) and renal perfusion by 1.6-fold (202 (67–599) to 129 (31–292) au/sec, p = 0.03) in control animals. Both did not restore 60 min post-CPB. This was paralleled by increased plasma creatinine (p < 0.01), neutrophil gelatinase-associated lipocalin (NGAL; p = 0.003) and kidney injury molecule-1 (KIM-1; p < 0.01). Aprotinin treatment preserved cremaster microcirculatory perfusion following CPB (12 (7–15) vs. 6 (2–10) perfused microvessels, p = 0.002), but not renal perfusion (96 (35–313) vs. 129 (31–292) au/s, p > 0.9) compared to untreated rats. Aprotinin treatment reduced endothelial gap formation (0.5 ± 0.5 vs. 3.1 ± 1.4 gaps, p < 0.0001), kidney wet/dry weight ratio (4.6 ± 0.2 vs. 4.4 ± 0.2, p = 0.046), and fluid requirements (3.9 ± 3.3 vs. 7.5 ± 3.0 ml, p = 0.006) compared to untreated rats. In addition, aprotinin treatment reduced tubulointerstitial neutrophil influx by 1.7-fold compared to untreated rats (30.7 ± 22.1 vs. 53.2 ± 17.2 neutrophil influx/section, p = 0.009). No differences were observed in renal PAR1 expression and plasma creatinine, NGAL or KIM-1 between groups.ConclusionsAprotinin did not improve renal perfusion nor reduce renal injury during the first hour following experimental CPB despite preservation of renal endothelial integrity and reduction of renal edema.

The Role of Protease-Activated Receptor 2 in Hepatocellular Carcinoma after Hepatectomy.

Background and Objectives: Protease activated receptor-2 (PAR2) is elevated in a variety of cancers and has been promoted as a potential therapeutic target. However, the clinical and prognostic values of PAR2 in hepatocellular carcinoma (HCC) are poorly characterized. This study aimed to evaluate the expression of PAR2 in HCC tissues and examine the prognostic value of PAR2 after resection in HCC. Materials and Methods: Two hundred and eight resected specimens were collected from HCC patients at Kaohsiung Chang Gung Memorial Hospital. PAR2 protein expression was assessed by western blotting in HCC tissues and matched normal tissues. The correlation between PAR2 expression and clinicopathological parameters was analyzed. Disease-free survival (DFS) and overall survival (OS) were compared using the log-rank test. A Cox regression model was used to identify independent prognostic factors. Results: PAR2 was expressed at higher levels in HCC tissues than the paired adjacent nontumor tissues. High expression of PAR2 was associated with advanced tumor, node, metastasis (TNM )stage and histological grade. Kaplan-Meier analysis indicated high PAR2 expression was associated with poorer DFS and OS compared to low PAR2 expression. Multivariate analyses indicated high PAR2 expression [hazard ratio (HR), 1.779, p = 0.006), α-fetoprotein (AFP) (HR, 1.696, p = 0.003), liver cirrhosis (HR, 1.735, p = 0.002), and advanced TNM stage (HR, 2.061, p < 0.001) were prognostic factors for DFS, and advanced TNM stage (HR, 2.741, p < 0.001) and histological grade (HR, 2.675, p = 0.002) and high PAR2 expression (HR, 1.832, p = 0.012) were significant risk factors for OS. In subgroup analyses, the combination of PAR2 expression and serum AFP provided improved prognostic ability for OS and DFS. Conclusion: Combination PAR2 and AFP predict HCC outcomes after resection. PAR2 represents a potentially clinically relevant biomarker for HCC.

Effects of norepinephrine on colonic tight junction protein expression during heat stress.

