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Psoralen is the main active component of Psoralea corylifolia and is used as a marker to assess its quality. The effects of psoralen on animals have been well characterized. However, the molecular pathway of its toxicity is not fully understood. In this study, the toxic effects of psoralen administration (60 mg/kg) for 7 days in Sprague-Dawley rats were observed. Serum biochemistry and liver histopathology were further investigated. Proton nuclear magnetic resonance was applied to characterize the metabolic profile of liver toxicity induced by psoralen and to find changed metabolites in rat serum and liver. It was revealed that visceral coefficients and serum biochemistry indexes were significantly changed in rats with psoralen-induced liver injury. Furthermore, the histopathological examination exhibited that the liver might be the target organ for psoralen. Metabolic analysis of both serum and liver samples further proved the liver was the target of toxicity of psoralen. Multivariate analysis identified 7 metabolites in serum samples and 15 in liver samples as potential biomarkers in liver injury induced by psoralen. In addition, our results suggest that psoralen can cause a disturbance in amino acid metabolism, especially valine, leucine, and isoleucine biosynthesis in both serum and liver samples. In conclusion, we combined the results of toxicity and metabolomics induced by psoralen and provide useful information about the safety and potential risks of psoralen and Psoralea corylifolia.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers. Recent research has demonstrated that chronic pancreatitis (CP) is associated with an increased risk of PDAC, partly due to acinar-to-ductal metaplasia (ADM). Baicalein has been shown to exert anti-inflammatory and anti-tumor effects for CP or PDAC, respectively. The aim of our study was to investigate the effect of baicalein, and the putative underlying mechanism, on inflammatory cytokines-induced ADM of rat pancreatic acinar cell line AR42J. To investigate ADM and baicalein effects in vitro, AR42J were treated with recombinant rat Tumor Necrosis Factor alpha (rTNFα) with or without baicalein for 5 days. Results showed that rTNFα-induced AR42J cells switched their phenotype from dominantly amylase-positive acinar cells to dominantly cytokeratin 19-positive ductal cells. Moreover, expression of the transcripts for TNFα or Hes-1, a Notch target, was up-regulated in these cells. Interestingly, baicalein reduced the population of ADM as well as cytokines gene expression but not Hes-1. Baicalein inhibited NF-κB activation induced by rTNFα in AR42J, but no effect on Notch 1activation. Moreover, baicalein suppressed the secretion of TNFα and Nitric Oxide (NO) in macrophages stimulated with LPS and further inhibited ADM of conditional medium-treated AR42J cells. Baicalein also suppressed the inflammatory response of LPS-activated macrophages, thereby inhibited ADM of AR42J by altering their microenvironment. Taken together, our study indicates that baicalein reduces rTNFα-induced ADM of AR42J cells by inhibiting NF-κB activation. It also sheds new light on Chinese material medica therapy of pancreatitis and thereby prevention of PDAC.

The study aims to evaluate the effectiveness and safety of Chinese herbal medicine granules Danzhi Qing'e formula (DZQE), Erzhi formula (EZ), and their combination (Combined formula) in the treatment of menopausal symptoms at different stages of menopause.Women between the ages of 40 to 60 years, who met menopausal symptoms diagnostic criteria and experienced hot flushes at least 14 times/week in the last 4 weeks, were recruited to participate in a stratified randomized, double-blind, placebo-controlled clinical trial (n = 389). They received a treatment period of 8 weeks and were followed up for 4 weeks. Participants were categorized into two subgroups: 197 in the perimenopausal subgroup (menstrual disorder to 1 y after amenorrhea) and 192 in the early postmenopausal subgroup (1-5 y after amenorrhea). Participants were randomly assigned to placebo or one of the three herbal formula treatments. The primary outcome instrument was the Menopause-Specific Quality of Life (MENQOL) questionnaire.When analyzing the two subgroups together, DZQE markedly decreased the MENQOL total score at the end of 12th week with statistical significance (P = 0.02) and improved vasomotor symptoms after 8 weeks treatment and 4 weeks follow-up (P 0.05). What is more, the combined formula also greatly improved the participants' vasomotor symptoms compared with placebo after the 4 weeks follow-up. No statistically meaningful difference was observed in any other outcomes among the groups. The results of subgroup analysis showed that DZQE and Combined formula were more effective than placebo in improving MENQOL total score for perimenopausal women at the end of week 12. For typical menopausal symptoms such as hot flushes and night sweats, DZQE displayed more favorable effects on early postmenopausal participants. Compared to placebo, the DZQE both showed statistically significant differences after 8 weeks treatment and 4 weeks follow-up. Although at the end of 12th week, DZQE also had better effects than placebo in the perimenopausal subgroup on vasomotor symptoms. Participants in the EZ group did not show a significant difference of any domains in MENQOL compared with participants in the placebo group.The DZQE formula improves the quality of life for menopausal women, especially for those with vasomotor symptoms during the whole menopausal period. The DZQE and EZ combination formula is effective only on perimenopausal symptoms.

