Diagnostic and Prognostic Ability of Pancreatic Stone Protein: A Scoping Review.

Pancreatic stone protein (PSP) is an acute-phase reactant mainly produced in response to stress. Its diagnostic and prognostic accuracy for several types of infection has been studied in several clinical settings. The aim of the current review was to assess all studies examining a possible connection of pancreatic stone protein levels with the severity and possible complications of patients diagnosed with infection. We performed a systematic search in PubMed, Scopus, the Cochrane Library and Clinicaltrials.gov to identify original clinical studies assessing the role of pancreatic stone protein in the diagnosis and prognosis of infectious diseases. We identified 22 eligible studies. Ten of them provided diagnostic aspects, ten studies provided prognostic aspects, and another two studies provided both diagnostic and prognostic information. The majority of the studies were performed in an intensive care unit (ICU) setting, five studies were on patients who visited the emergency department (ED), and three studies were on burn-injury patients. According to the literature, pancreatic stone protein has been utilized in patients with different sites of infection, including pneumonia, soft tissue infections, intra-abdominal infections, urinary tract infections, and sepsis. In conclusion, PSP appears to be a useful point-of-care biomarker for the ED and ICU due to its ability to recognize bacterial infections and sepsis early. Further studies are required to examine PSP's kinetics and utility in specific populations and conditions.

Identification of biomarkers and potential therapeutic targets for pancreatic cancer by proteomic analysis in two prospective cohorts.

Pancreatic cancer (PC) is the deadliest malignancy due to late diagnosis. Aberrant alterations in the blood proteome might serve as biomarkers to facilitate early detection of PC. We designed a nested case-control study of incident PC based on a prospective cohort of 38,295 elderly Chinese participants with ∼5.7 years' follow-up. Forty matched case-control pairs passed the quality controls for the proximity extension assay of 1,463 serum proteins. With a lenient threshold of p < 0.005, we discovered regenerating family member 1A (REG1A), REG1B, tumor necrosis factor (TNF), and phospholipase A2 group IB (PLA2G1B) in association with incident PC, among which the two REG1 proteins were replicated using the UK Biobank Pharma Proteomics Project, with effect sizes increasing steadily as diagnosis time approaches the baseline. Mendelian randomization analysis further supported the potential causal effects of REG1 proteins on PC. Taken together, circulating REG1A and REG1B are promising biomarkers and potential therapeutic targets for the early detection and prevention of PC.

Discriminative performance of pancreatic stone protein in predicting ICU mortality and infection severity in adult patients with infection: a systematic review and individual patient level meta-analysis.

BACKGROUND: Several studies suggested pancreatic stone protein (PSP) as a promising biomarker to predict mortality among patients with severe infection. The objective of the study was to evaluate the performance of PSP in predicting intensive care unit (ICU) mortality and infection severity among critically ill adults admitted to the hospital for infection. METHODS: A systematic search across Cochrane Central Register of Controlled Trials and MEDLINE databases (1966 to February 2022) for studies on PSP published in English using 'pancreatic stone protein', 'PSP', 'regenerative protein', 'lithostatin' combined with 'infection' and 'sepsis' found 46 records. The search was restricted to the five trials that measured PSP using the enzyme-linked immunosorbent assay technique (ELISA). We used Bayesian hierarchical regression models for pooled estimates and to predict mortality or disease severity using PSP, C-Reactive Protein (CRP) and procalcitonin (PCT) as main predictor. We used statistical discriminative measures, such as the area under the receiver operating characteristic curve (AUC) and classification plots. RESULTS: Among the 678 patients included, the pooled ICU mortality was 17.8% (95% prediction interval 4.1% to 54.6%) with a between-study heterogeneity (I-squared 87%). PSP was strongly associated with ICU mortality (OR = 2.7, 95% credible interval (CrI) [1.3-6.0] per one standard deviation increase; age, gender and sepsis severity adjusted OR = 1.5, 95% CrI [0.98-2.8]). The AUC was 0.69 for PSP 95% confidence interval (CI) [0.64-0.74], 0.61 [0.56-0.66] for PCT and 0.52 [0.47-0.57] for CRP. The sensitivity was 0.96, 0.52, 0.30 for risk thresholds 0.1, 0.2 and 0.3; respective false positive rate values were 0.84, 0.25, 0.10. CONCLUSIONS: We found that PSP showed a very good discriminative ability for both investigated study endpoints ICU mortality and infection severity; better in comparison to CRP, similar to PCT. Combinations of biomarkers did not improve their predictive ability.

