Background:Zornia diphylla (L.) Pers. (ZDP) is a traditional Chinese herbal medicine that has been used for several decades to treat patients with liver diseases. Whether ZDP is best administered as a single agent or adjunctive therapy has yet to be determined as does the mechanism whereby it exerts its effects on antagonizing acute liver injury (ALI).Aim of the study: To investigate the protective effects of ZDP on ALI induced by carbon tetrachloride (CCl4) and the potential underlying mechanisms.Materials and Methods: Sixty adult mice were randomized into six study groups (n = 10/group). Three groups were treated with different concentrations of ZDP , one with bifendate (0.0075 g/kg) alone (positive control) and one with physiologic saline . All groups were treated for 14 days. Two hours after the last administration, the normal group received an intraperitoneal injection of peanut oil, and the other five groups received an intraperitoneal injection of an equal dose of CCl4 peanut oil solution. At 24 h, the liver index, histology and serum or tissue levels and/or protein expression of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), alkaline phosphatase (ALP), superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), glutathione (GSH), Akt, phosphorylated Akt (p-Akt), nuclear factor kappa B p65 (NF-κB p65), inhibitor of NF-κB α (IκB-α), interleukin-1 β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), E-cadherin and vimentin were determined.Results: Compared to the model controls, the degree of inflammatory cell infiltration and hepatocyte injury of liver tissue was relieved in the bifendate and three ZDP groups; liver index in the ZDP  groups and serum liver function indices in the ZDP  groups were decreased; antioxidants SOD, CAT and GSH in liver tissue were increased but the lipid peroxidation index MDA was decreased; protein expression of inflammatory cytokines Akt, p-Akt, NF-κB p65, IκB-α, IL-1β, IL-6 and TNF-α in the liver was ameliorated, and E-cadherin expression was increased. The results of liver histopathology also showed that ZDP had a significant effect on ALI.Conclusion: ZDP has obvious protective effects on CCl4-induced ALI as a single therapy and appears to act by inhibiting oxidation, reducing the release of inflammatory factors and promoting hepatocyte repair. In recent years, due to the emergence of drug abuse, and the widespread use of various types of therapeutic drugs with many side effects, clinical drug-induced liver damage is more common, and to date, there is no effective prevention and treatment measures in clinical practice. Acute liver injury (ALI) refers to sudden hepatocyte damage and abnormal liver function due to various reasons within a short period of time. Drug poisoning, viral infection, immune response, and ischemia reperfusion are the most common ALI inducing factors , of whicZornia diphylla (L.) Pers. (ZDP), known as “Dingkuicao” in Chinese, is the dry whole herb of Zornia diphylla (L.) Pers. from the Fabaceae family, which grows in tropical areas, mainly in Zhejiang, Fujian, Guangxi, Yunnan and other regions in China -induced ALI model was established in mice. In addition to biochemical, molecular biological protein analysis and pathomorphological observations, the hepatoprotective effect of ZDP was confirmed and its possible mechanism was discussed, which not only explains the efficacy of TCM using modern scientific theory, but also provides the necessary experimental data for further development of ZDP in the treatment of liver disease.In order to verify the hepatoprotective activity of ZDP, a carbon tetrachloride  aged 4–6 weeks, weighing 20 ± 2 g, were purchased from Minhou Wushi Experimental Animal Trading Co., Ltd. , which has the Experimental Animal Production License Number: SCXK (Min) 2016-0002. All the experimental mice [Experimental Animal Usage License Number: SYXK (Min) 2018-0009] were randomly divided into six groups with ten mice in each group, and the experiment was carried out after 7 days of adaptive feeding. The room temperature was kept at 22 ± 2°C, the air humidity