Stress induced by changes in the internal or external environment in humans and animals leads to intestinal epithelial damage, in a manner that is associated with impaired intestinal barrier function. However, the role of the stress hormone norepinephrine (NE) in impairments in barrier function remains poorly understood. In the present study, a rat heat-exposed model was used to observe changes in the tight junction proteins Occludin and zonula occludens-1 (ZO-1), in addition to those in protease-activated receptor 2 (PAR-2) and transient receptor potential ankyrin 1 channel (TRPA1) in colon. The levels of plasma NE were detected using an ELISA kit. Different concentrations of NE were used to culture the human colon cell line Caco-2 for 6 and 24 h to investigate the cell viability using Cell Counting Kit-8 assay, whilst the expression levels of Occludin, ZO-1, PAR-2 and TRPA1 were examined using western blotting and immunofluorescence in Caco-2 cells and immunohistrochemistry in rat colon tissues. Although there was no clear histological damage to the rat colonic mucosa, there were decreased expression levels of tight junction proteins Occludin and ZO-1 after heat exposure. In addition, PAR-2 expression was increased by heat exposure. It was found that TRPA1 expression was concentrated to the luminal surface of the colon in the heat exposed group compared with that in the control group. After the administration of increasing concentrations of NE for 6 h, treatment did not affect cell viability. Furthermore, after application of NE for 24 h, cell viability gradually increased as the NE concentration was elevated from 10 to 100 µM. However, no significant increase in viability was observed when the cells were treated with 120 and 160 µM NE. Occludin expression was decreased when 10 µM NE was applied for 6 or 24 h. By contrast, 60 µM NE significantly downregulated Occludin expression in the 6 h group, but caused an insignificant decrease in the 24 h group. It was found that ZO-1 expression was upregulated after treatment with 10 µM NE for 6 h, whilst downregulation was observed after treatment with 10 µM NE for 24 h. PAR-2 protein expression was increased after application of NE for both 6 and 24 h, but not after treatment with 60 µM NE. In addition, TRPA1 expression was not affected by the treatment of NE, but increased positive staining was observed on the luminal side of the mucosa, which appeared to be concentrated in the cells of the luminal side in the rat colon after heat exposure. Collectively, the present results suggested that expression of tight junction proteins Occludin and ZO-1, in addition to that of PAR-2, can be regulated by NE, which may contribute to impairments in barrier function observed during heat stress.

Activation of protease-activated receptor 2 is associated with blood pressure regulation and proteinuria reduction in metabolic syndrome.

Metabolic syndrome (MetS) increases the risk of kidney disease. In SHRSP.Z-Leprfa /IzmDmcr (SHRSP.ZF) rats with MetS, protease-activated receptor 2 (PAR2)-mediated vasorelaxation is preserved in the aorta at 20weeks of age (weeks) via enhancement of nitric oxide production but impaired at 30weeks by oxidative stress. However, impairment of PAR2-mediated vasorelaxation of renal arteries and its possible implications for kidney disease are unclear. We used organ baths to assess PAR2-mediated vasorelaxation of isolated renal arteries, colorimetric methods to measure urinary protein levels as an index of renal function, and western blot to determine expression of PAR2 and nephrin proteins in the kidneys of SHRSP.ZF rats at 10, 20, and 30weeks. We assessed renal arteries and kidney function for effects of orally administered GB88, a pathway-dependent PAR2 antagonist, from 10 to 18weeks, and azilsartan, an angiotensin II type 1 receptor blocker, from 13 to 23weeks. PAR2-mediated vasorelaxation was slightly lower at 20weeks and attenuated significantly at 30weeks compared with those at 10weeks. Urinary protein levels were increased at 20 and 30weeks. Decreased protein expression of PAR2 and nephrin in the kidney were observed at 30weeks. Administration of GB88 increased blood pressure (BP) and proteinuria. Azilsartan reduced the high BP and the impaired PAR2-mediated vasorelaxation, but did not restore the increase in urinary protein levels and decreased PAR2 and nephrin protein expression in the kidney. PAR2 activation in the kidney may be associated with maintenance of BP and urinary protein excretion in MetS.