Reg family proteins contribute to inflammation and pancreatic stellate cells activation in chronic pancreatitis.

Chronic pancreatitis (CP) is a disease characterized by the inflammation and destruction of pancreatic tissue, leading to the replacement of functional tissue with fibrotic tissue. The regenerating gene (Reg) family proteins have recently been implicated in the repair and regeneration of inflamed pancreatic tissue, though the exact mechanisms of their involvement in the pathogenesis of CP are not yet fully understood. To investigate the role of Reg family proteins in CP, we generated global knockout mice (Reg-/-) for Reg1-3 (Reg1,2,3a,3b,3d,3g) genes using the CRISPR/Cas9 system. We then investigated the effect of Reg family protein deficiency in a genetic model of CP (X-SPINK1) mice by knocking out Reg1-3 genes. We examined pancreatic morphology, inflammatory cytokines expression, and activation of pancreatic stellate cells (PSCs) at different ages. Reg-/- mice showed no abnormalities in general growth and pancreas development. Deficiency of Reg1-3 in CP mice led to a reduction in pancreatic parenchymal loss, decreased deposition of collagen, and reduced expression of proinflammatory cytokines. Additionally, Reg proteins were found to stimulate PSCs activation. Overall, our study suggests that Reg1-3 deficiency can lead to the remission of CP and Reg family proteins could be a potential therapeutic target for the treatment of CP.

Keratinocyte Regnase-1, a Downregulator of Skin Inflammation, Contributes to Protection against Tumor Promotion by Limiting Cyclooxygenase-2 Expression.

We previously showed that the ribonuclease Regnase-1 (Reg1) in keratinocytes plays a role in mitigating skin inflammation by downregulating proinflammatory cytokines. In this study, we explored whether Reg1 also has a protective role against skin carcinogenesis. The chemically induced two-stage carcinogenesis protocol revealed that epidermis-specific Reg1-deficient (Reg1-knockout [Reg1-cKO]) mice developed skin tumors with shorter latency and more multiplicity than control mice. In addition, repeated UVB irradiation readily provoked solar keratosis-like lesions in Reg1-cKO mice. Increased levels of cyclooxygenase 2, whose mRNA (Ptgs2) is reportedly a target of Reg1, have been known to be associated with the development of squamous cell carcinomas. Indeed, Ptgs2 mRNA levels were upregulated in the skin of Reg1-cKO mice after treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. The level of prostaglandin E2 was higher in 12-O-tetradecanoylphorbol-13-acetate‒treated Reg1-cKO mouse skin than in control mice skin. Moreover, in vivo inhibition of cyclooxygenase 2 attenuated the 12-O-tetradecanoylphorbol-13-acetate‒induced epidermal thickening in Reg1-cKO mice. Finally, REG1 knockdown in human squamous cell carcinomas lines enhanced PTGS2 mRNA levels after 12-O-tetradecanoylphorbol-13-acetate treatment. In conclusion, epidermal Reg1 plays a regulatory role not only in skin inflammation but also in tumor promotion through the downregulation of cyclooxygenase 2. Therefore, forced expression of Reg1 under inflammatory conditions may be relevant to preventing skin cancer.

The role of pancreatic stone protein as a prognostic factor for COVID-19 patients.