Acute pancreatitis (AP) is a common acute abdominal disease with local or systemic inflammatory response, caused by abnormal activation of digestive enzymes. Baicalein has been shown to exert anti-inflammatory effects and to attenuate the pathological changes of AP. The aim of the research was to investigate the effects of baicalein on caerulein induced pancreatitis, and to elucidate the putative underlying mechanism. In this study, the therapeutic potential of baicalein and its mechanism were investigated in a caerulein-induced AP in vivo and in vitro model. The results indicate that baicalein treatment alleviates the caerulein-induced pathological damage in the pancreas. Baicalein decreased the expression level of pro-inflammatory cytokines and chemokines of the pancreas in caerulein treated mice and of isolated pancreatic acinar cells. Moreover, baicalein inhibited the expression of NF-κB p65 and the phosphorylation of p38 MAPK, ERK (extracellular signal-regulated kinase) as well as STAT 3, which indicates that baicalein exerts its anti-inflammatory effects via dampening the NF-κB, MAPK and STAT 3 signaling pathways. Together, this study provides experimental evidence for the clinical application of Scutellaria baicalensis Georgi or baicalein and indicates that baicalein may be a promising candidate for treatment of AP patients in the future.

We investigated the beneficial effects and underlying mechanisms of Zhuanggu Guanjie (ZGGJ) pill in osteoporosis in vitro and in vivo. Bone marrow macrophages from 4–6-week-old mice were cultured in the presence of macrophage colony-stimulating factor (15 ng/mL) and receptor activator of nuclear factor-κB ligand (30 ng/mL). Osteoclast differentiation was determined by quantification of tartrate-resistant acid phosphatase activity. Gelatin zymography was used to detect the activity of matrix metalloproteinases in osteoclasts. Ovariectomized rats were administered orally with estradiol valerate or ZGGJ for 8 weeks. Blood was collected to measure serum indices. Tibiae were harvested to carry out bone microcomputed tomography scanning, histomorphological analysis, and bone strength determination. ZGGJ inhibited tartrate-resistant acid phosphatase activity, matrix metalloproteinase 9 expression, and bone resorption in vitro. At doses of 0.55, 1.1, and 2.2 g/kg, ZGGJ exerted significant osteoprotective effects including inhibition of bone turnover markers and improved tibia bone strength in ovariectomized rats. Microcomputed tomographic analysis showed that ZGGJ improved the trabecular architecture with increased connectivity density and trabecular thickness and decreased trabecular spacing. These results revealed that ZGGJ prevents bone loss induced by ovariectomy in rats and that inhibition of bone resorption is involved in the bone-protective effects of ZGGJ.

Isopsoralen is a major active and quality-control component of Fructus Psoraleae, but lacks a full safety evaluation. We evaluated the oral toxicity of isopsoralen in Wistar rats treated for 3 months at doses of 0, 3.5, 7.0, and 14 mg/kg. Additionally, the plasma metabolomics of isopsoralen in male and female rats treated for 3 months at doses of 0 and 14 mg/kg were investigated by gas chromatography-mass spectrometry. Many abnormalities were observed in the isopsoralen-treated rats, including suppression of body weight gain, and changes in serum biochemical parameters and visceral coefficients. Histopathological changes in liver, pancreatic, and reproductive system tissues were also observed in the isopsoralen-treated rats. The metabolomic analyses showed alterations in many metabolites (19 in female rats; 28 in male rats) after isopsoralen administration. The significant changes in these metabolites revealed metabolomic alterations in the isopsoralen-treated rats, especially in amino acid metabolism regardless of sex, including phenylalanine, tyrosine, and tryptophan biosynthesis and glycine, serine, and threonine metabolism. Furthermore, fatty acid metabolism comprised the main affected pathways in female rats, while lipid metabolism and energy metabolism were the main affected pathways in male rats.