Expression analysis of protease-activated receptor-2 in cats

Chronic kidney disease (CKD) is a common disease in geriatric cats. Despite its high prevalence, the pathogenesis of feline CKD is poorly understood. Recently, there has been increasing evidence for the role of protease-activated receptor-2 (PAR-2) in the progression of CKD in humans and rodents. However, the role of PAR-2 in feline CKD has not been evaluated. In this study, we determined nucleotide sequence of feline PAR-2 from the kidney, evaluated PAR-2 mRNA and protein expression in normal feline tissues, and analyzed functional expression in the feline kidney epithelial cell line Crandell-Rees Feline Kidney (CRFK). The open reading frame of feline PAR-2 comprised 1,194 bp and encoded 397 amino acids, showing 90%, 90%, and 85% identities to human, dog, and mouse PAR-2, respectively. In healthy cats, expression levels of the PAR-2 mRNA and protein were relatively higher in the gastrointestinal tract and kidney, and was lowest in the heart. The feline PAR-2 protein expression was confirmed, and stimulation of trypsin and PAR-2 agonists induced a prompt increase in the intracellular calcium ion concentration in CRFK cells. The present study will provide fundamental information for investigation of the involvement of PAR-2 in the pathogenesis of CKD in cats.

他克莫司对人角质形成细胞蛋白酶活化受体2表达及功能的影响

Objective To evaluate the in vitro effect of tacrolimus on the expression and function of protease-activated receptor 2 (PAR-2) in cultured human keratinocytes. Methods After 24-hour co-culture of human keratinocytes with 10-9 - 10-5 mol/L tacrolimus, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine the mRNA expression of PAR-2, immunofluorescence (IF) staining and Western blot analysis were performed to determine the protein expression of PAR-2 in the keratinocytes, and the fluorescent calcium probe fluo-4 was used to evaluate the effect of tacrolimus at different concentrations on the intracellular calcium concentration after the activation of PAR-2 in the keratinocytes. The group treated without tacrolimus served as control group. One-way analysis of variance was used to compare the PAR-2 expression and calcium concentration in the human keratinocytes in different groups, and least significant difference (LSD) -t test was carried out for multiple comparisons. Results PAR-2 was expressed on both the membrane and cytoplasm of keratinocytes. After 24-hour co-culture of keratinocytes with 10-9 - 10-5 mol/L tacrolimus, the PAR-2 mRNA expression significantly decreased in these cells compared with the control group (all P 0.05) . No significant difference in the PAR-2 protein expression was observed between the 10-8- or 10-9-mol/L tacrolimus group and the control group (both P > 0.05) . After 24-hour co-culture, the peak concentration of intracellular calcium after PAR-2 activation was significantly lower in the 10-5-, 10-6- and 10-7-mol/L tacrolimus groups (peak absorbance: 1 463 ± 283, 1 455 ± 270, 1 423 ± 291 respectively) than in the control group (1 602 ± 407; t = 2.582, 2.821, 2.923, P = 0.032, 0.022, 0.019, respectively) , while there was no significant difference between the 10-8- or 10-9-mol/L tacrolimus group (1 649 ± 379, 1 633 ± 415 respectively) and the control group (t = 0.846, 0.462, P = 0.422, 0.657, respectively) . Conclusion Tacrolimus can inhibit PAR-2 expression and suppress calcium mobilization induced by a PAR-2 agonist in keratinocytes. Key words: Tacrolimus; Keratinocytes; Calcium signaling; Receptors, thrombin; Receptor, PAR-2

Effect of epigallocatechin gallate on ultraviolet B-induced transfer and degradation of melanosomes in mice.