OBJECTIVE: The outbreak of Severe Acute Respiratory Syndrome-CoronaVirus 2 (SARS-CoV-2) has rapidly spread throughout the world straining health care systems. Several biomarkers indicate the presence of hyper-inflammation and evaluate the severity of the disease. Our aim was to investigate the prognostic value of pancreatic stone protein plasma concentration in patients with SARS-CoV-2 pneumonia. PATIENTS AND METHODS: We prospectively studied 55 patients with acute SARS-CoV-2 pneumonia admitted to our tertiary hospital. Sepsis biomarkers, including pancreatic stone protein (PSP), were measured on admission. The role of these biomarkers in the prediction of in-hospital mortality (28 day) and length of hospital stay was investigated. RESULTS: Although Pancreatic stone protein did not have significant prognostic value for in-hospital mortality, there was a moderate accuracy for prolonged length of stay. The optimal cut-off value for prolonged hospital stay was 51 ng/dL (Sensitivity: 0.65, Specificity: 0.913). CONCLUSIONS: Pancreatic Stone Protein on admission could accurately identify patients requiring prolonged hospitalization. The results of this study can serve as a strong early basis for future validation studies of such an innovative approach.

Reg-1α, a New Substrate of Calpain-2 Depending on Its Glycosylation Status.

Reg-1α/lithostathine, a protein mainly associated with the digestive system, was previously shown to be overexpressed in the pre-clinical stages of Alzheimer's disease. In vitro, the glycosylated protein was reported to form fibrils at physiological pH following the proteolytic action of trypsin. However, the nature of the protease able to act in the central nervous system is unknown. In the present study, we showed that Reg-1α can be cleaved in vitro by calpain-2, the calcium activated neutral protease, overexpressed in neurodegenerative diseases. Using chemical crosslinking experiments, we found that the two proteins can interact with each other. Identification of the cleavage site using mass spectrometry, between Gln4 and Thr5, was found in agreement with the in silico prediction of the calpain cleavage site, in a position different from the one reported for trypsin, i.e., Arg11-Ile12 peptide bond. We showed that the cleavage was impeded by the presence of the neighboring glycosylation of Thr5. Moreover, in vitro studies using electron microscopy showed that calpain-cleaved protein does not form fibrils as observed after trypsin cleavage. Collectively, our results show that calpain-2 cleaves Reg-1α in vitro, and that this action is not associated with fibril formation.

Upregulation of REG IV gene in human intestinal epithelial cells by lipopolysaccharide via downregulation of microRNA-24.

The pathophysiology of inflammatory bowel diseases (IBD) reflects a balance between mucosal injury and reparative mechanisms. Some regenerating gene (Reg) family members (REG Iα, REG Iß and REG IV) are expressed in Crohn's disease (CD) and ulcerative colitis (UC) and involved as proliferative mucosal factors in IBD. We revealed that REG Iα and REG Iß were induced in cell culture system by IL-6/IL-22. Although REG IV was upregulated in IBD biopsy samples, the upregulation of REG IV was not at all induced in cell culture by autoimmune-related cytokines such as IL-6, IL-22 and TNFα. Here, we analysed REG IV expression in LS-174 T and HT-29 human intestinal epithelial cells by real-time RT-PCR and elisa. REG IV expression was induced by lipopolysaccharide (LPS). However, LPS did not activate REG IV promoter activity. As the LPS-induced upregulation of REG IV was considered to be regulated post-transcriptionally, we searched targeted microRNA (miR), which revealed that REG IV mRNA has a potential target sequence for miR-24. We measured the miR-24 level of LPS-treated cells and found that the level was significantly lower. The LPS-induced increase of REG IV mRNA was abolished by the introduction of miR-24 mimic but not by non-specific control RNA.

The Potential Role of REG Family Proteins in Inflammatory and Inflammation-Associated Diseases of the Gastrointestinal Tract.