The pathophysiology of human chronic pancreatitis is not well understood and difficult to follow on a molecular basis. Therefore, we used a rat model [Wistar-Bonn/Kobori (WBN/Kob)] that exhibits spontaneous chronic inflammation and fibrosis in the pancreas. Using microarrays we compared gene expression patterns in the pancreas during development of inflammation and fibrosis of WBN/Kob rats with age-matched healthy Wistar rats. The extracellular matrix protein SPARC (secreted protein, acidic, and rich in cysteines) and other transcripts of inflammatory genes were quantified by real-time PCR, and some were localized by immunohistochemistry. When pancreatic inflammation becomes obvious at the age of 16 wk, several hundred genes are increased between 3- and 50-fold in WBN/Kob rats compared with healthy Wistar rats. Proteins produced by acinar cells and characteristic for inflammation, e.g., pancreatitis-associated protein, are highly upregulated. Other proteins, derived from infiltrating inflammatory cells and from activated stellate cells (fibrosis) such as collagens and fibronectins are also significantly upregulated. SPARC was localized to acinar cells where it increased in the vicinity of inflammatory foci. However, acinar expression of SPARC was lost during destruction of acinar cells. In human pancreatic specimens with chronic pancreatitis, SPARC exhibited a similar expression profile. During chronic inflammation and fibrosis in the WBN/Kob rat, inflammatory genes, growth factors, and structural genes exhibit a high increase of expression. A temporal profile including pre- and postinflammatory phases indicates a concurrent activation of inflammatory and fibrotic changes. Inflammation dependent expression of SPARC appears to be lost during acinar-to-duct metaplasia both in rat and human pancreas.

Background Regenerating protein ( reg ) and pancreatic stone protein (PSP) have been discovered independently in the fields of diabetes and pancreatitis. Materials and methods These proteins are identical; however, because of the gap between the endocrine and exocrine field, there was never a consensus and the nomenclature has not been rectified. Since the time of the initial discovery, more isoforms have been unified. Historically, PSP was discovered long before reg , yet, in many areas outside of the pancreatitis research field, reg is being used. Results For PSP/ reg , a role in proliferation and regeneration of islet cells has been postulated. A hitherto insufficiently understood phenomenon is the massive up-regulation of PSP/ reg in pancreatic tissue and juice under conditions of stress. Similarly, PAP (pancreatitis-associated protein)/ reg III has been attributed various functional roles. Structurally, the ability to form fibrils after tryptic cleavage is a striking common features of both proteins. However, this biochemical transformation is in itself not enough to gain functional insight. Thus, physiological and genetic approaches are required to further characterize the role of these proteins in the pancreas. Recently, more evidence has been presented in support of the theory that PSP/ reg plays a key role in islet neogenesis/regeneration. Conclusions In this review we discuss the debate on the localization and functional roles of PSP/ reg and PAP/ regIII . Therefore, we have summarized hypotheses and experimental results supporting such hypotheses.