Objective To evaluate the effect of tea polyphenol epigallocatechin gallate (EGCG) on ultraviolet B (UVB) -induced skin pigmentation, transfer and degradation of melanosomes in mice, and to explore the role of autophagy in the mechanism of melanosome degradation. Methods A total of 32 ears from 16 female C57/BL6 mice were randomly and equally divided into 4 groups: acetone control group topically treated with acetone solution daily, EGCG group topically treated with 10 g/L EGCG acetone solution daily, UVB irradiation group irradiated with 500 mJ/cm2 UVB once a day and 2 hours later topically treated with acetone solution, UVB + EGCG group irradiated with 500 mJ/cm2 UVB once a day and 2 hours later topically treated with EGCG acetone solution. Ten days later, all the mice were sacrificed, and skin tissue samples were collected from the ears. Transmission electron microscopy was performed to observe ultrastructural changes of melanosomes and autophagosomes, immunohistochemical study to measure expression of protease-activated receptor 2 (PAR2) and microtubule-associated protein light chain 3 (LC3) in the epidermis, and Western blot analysis to determine the protein expression of PAR2, Ras-related protein Rab27a and LC3 in the epidermis. Results There was a significant difference in the number of melanosomes and autophagosomes among the acetone control group, EGCG group, UVB irradiation group and UVB + EGCG group (H= 12.249, 13.888, respectively, both P 0.05) . Western blot analysis revealed significant differences in the protein expression of PAR2 and Rab27a, as well as in the LC3-Ⅱ/LC3-Ⅰratio, among the 4 groups (F = 18.739, 25.967, 24.022, respectively, all P < 0.05) . Compared with the UVB irradiation group, the UVB + EGCG group showed significantly decreased expression of PAR2 (0.91 ± 0.54 vs. 3.12 ± 0.61, P < 0.05) and Rab27a (0.99 ± 0.16 vs. 1.42 ± 0.07, P < 0.05) , but significantly increased LC3-Ⅱ/LC3-Ⅰratio (1.67 ± 0.08 vs. 1.24 ± 0.07, P < 0.05) . Conclusion Topical EGCG treatment can effectively suppress UVB-induced skin pigmentation, which may be related to the inhibition of melanosome transfer and promotion of melanosome autophagy. Key words: Polyphenols; Ultraviolet rays; Autophagy; Melanins; Epigallocatechin gallate

Astragaloside Alleviates Hepatic Fibrosis Function via PAR2 Signaling Pathway in Diabetic Rats

Background/Aims: Astragaloside (AGS) extracted from radix astragalin (Huangqi) has been considered to be beneficial to liver diseases. In this study, we examined the role played by AGS in alleviating hepatic fibrosis function via protease-activated receptor-2 (PAR2) mechanisms. We hypothesized that AGS affects PAR2 signaling pathway thereby improving hepatic function in rats with hepatic fibrosis induced by carbon tetrachloride (CCl₄). We further hypothesized that AGS attenuates impaired hepatic function evoked by CCl₄ to a greater degree in diabetic animals. Methods: ELISA and Western Blot analysis were used to examine PAR2 signaling pathway in diabetic CCl₄-rats and non-diabetic CCl₄-rats. Results: AGS inhibited the protein expression of PAR2 and its downstream pathway PKA and PKCɛ in CCl₄-rats. Notably, the effects of AGS were greater in CCl₄-rats with diabetes. AGS also significantly attenuated the CCl₄-induced upregulations of pro-inflammatory cytokines, namely interleukin-1β, interleukin-6 and tumor necrosis factor-α accompanied with decreases of collagenic parameters such as hexadecenoic acid, laminin and hydroxyproline. Additionally, AGS improved the CCl₄-induced exaggerations of liver index and functions including alanine aminotransferase, aspartate aminotransferase. Moreover, TGF-β1, a marker of hepatic fibrosis, was increased in CCl₄-rats and AGS inhibited increases in TGF-β1 induced by CCl₄. Conclusions: AGS alleviates hepatic fibrosis by inhibiting PAR2 signaling expression and its effects are largely enhanced in diabetic animals. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of hepatic fibrosis; and results of our study are likely to shed light on strategies for application of AGS because it has potentially greater therapeutic effectiveness for hepatic fibrosis in diabetes.

Low-concentration hydrogen peroxide can upregulate keratinocyte intracellular calcium and PAR-2 expression in a human keratinocyte-melanocyte co-culture system.

Hydrogen peroxide (H2O2) may have a biphasic effect on melanin synthesis and melanosome transfer. High H2O2 concentrations are involved in impaired melanosome transfer in vitiligo. However, low H2O2 concentration promotes the beneficial proliferation and migration of melanocytes. The aim of this study was to explore low H2O2 and its mechanism in melanosome transfer, protease-activated receptor-2 (PAR-2) expression and calcium balance. Melanosomes were fluorescein-labeled for clear visualization of their transfer. The expression of protease-activated receptor-2 (PAR-2) in keratinocytes was determined by western blot analysis. Flow cytometry was employed to evaluate the effects of H2O2 on calcium levels in keratinocytes. Fluorescence microscopy showed the upregulation of melanosome transfer into keratinocytes following 0.3mM H2O2 treatment in the co-cultures rather than in the untreated control groups, which was associated with higher expression of PAR-2 protein and increased calcium concentration. The addition of a PAR-2 antagonist inhibited the positive activity of H2O2 and calcium flow in keratinocytes. When calcium flow was blocked by a calcium chelator, the addition of H2O2 did not increase the PAR-2 expression level in keratinocytes, therefore, inhibiting dendrite formation and melanosome transfer. Low H2O2 concentration promotes melanosome transfer with increased PAR-2 expression level and calcium concentration in keratinocytes. In addition, the interaction between melanocytes and keratinocytes is more beneficial to enhance calcium levels in keratinocytes which mediate melanin transfer. Moreover, low H2O2 concentration promotes dendrite formation, in which extracellular calcium and Par-2 were involved.