Regenerating gene (REG) family proteins serve as multifunctional secretory molecules with trophic, antiapoptotic, anti-inflammatory, antimicrobial and probably immuno-regulatory effects. Since their discovery, accumulating evidence has clarified the potential roles of the REG family in the occurrence, progression and development of a wide range of inflammatory and inflammation-associated diseases of the gastrointestinal (GI) tract. However, significant gaps still exist due to the undefined nature of certain receptors, regulatory signaling pathways and possible interactions among distinct Reg members. In this narrative review, we first describe the structural features, distribution pattern and purported regulatory mechanisms of REG family proteins. Furthermore, we summarize the established and proposed roles of REG proteins in the pathogenesis of various inflammation-associated pathologies of the GI tract and the body as a whole, focusing particularly on carcinogenesis in the ulcerative colitis-colitic cancer sequence and gastric cancer. Finally, the clinical relevance of REG products in the context of diagnosis, treatment and prognostication are also discussed in detail. The current evidence suggests a need to better understanding the versatile roles of Reg family proteins in the pathogenesis of inflammatory-associated diseases, and their broadened future usage as therapeutic targets and prognostic biomarkers is anticipated.

Protective and anti-inflammatory role of REG1A in inflammatory bowel disease induced by JAK/STAT3 signaling axis.

Regenerating islet-derived protein 1-alpha (REG1A) was abnormally upregulated in a series of gastrointestinal inflammatory disorders. However, the potential biological function and underlying regulatory mechanisms of the increased REG1A in inflammatory bowel disease (IBD) pathogenesis remain to be fully elucidated. In this study, we uncovered that REG1A was substantially increased in the inflamed colorectal tissues of IBD patients. And the aberrantly expressed REG1A in intestinal epithelial cells (IEC) prominently inhibited inflammatory responses, promoted cell proliferation and suppressed epithelial apoptosis. Mechanically, IL-6 and IL-22 markedly activated REG1A transcription through triggering JAK/STAT3 signaling pathway. In addition, overexpression of REG1A in mice by systematic delivery of REG1A lentivirus remarkably alleviated DSS-induced inflammatory injury and maintained the integrity of intestinal mucosal barrier. Taken together, our data demonstrated that the novel proliferative factor REG1A controlled by IL-6/IL-22-JAK-STAT3 signaling may provide a promising therapeutic target for patients with IBD.

Fluid shear stress enhances differentiation of jejunal human enteroids in Intestine-Chip.

There is increasing evidence that the study of normal human enteroids duplicates many known aspects of human intestinal physiology. However, this epithelial cell-only model lacks the many nonepithelial intestinal cells present in the gastrointestinal tract and exposure to the mechanical forces to which the intestine is exposed. We tested the hypothesis that physical shear forces produced by luminal and blood flow would provide an intestinal model more closely resembling normal human jejunum. Jejunal enteroid monolayers were studied in the Emulate, Inc. Intestine-Chip under conditions of constant luminal and basolateral flow that was designed to mimic normal intestinal fluid flow, with human umbilical vein endothelial cells (HUVECs) on the basolateral surface and with Wnt3A, R-spondin, and Noggin only on the luminal surface. The jejunal enteroids formed monolayers that remained confluent for 6-8 days, began differentiating at least as early as day 2 post plating, and demonstrated continuing differentiation over the entire time of the study, as shown by quantitative real-time polymerase chain reaction and Western blot analysis. Differentiation impacted villus genes and proteins differently with early expression of regenerating family member 1α (REG1A), early reduction to a low but constant level of expression of Na+-K+-2Cl- cotransporter 1 (NKCC1), and increasing expression of sucrase-isomaltase (SI) and downregulated in adenoma (DRA). These results were consistent with continual differentiation, as was shown to occur in mouse villus enterocytes. Compared with differentiated enteroid monolayers grown on Transwell inserts, enteroids exposed to flow were more differentiated but exhibited increased apoptosis and reduced carbohydrate metabolism, as shown by proteomic analysis. This study of human jejunal enteroids-on-chip suggests that luminal and basolateral flow produce a model of continual differentiation over time and NaCl absorption that mimics normal intestine and should provide new insights in intestinal physiology.NEW & NOTEWORTHY This study showed that polarized enteroid models in which there is no basolateral Wnt3a, are differentiated, regardless of the Wnt3a status of the apical media. The study supports the concept that in the human intestine villus differentiation is not an all or none phenomenon, demonstrating that at different days after lack of basolateral Wnt exposure, clusters of genes and proteins exist geographically along the villus with different domains having different functions.