Therapeutic strategies to treat chronic pancreatitis (CP) are very limited. Other chronic inflammatory diseases can be successfully suppressed by selective cyclooxygenase 2 (COX-2) inhibitors. As COX-2 is elevated in CP, we attempted to inhibit COX-2 activity in an animal model of CP (WBN/Kob rat). We then analysed the effect of COX-2 inhibition on macrophages, important mediators of chronic inflammation.Male WBN/Kob rats were continuously fed the COX-2 inhibitor rofecoxib, starting at the age of seven weeks. Animals were sacrificed 2, 5, 9, 17, 29, 41, and 47 weeks later. In some animals, treatment was discontinued after 17 weeks, and animals were observed for another 24 weeks.Compared with the spontaneous development of inflammatory injury and fibrosis in WBN/Kob control rats, animals treated with rofecoxib exhibited a significant reduction and delay (p0.0001) in inflammation. Collagen and transforming growth factor beta synthesis were significantly reduced. Similarly, prostaglandin E(2) levels were markedly lower, indicating strong inhibition of COX-2 activity (p0.003). If treatment was discontinued at 24 weeks of age, all parameters of inflammation strongly increased comparable with that in untreated rats. The correlation of initial infiltration with subsequent fibrosis led us to determine the effect of rofecoxib on macrophage migration. In chemotaxis experiments, macrophages became insensitive to the chemoattractant fMLP in the presence of rofecoxib.In the WBN/Kob rat, chronic inflammatory changes and subsequent fibrosis can be inhibited by rofecoxib. Initial events include infiltration of macrophages. Cell culture experiments indicate that migration of macrophages is COX-2 dependent.

Osteopontin (Opn) is a potent chemoattractant for mononuclear cells that is upregulated in various inflammatory states of the kidney. Opn is believed to contribute to mononuclear cell infiltration and renal injury. The importance of Opn was examined in vivo in rapidly progressive glomerulonephritis in Opn knockout mice. Glomerulonephritis was induced by intravenous injection of rabbit anti-mouse glomerular basement membrane antiserum in mice that had been presensitized to rabbit IgG. Immunologic responsiveness to rabbit IgG (assessed by cutaneous delayed-type hypersensitivity and antibody titers) showed no significant difference between wild-type and Opn -/- mice. Proteinuria was also similar in both groups. Glomerular crescent formation was not different in Opn +/+ and -/- groups (26 ± 6% versus 29 ± 7%). Tubulointerstitial infiltration was assessed qualitatively and showed no significant difference between the two genotypes. Formation of thrombi in the glomerular capillaries on a scale from 0 to 3 also showed no significant difference (1.3 ± 0.3 for Opn +/+ and 1.4 ± 0.3 for Opn -/- mice). Northern blot analysis of total kidney RNA showed a 5.1-fold increase of Opn expression in Opn +/+ mice compared with untreated controls and the absence of expression in Opn -/- mice, as expected. Regulated upon activation, normal T cell expressed and secreted (RANTES) and monocyte chemoattractant protein-1 mRNA levels were also markedly upregulated with no significant difference between the two strains, excluding compensatory overexpression of these two chemokines in Opn -/- mice. It is concluded that the known upregulation of Opn in murine anti-glomerular basement membrane nephritis does not significantly contribute to the glomerular and tubulointerstitial mononuclear cell infiltration in this model.

Background/Aim: Pathological accumulation of the extracellular matrix component hyaluronan (HA) occurs in the kidney cortex in immune-system mediated tissue injury. The purpose of the present study was to examine the pattern of HA deposition and the mechanisms of HA synthesis in the MRL-Faslpr mouse model of lupus nephritis. Methods: Kidneys from normal and autoimmune mice were examined for HA content by immunofluorescence staining. Steady state mRNA levels for key enzymes involved in HA synthesis – uridine diphosphate-glucose dehydrogenase (UDPGDH) and HA synthases (HAS) 1, 2 and 3 – were assessed by reverse-transcriptase polymerase chain reaction (RT-PCR). Using cultured mouse tubular epithelial cells, the regulation of the HA production in vitro in response to tumor necrosis factor alpha and interferon gamma was also examined. Results: By immunofluorescence staining, large amounts of HA were detected in the cortical interstitium of MRL-Faslpr mice with autoimmune renal injury, but not in congenic MRL-++ mice. By RT-PCR the presence of transcripts for several genes involved in the synthesis of HA in normal and autoimmune kidneys could be demonstrated, including mRNA for UDPGDH and HAS1 and HAS2, but not for HAS3. Except for HAS2, steady state mRNA levels for these enzymes did not correlate with disease activity. Analyzing a kidney tubular epithelial cell line in vitro, it was found that tumor necrosis factor alpha and interferon gamma, and particularly the combination of these two cytokines, markedly enhanced the synthesis of HA. The expression of HAS2 mRNA was also enhanced in response to cytokine treatment. Conclusions: HA deposition is prominent in MRL-Faslpr mice with renal disease and could be mediated by local synthesis through HAS1 and HAS2. We hypothesize that the enhanced synthesis of HA could be promoted by proinflammatory cytokines in vivo. The functional significance of HA accumulation in autoimmune renal injury remains to be determined.