PAR‐2 is involved in melanogenesis by mediating stem cell factor production in keratinocytes

<scp>PAR</scp>‐2 is involved in melanogenesis by mediating stem cell factor production in keratinocytes

Protective effects of activated protein C on neurovascular unit in a rat model of intrauterine infection-induced neonatal white matter injury.

Activated protein C (APC), a natural anticoagulant, has been reported to exert direct vasculoprotective, neural protective, anti-inflammatory, and proneurogenic activities in the central nervous system. This study was aimed to explore the neuroprotective effects and potential mechanisms of APC on the neurovascular unit of neonatal rats with intrauterine infection-induced white matter injury. Intraperitoneal injection of 300 μg/kg lipopolysaccharide (LPS) was administered consecutively to pregnant Sprague-Dawley rats at embryonic days 19 and 20 to establish the rat model of intrauterine infection- induced white matter injury. Control rats were injected with an equivalent amount of sterile saline on the same time. APC at the dosage of 0.2 mg/kg was intraperitoneally injected to neonatal rats immediately after birth. Brain tissues were collected at postnatal day 7 and stained with hematoxylin and eosin (H&E). Immunohistochemistry was used to evaluate myelin basic protein (MBP) expression in the periventricular white matter region. Blood-brain barrier (BBB) permeability and brain water content were measured using Evens Blue dye and wet/dry weight method. Double immunofluorescence staining and real-time quantitative PCR were performed to detect microglial activation and the expression of protease activated receptor 1 (PAR1). Typical pathological changes of white matter injury were observed in rat brains exposed to LPS, and MBP expression in the periventricular region was significantly decreased. BBB was disrupted and the brain water content was increased. Microglia were largely activated and the mRNA and protein levels of PAR1 were elevated. APC administration ameliorated the pathological lesions of the white matter and increased MBP expression. BBB permeability and brain water content were reduced. Microglia activation was inhibited and the PAR1 mRNA and protein expression levels were both down-regulated. Our results suggested that APC exerted neuroprotective effects on multiple components of the neurovascular unit in neonatal rats with intrauterine infection- induced white matter injury, and the underlying mechanisms might involve decreased expression of PAR1.

Thrombin may modulate dendritic cell activation in kidney transplant recipients with delayed graft function.

Coagulation and complement activation represent key events in ischaemia-reperfusion-induced renal injury leading to delayed graft function (DGF). It is still unclear whether the coagulation cascade may also influence the acquired immunity. The aim of the present study was to investigate the expression of protease-activated receptor 1 (PAR-1), the main thrombin receptor, by graft-infiltrating dendritic cells (DCs), and to evaluate whether thrombin may influence DCs complement production and T-cell response. PAR-1, BDCA1, CD11c, BDCA4, fibrin, C3c and C3d protein expression were evaluated by confocal microscopy. Cultured DCs were obtained incubating monocytes (Ms) with IL-4 and GM-CSF. DC maturation was obtained with IFN-g+sCD40L or with a cytokine cocktail (IL-1b, TNF-a, PGE2, IL-6). PAR1 protein expression on cultured DC was evaluated by flow-cytometry. Complement receptors, C3, IL12/IL17p40 and IL10 gene expression was evaluated by qPCR. T cell phenotype was evaluated by ELISPOT. IFN-g protein presence was evaluated by ELISA. PAR-1 was expressed by infiltrating myeloid DCs in pre-transplant and in DGF biopsies. In DGF grafts, myeloid DCs localized within fibrin and C3d deposits and expressed C3c. In vitro, PAR-1 protein expression was increased in monocyte-derived immature DCs and in cytokine-induced mature DCs compared to monocytes. PAR-1 activation caused a time-dependent increase in C3 and complement receptors expression. Moreover, thrombin stimulation, while reducing interleukin-10 mRNA abundance, induced interleukin-12/IL-17 p40 gene expression, and promoted C3a ability to increase interleukin-12/IL17 mRNA abundance. These changes in the DCs' cytokine pattern influenced their ability to induce interferon-g production by T cells, suggesting the activation of a T helper-1 bias. Our data suggest that PAR-1 is expressed by DCs in DGF grafts and its activation may induce complement production and a Th1 bias. This observation suggests a potential pathogenic link between DGF and acquired allo-response leading to graft damage.