Pancreatic stone protein - sepsis and the riddles of the exocrine pancreas.

Pancreatic stone protein (PSP), discovered in the 1970ies, was first associated with stone formation during chronic pancreatitis. Later, the same protein was independently detected in islet preparations and named regenerating protein 1 (REG1). Additional isoforms of PSP, including pancreatitis-associated protein (PAP), belong to the same protein family. Although the names indicate a potential function in stone formation or islet regeneration, involvements in cellular processes were only suggestive and never unequivocally proven. We established an association between PSP levels in patient blood samples and the development of sepsis. In this review, written in connection with receiving the Lifetime Achievement Award of the European Pancreatic Club, the evolution of the sepsis aspect of PSP is described. We conclude that the true functional properties of this fascinating pancreatic protein still remain an enigma.

MicroRNA-7 directly targets Reg1 in pancreatic cells.

Regenerating islet-derived (Reg) proteins, which were first discovered in the pancreas, are associated with increased proliferation, prevention of apoptosis, and enhanced differentiation in normal and disease states, but very little is known about the regulation of their expression. We hypothesized that Reg expression is influenced by microRNAs. Bioinformatic analysis predicted Reg1 to be a target of microRNA-7 (miR-7), which influences pancreatic ß-cell function. To this end, we investigated the effects of miR-7 on Reg1 expression in pancreatic acinar and islet ß-cells. High levels of Reg1 were noted by immunostaining and Western blotting in acinar cells in contrast to islet cells. A reciprocal expression pattern was observed for miR-7. Overexpression of miR-7 resulted in Reg1 mRNA suppression and reduction of secreted Reg1 protein. Conversely, miR-7 knockdown led to increases in Reg1. Targeting of Reg1 by miR-7 was confirmed via luciferase activity assays. In contrast, miR-7 did not directly repress the human ortholog of Reg1 REG1A as well as REG1B indicating species differences in the regulation of Reg expression. This is the first account of microRNA modulation of any Reg member warranting studies to fill gaps in our knowledge of Reg protein biology, particularly in disease contexts.

Knockdown of REG Iα Enhances the Sensitivity to 5-Fluorouracil of Colorectal Cancer Cells via Cyclin D1/CDK4 Pathway and BAX/BCL-2 Pathways.

Objective: The reverse of chemoresistance and the improvement of sensitivity to chemotherapeutic agents of colorectal cancer cells have great clinical significance and the mechanism underlying the drug resistance is still unclear. REG Iα was reported to be upregulated in colorectal cancer tissues, but the roles of chemoresistance are still unclear. Materials and Methods: The expression of REG Iα in colorectal cancer cell lines was assessed by quantitative real-time polymerase chain reaction (Q-PCR). The expression of REG Iα in HCT116 and LOVO cells was knockdown by siRNA. The cell viability and IC50 (half maximal inhibitory concentration) values were analyzed by the CCK8 assay. The proportion of apoptosis and cell cycles were analyzed by flow cytometry. The migration potency of HCT116 and LOVO cells was analyzed by cell migration assay. The protein level of Cyclin D1, CDK4 (cyclin-dependent kinase 4), Bax and Bcl-2 were analyzed by western blot. Results: Knockdown of REG Iα enhances the sensitivity to 5-Fu of colorectal cancer cells. REG Iα knockdown promoted the cell apoptosis of HCT116 and LOVO under the 5-Fu treatment. The cell migration and cycle of colorectal cancer cells was also inhibited by REG Iα knockdown. We also found that REG Iα knockdown induced cell cycle arrest and cell apoptosis by Cyclin D1/CDK4 pathway and BAX/BCL-2 pathways. Conclusions: Knockdown of REG Iα enhances the sensitivity to 5-Fu of colorectal cancer cells via cyclin D1/CDK4 pathway and BAX/BCL-2 pathways.