CBA/CaH-kdkd mice develop hereditary tubulointerstitial disease with mononuclear cell infiltration and cyst formation, possibly representing a model of human nephronophthisis. The purpose of the present investigation was to examine the components of the fibrotic changes which typically develop in the kidneys of these mice. By conventional histology, kdkd mice displayed progressive interstitial fibroblast and matrix accumulation. Immunohistological analysis of kdkd kidneys showed marked deposition of fibronectin in the tubulointerstitial space and revealed prominent irregularities for laminin and collagen type IV in the tubular basement membrane (TBM), including thickening, widening and folding. Electron microscopy confirmed the TBM abnormalities and showed marked undulation and thickening in areas of proximal tubular (PT) degeneration. Immunofluorescence staining analysis for the fibronectin receptors VLA-4 and VLA-5 showed no expression on injured proximal tubules, whereas the expression of the laminin receptor VLA-6 was increased and irregular on altered PT. Analysis of RNA derived from kdkd kidneys revealed marked upregulation of steady-state mRNA levels for the fibrogenic growth factor TGF-β. We conclude that TBM alterations, matrix accumulation and changes in integrin expression together with enhanced TGF-β production are typical features of kdkd tubulointerstitial disease and suggest that characteristic TBM or matrix alterations could contribute to the pathogenesis of the disease in these mice.

The accumulation of hyaluronan (HA) in the renal cortex is a characteristic feature of inflammatory renal diseases. Fragments of HA derived from high molecular weight precursors are known to display inflammatory effects in vitro and could, therefore, participate in immune renal injury. To understand the mechanisms of HA fragmentation in vivo we examined the expression of recently characterized mammalian hyaluronidases in normal and autoimmune kidneys and in cell lines. We found that transcripts for the lysosomal–type hyaluronidases Hyal1 and Hyal2 were constitutively expressed in normal and autoimmune kidneys as well as in tubular epithelial cells and in a macrophage cell line. The expression of hyaluronidase genes in the cell lines did not increase in response to treatment with tumor necrosis factor alpha or gamma interferon. Interestingly, transcripts for the testicular–type hyaluronidase PH–20 (Spam1, Hyal3) were also detected in normal and autoimmune kidneys as well as in tubular cells and macrophages. Transcript levels were higher in kidneys from male mice as compared with age–matched females. Again, transcript levels did not change in vitro in response to cytokines. We conclude that mRNA for three different hyaluronidases is found in murine kidneys. The functional role played by these hyaluronidases in the degradation and metabolism of HA remains to be investigated further.

Background & Aims Little is known about the pathogenic mechanisms of autoimmune pancreatitis (AIP), an increasingly recognized, immune-mediated form of chronic pancreatitis. Current treatment options are limited and disease relapse is frequent. We investigated factors that contribute to the development of AIP and new therapeutic strategies. Methods We used quantitative polymerase chain reaction, immunohistochemical, and enzyme-linked immunosorbent analyses to measure the expression of cytokines and chemokines in tissue and serum samples from patients with and without AIP. We created a mouse model of human AIP by overexpressing lymphotoxin (LT)α and β specifically in acinar cells ( Ela1-LTab mice). Results Messenger RNA levels of LTα and β were increased in pancreatic tissues from patients with AIP, compared with controls, and expression of chemokines ( CXCL13 , CCL19, CCL21 , CCL1 , and B-cell–activating factor ) was increased in pancreatic and serum samples from patients. Up-regulation of these factors was not affected by corticosteroid treatment. Acinar-specific overexpression of LTαβ (Ela1-LTαβ) in mice led to an autoimmune disorder with various features of AIP. Chronic inflammation developed only in the pancreas but was sufficient to cause systemic autoimmunity. Acinar-specific overexpression of LTαβ did not cause autoimmunity in mice without lymphocytes ( Ela1-LTab/Rag1 −/− ); moreover, lack of proinflammatory monocytes ( Ela1-LTab/Ccr2 −/− ) failed to prevent AIP but prevented early pancreatic tissue damage. Administration of corticosteroids reduced pancreatitis but did not affect production of autoantibodies, such as antipancreatic secretory trypsin inhibitor in Ela1-LTab mice. In contrast, inhibition of LTβR signaling reduced chemokine expression, renal immune-complex deposition, and features of AIP in Ela1-LTab mice. Conclusions Overexpression of LTαβ specifically in acinar cells of mice causes features of AIP. Reagents that neutralize LTβR ligands might be used to treat patients with AIP.