Heat stress-induced disruption of endothelial barrier function is via PAR1 signaling and suppressed by Xuebijing injection.

Increased vascular permeability leading to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) is central to the pathogenesis of heatstroke. Protease-activated receptor 1 (PAR1), the receptor for thrombin, plays a key role in disruption of endothelial barrier function in response to extracellular stimuli. However, the role of PAR1 in heat stress-induced endothelial hyper-permeability is unknown. In this study, we measured PAR1 protein expression in heat-stressed human umbilical venous endothelial cells (HUVECs), investigated the influences of PAR1 on endothelial permeability, F-actin rearrangement, and moesin phosphorylation by inhibiting PAR1 with its siRNA, neutralizing antibody (anti-PAR1), specific inhibitor(RWJ56110), and Xuebijing injection (XBJ), a traditional Chinese medicine used for sepsis treatment, and evaluated the role of PAR1 in heatstroke-related ALI/ARDS in mice by suppressing PAR1 with RWJ56110, anti-PAR1and XBJ. We found that heat stress induced PAR1 protein expression 2h after heat stress in endothelial cells, caused the release of endothelial matrix metalloprotease 1, an activator of PAR1, after 60 or 120 min of heat stimulation, as well as promoted endothelial hyper-permeability and F-actin rearrangement, which were inhibited by suppressing PAR1 with RWJ56110, anti-PAR1 and siRNA. PAR1 mediated moesin phosphorylation, which caused F-actin rearrangement and disruption of endothelial barrier function. To corroborate findings from in vitro experiments, we found that RWJ56110 and the anti-PAR1 significantly decreased lung edema, pulmonary microvascular permeability, protein exudation, and leukocytes infiltrations in heatstroke mice. Additionally, XBJ was found to suppress PAR1-moesin signal pathway and confer protective effects on maintaining endothelial barrier function both in vitro and in vivo heat-stressed model, similar to those observed above with the inhibition of PAR1. These results suggest that PAR1 is a potential therapeutic target in heatstroke.

Protease-activated receptor 2 agonist increases cell proliferation and invasion of human pancreatic cancer cells.

The aim of this study was to determine the expression of protease-activated receptor 2 (PAR-2) in the human pancreatic cancer cell line SW1990, and to evaluate its effect on cell proliferation and invasion. The expression of PAR-2 protein and mRNA in SW1990 cells was determined by immunocytochemistry and reverse transcription polymerase chain reaction (PCR), respectively. MTT and cell invasion and migration assays, as well as semi-quantitative PCR and zymography analysis, were additionally performed. PAR-2 mRNA was significantly upregulated in the cells treated with trypsin or the PAR-2 activating peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV) (P<0.01), but not in the Val-Lys-Gly-Ile-Leu-Ser group (P>0.05). Trypsin and SLIGKV significantly promoted SW1990 cell proliferation in a dose- and time-dependent manner (P<0.05). Compared with the control group, trypsin and SLIGKV significantly increased the mRNA expression (P<0.01) and gelatinolytic activity (P<0.01) of matrix metalloproteinase (MMP)-2. In conclusion, PAR-2 is expressed in SW1990 cells. PAR-2 activation may promote the invasion and migration of human pancreatic cancer cells by increasing MMP-2 expression.