METTL14 is essential for ß-cell survival and insulin secretion.

Defects in the development, maintenance or expansion of ß-cell mass can result in impaired glucose metabolism and diabetes. N6-methyladenosine affects mRNA stability and translation efficiency, and impacts cell differentiation and stress response. To determine if there is a role for m6A in ß-cells, we investigated the effect of Mettl14, a key component of the m6A methyltransferase complex, on ß-cell survival and function using rat insulin-2 promoter-Cre-mediated deletion of Mettl14 mouse line (ßKO). We found that ßKO mice with normal chow exhibited glucose intolerance, lower levels of glucose-stimulated insulin secretion, increased ß-cell death and decreased ß-cell mass. In addition, HFD-fed ßKO mice developed glucose intolerance, decreased ß-cell mass and proliferation, exhibited lower body weight, increased adipose tissue mass, and enhanced insulin sensitivity due to enhanced AKT signaling and decreased gluconeogenesis in the liver. HFD-fed ßKO mice also showed a decrease in de novo lipogenesis, and an increase in lipolysis in the liver. RNA sequencing in islets revealed that Mettl14 deficiency in ß-cells altered mRNA expression levels of some genes related to cell death and inflammation. Together, we showed that Mettl14 in ß-cells plays a key role in ß-cell survival, insulin secretion and glucose homeostasis.

Pancreatic stone protein/regenerating protein is a potential biomarker for endoplasmic reticulum stress in beta cells.

Endoplasmic reticulum (ER) stress in beta cells is an important pathogenic component of both type 1 and type 2 diabetes mellitus, as well as genetic forms of diabetes, especially Wolfram syndrome. However, there are currently no convenient ways to assess ER stress in beta cells, raising the need for circulating ER stress markers indicative of beta cell health. Here we show that pancreatic stone protein/regenerating protein (PSP/reg) is a potential biomarker for ER stressed beta cells. PSP/reg levels are elevated in cell culture and mouse models of Wolfram syndrome, a prototype of ER stress-induced diabetes. Moreover, PSP/reg expression is induced by the canonical chemical inducers of ER stress, tunicamycin and thapsigargin. Circulating PSP/reg levels are also increased in some patients with Wolfram syndrome. Our results therefore reveal PSP/reg as a potential biomarker for beta cells under chronic ER stress, as is the case in Wolfram syndrome.

Measurement of pancreatic stone protein in the identification and management of sepsis.

Sepsis is a life-threatening syndrome characterized by a dysregulated host response to an infection resulting in multiple organ dysfunctions. Early diagnosis and management of sepsis is key to improve patient outcome but remains challenging. Despite extensive research, only few biomarkers have so far proven to be helpful in the diagnosis of sepsis. A novel protein biomarker, the pancreatic stone protein (PSP), is showing great promises. Several lines of evidences suggest that PSP has a higher diagnostic performance for the identification of sepsis than procalcitonin and C-reactive protein, and a strong prognostic value to predict unfavorable outcome at admission to intensive care unit. This review summarizes the current knowledge on the molecular mechanisms of PSP function and the clinical evidences available to highlight the relevance of this protein in the diagnosis and prognosis of sepsis.

Comparative Analysis of Expression Profiles of Reg Signaling Pathways-Related Genes Between AHF and HCC.