was controlled at 55 ± 20%, light was 12 h/day, and the mice were free to drink pure water and eat freely. The experiment was carried out in strict accordance with the standards of the Laboratory Animal Management and Ethics Committee of Xiamen University  (No. XMULAC2012-0039).http://sweetgum.nybg.org/science/ih/herbarium-details/?irn=249232) (Herbarium Code: XMBG) for future reference. It was crushed, sieved through a 50 mesh, and weighed. ZDP powder was extracted three times with 10× distilled water, for 2 h each time, and the filtrate was concentrated and dried in a vacuum drier. The weight of the final freeze-dried powder of ZDP (50 g) was 5.3 g, and the extraction yield was 10.6%. Distilled water was used to prepare solutions containing 0.25, 0.125 and 0.0625 g of extract per ml, respectively, labeled as high, medium and low dose extracts of ZDP, and placed in a refrigerator at 4°C until used. Biphenyl diester drop pills (Lot No.: A02180903) (Specification: 15 mg × 250 pills) were purchased from Wanbang Pharmaceutical Group Co., Ltd. , diluted in distilled water and an aqueous solution was prepared at the concentration of 0.75 mg/ml for the experiments.One kilogram of dried whole herb of ZDP (Voucher Number: 181012) was purchased from Guangdong Kangmei Pharmaceutical Co., Ltd., Puning, China. The voucher specimen was deposited at the Xiamen Botanical Garden (4 solution (Lot No.: C805332) was purchased from Shanghai Macklin Biochemical Co., Ltd. .In this study, the following reagents were used: an alanine aminotransferase (ALT) assay kit (Lot No.: C009-2-1), aspartate aminotransferase (AST) assay kit (Lot No.: C010-2-1), alkaline phosphatase (ALP) assay kit (Lot No.: A059-2-2), total bilirubin (TBIL) assay kit (Lot No.: C019-1-1), catalase (CAT) assay kit (Lot No.: A007-1), glutathione (GSH) assay kit (Lot No.: A006-2-1), malondialdehyde (MDA) assay kit (Lot No.: A003-4-1) and superoxide dismutase (SOD) assay kit (Lot No.: A001-3), which were purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd. . An interleukin-1 β (IL-1β) ELISA kit (Lot No.: M0037c) and tumor necrosis factor α (TNF-α) ELISA kit (Lot No.: M0049c) were purchased from Elabscience Biotechnology Co., Ltd. . Akt antibody (Lot No.: 4691), p-Akt antibody (Lot No.: 13038), nuclear factor kappa B p65 (NF-κB p65) antibody (Lot No.: 3033), inhibitor of NF-κB α (IκB-α) antibody (Lot No.: 4814), interleukin-6 (IL-6) antibody (Lot No.: 12912) and TNF-α antibody (Lot No.: 11948) were purchased from Cell Signaling Technology, Inc. . IL-1β antibody (Lot No.: 16806-1-AP), E-cadherin antibody (Lot No.: 20874-1-AP) and rabbit secondary antibody (Lot No.: SA00001), were purchased from Proteintech Group, Inc. . Vimentin antibody (Lot No.: ARG65682) was purchased from Arigo Biolaboratories Corp. . A BCA kit (Lot No.: QB214754) was purchased from Thermo Fisher Scientific Technology Co., Ltd. . RIPA buffer (Lot No.: R0010) was purchased from Beijing Solarbio Science and Technology Co., Ltd. . CClThe following instruments were employed: D3024R High-speed freezing microcentrifuge , GloMax GM3030 Microplate reader , DXC800 Beckman Automatic Biochemistry Analyzer , 10RTEX-5 Oscillation apparatus and TSB-108 Shaking table , 041BR126545 Electrophoresis apparatus , HJ-4A Magnetic constant temperature stirrer , HLD-10002 Electronic balance , SB-1100 Water bath pot , DK-8D Electric thermostatic bath , LeicaRM2016 histotome , Olympus CX41 Microscope  and Thermo Q-Exactive Orbitrap Mass spectrometer equipped with a Thermo UltiMate 3000 HPLC system .m/z scan range from 100 to 1000, was acquired both in positive and in negative mode. The ionization process was operated with a spray voltage of 3.8 and 3.5 kV for the positive and negative mode, respectively.The major chemical constituents of ZDP were profiled by high performance liquid chromatography (HPLC) coupled with a high resolution electrospray ionization mass detector. The freeze-dried powder of ZDP dissolved in water at a concentration of 10 mg/ml was used as the test solution. The solution was filtered with a 0.22 µm nylon filter membrane