OBJECTIVES: The level of pancreatic stone protein/regenerating protein (PSP/reg), a secretory protein produced in the pancreas, increases dramatically during pancreatic disease. However, after stress (e.g., anesthesia), PSP/reg levels are increased transiently in animals without pancreatic injury. Therefore, we aimed to determine whether PSP/reg is an acute-phase protein after nonpancreatic trauma. PATIENTS: Eighty-three polytraumatic patients without pancreatic damage. MEASUREMENTS AND MAIN RESULTS: We compared serum PSP/reg levels from polytraumatic patients without pancreatic damage with those in healthy controls (n = 38). C-reactive protein, interleukin-6, procalcitonin, and leukocyte numbers were also compared. The expression of CD62L and CD11b on neutrophils after exposure to PSP/reg was analyzed by flow cytometry. Thirty-three patients (39%) developed sepsis, 32 (38%) had local infections, and 18 (21%) had no infections. At admission, PSP/reg serum levels (10.2 [6.2-14.5] ng/mL; median [interquartile range]) were comparable with those in healthy controls (10.4 [7.5-12.3] ng/mL). During hospital stay, PSP/reg levels were elevated significantly in patients with sepsis (146.4 ng/mL) and in patients with infections (111.4 ng/mL) compared with patients without infections (22.8 ng/mL). Furthermore, binding of fluorescein isothiocyanate-labeled recombinant PSP/reg to human neutrophils was demonstrated. Recombinant PSP/reg elicited a dose-dependent shedding of L-selectin (CD62L) and upregulation of beta2-integrin (CD11b) in neutrophils, which indicates that PSP/reg activates neutrophils. CONCLUSIONS: We conclude that PSP/reg is up-regulated in blood after trauma, and the PSP/reg level is related to the severity of inflammation. Furthermore, PSP/reg binds to and activates neutrophils. Therefore, PSP/reg might be an acute-phase protein that could serve as a marker for posttraumatic complications.

Cyclooxygenase (COX)-2 is increased in human chronic pancreatitis. We recently demonstrated in a model of chronic pancreatitis (WBN/Kob rat) that inhibition of COX-2 activity reduces and delays pancreatic inflammation and fibrosis. Monocyte chemoattractant protein (MCP)-1 mRNA and PGE2were significantly reduced, correlating with a decreased infiltration of macrophages. MCP-1 plays an important role in the recruitment of macrophages to the site of tissue injury. The aim of our study is to identify mechanisms by which macrophages and acinar cells maintain an inflammatory reaction. The expression profile of E prostanoid receptors EP1-4and MCP-1 was analyzed by RT-PCR from pancreatic specimens and AR42J cells. MCP-1 secretion was detected by ELISA from rat pancreatic lobuli. We determined EP1-4mRNA levels in WBN/Kob rats with chronic pancreatic inflammation. Individual isoforms were highly increased in rat pancreas, concurrent with MCP-1 mRNA expression. In supernatants of pancreatic lobuli and AR42J cells, MCP-1 was detectable by ELISA. In the presence of TNF-α, MCP-1 was upregulated. Coincubation with PGE2enhanced the TNF-α-induced MCP-1 synthesis significantly. Similarly, TNF-α mRNA was synergistically upregulated by TNF-α and PGE2. Furthermore, the synergistic effect of TNF-α and PGE2was abolished by inhibition of PKA but not of PKC. We conclude that EP receptors are upregulated during chronic pancreatic inflammation. PGE2modulates the TNF-α-induced MCP-1 synthesis and secretion from acinar cells. This synergistic effect is controlled by PKA. This mechanism might explain the COX-2-dependent propagation of pancreatic inflammation.