Regenerating islet-derived protein (Reg) could participate in the occurrence of diabetes mellitus, inflammation, tumors, and other diseased or damaged tissues. However, the correlation of Reg with acute hepatic failure (AHF) and hepatocellular carcinoma (HCC) is poorly defined. To reveal the expression profiles of Reg family and their possible regulatory roles in AHF and HCC, rat models of HCC and AHF were separately established, and Rat Genome 230 2.0 was used to detect expression profiles of Reg-mediated signaling pathways-associated genes from liver tissues in AHF and HCC. The results showed that a total of 79 genes were significantly changed. Among these genes, 67 genes were the AHF-specific genes, 45 genes were the HCC-specific genes, and 33 genes were the common genes. Then, K-means clustering classified these genes into 4 clusters based on the gene expression similarity, and DAVID analysis showed that the above altered genes were mainly associated with stress response, inflammatory response, and cell cycle regulation. Thereafter, IPA software was used to analyze potential effects of these genes, and the predicted results suggested that the Reg-mediated JAK/STAT, NF-κB, MAPK (ERK1/2, P38 and JNK), PLC, and PI3K/AKT signaling pathways may account for the activated inflammation and cell proliferation, and the attenuated apoptosis and cell death during the occurrence of AHF and HCC.

Crucial role of Reg I from acinar-like cell cluster touching with islets (ATLANTIS) on mitogenesis of beta cells in EMC virus-induced diabetic mice.

Recently, we reported the presence of distinct cell clusters named acinar-like cell clusters touching Langerhans islets with thin interstitial surrounding (ATLANTIS) in human pancreas. A morphological study in humans demonstrated that ATLANTIS and islet cell clusters are found together in the microenvironment enclosed by a common basement membrane, and ATLANTIS releases vesicles containing Regenerating gene protein (REG Iα) to islet cell clusters. We examined 1) the presence or absence of ATLANTIS in homozygous Reg I (mouse homologue of human REG Iα) deficient (Reg I-/-) and wild-type mice, and 2) the possible role of ATLANTIS in the regeneration of beta cell clusters after encephalomyocarditis (EMC) virus (D-variant) infection in Reg I-/- and wild-type mice. ATLANTIS was found in both wild-type and Reg I-/- mice. In both groups, mean blood glucose increased transiently to greater than 14.0 mmol/L at 5 days after EMC virus infection and recovered to baseline at 12 days. At 12 days after EMC virus infection, lower BrdU labeling indices were observed in islet beta cells of Reg I-/- mice compared to wild-type mice. Beta cell volume 12 days after EMC virus infection in Reg I-/- mice did not differ from that of wild-type mice. These results suggest that Reg I, which is released from ATLANTIS to islet beta cell clusters, has a crucial role in beta cell regeneration in EMC virus-induced diabetes. The presence of mechanism(s) other than that mediated by Reg I in beta cell restoration after destruction by EMC virus was also suggested.

Uncovering the anticancer mechanism of Compound Kushen Injection against HCC by integrating quantitative analysis, network analysis and experimental validation.

Compound Kushen Injection (CKI) is a Traditional Chinese Medicine (TCM) preparation that has been clinically used in China to treat various types of solid tumours. Although several studies have revealed that CKI can inhibit the proliferation of hepatocellular carcinoma (HCC) cell lines, the active compounds, potential targets and pathways involved in these effects have not been systematically investigated. Here, we proposed a novel idea of "main active compound-based network pharmacology" to explore the anti-cancer mechanism of CKI. Our results showed that CKI significantly suppressed the proliferation and migration of SMMC-7721 cells. Four main active compounds of CKI (matrine, oxymatrine, sophoridine and N-methylcytisine) were confirmed by the integration of ultra-performance liquid chromatography/mass spectrometry (UPLC-MS) with cell proliferation assays. The potential targets and pathways involved in the anti-HCC effects of CKI were predicted by a network pharmacology approach, and some of the crucial proteins and pathways were further validated by western blotting and metabolomics approaches. Our results indicated that CKI exerted anti-HCC effects via the key targets MMP2, MYC, CASP3, and REG1A and the key pathways of glycometabolism and amino acid metabolism. These results provide insights into the mechanism of CKI by combining quantitative analysis of components, network pharmacology and experimental validation.