before injection into the HPLC system. The 1D-HPLC separations were performed on a Kinetex C18 column . The mobile phase was water (A) and methanol (B), gradiently eluted according to the following elution program: 0–25 min, 20% B to 100% B and 100% B was kept until 30 min. The column was maintained at 30°C and eluted at a flow rate of 0.3 ml/min. Sample loading of the test solution was 10 µl (10 mg/ml). The high resolution electrospray ionization detector was used to record the HPLC chromatograms. The MS spectra, with an n = 10/group) according to a random number table, namely, normal (negative control), model, western medicine control (biphenyl diester), and high, medium and low-dose water extracts of ZDP . The normal and model groups were given 0.9% physiological saline by gavage, once a day. The western medicine control group was given an aqueous solution of bifendate dropping pills by gavage at 0.0075 g/kg (0.00075 g/ml), once a day. Based on clinical experience, the commonly-used daily dose of ZDP (dried whole herb) for adults with a body weight of 60 kg is 12 g (Km/Human Km (Km)/3 (Mouse Km) = 2.5 g/kg, which was used in the ZDP high-dose group in this study. The three ZDP groups were given the aqueous extract solution of ZDP by gavage at 2.5 g/kg (0.25 g/ml), 1.25 g/kg (0.125 g/ml) and 0.625 g/kg (0.0625 g/ml), respectively, once a day. Each group was treated for 14 consecutive days. CCl4 solution was prepared into a 0.1% peanut oil diluent. Two hours after the last administration, mice in the normal control group were intraperitoneally injected with an equal dose of peanut oil (10 ml/kg), whereas mice in the other five groups were intraperitoneally injected with CCl4 peanut oil solution  to induce the murine model of ALI . After weighing, half of the liver tissue was routinely fixed and embedded in wax blocks, and the other half was stored at −80°C.Using a sterile coagulation promoting tube, peripheral blood (0.8 ml) was collected from the orbital sinus of each mouse prior to sacrifice. After standing for 2 h at room temperature, the blood samples were centrifuged at 3,000 g for 10 min. The supernatant was collected and stored in a refrigerator at −80°C. Serum ALT, AST, TBIL and ALP levels were determined using their respective assay kits and the automatic biochemistry analyzer according to the manufacturer’s instructions.At the time of sacrifice, some of the hepatic tissue not required for histology was washed with PBS, cut into small pieces using ophthalmic scissors, transferred to a homogenizer, exposed to protein extraction solution, incubated in an ice bath and then centrifuged at 15,000 g. The supernatants were collected and stored at −20°C for protein quantification. SOD, MDA, CAT, GSH, IL-1β and TNF-α ELISA kits were used to determine the concentrations of these parameters according to the manufacturer’s instructions . The detFollowing blood collection, the mice were immediately sacrificed by cervical dislocation. The whole liver was completely removed and placed on sterile ice. After washing with normal saline, filter paper was used to remove excess saline. The liver was weighed and the value recorded. The liver index (liver weight/body weight × 100%) was calculated .Liver tissue 1.0 cm × 1.0 cm × 0.3 cm in size was quickly cut and fixed in 4% paraformaldehyde for 24 h. A tissue block 0.4 cm × 0.4 cm × 0.3 cm in size was cut with a blade and placed into a disposable plastic embedding frame, and then dehydrated in low to high concentration ethanol. After dehydration, the tissue was made transparent with xylene. The embedded wax blocks were sliced into 5 μm wax strips, spread on the water surface at 40–45°C and baked in an incubator for 2 h at 60°C. The paraffin strips were dewaxed with xylene and washed with ethanol and distilled water. Following hematoxylin and eosin (HE) staining, the stained sections were dehydrated using a low to high concentration ethanol gradient, and xylene added; the sections were removed, excess xylene was wiped off and a drop of 50% neutral gum was added. The slides were covered and sealed using neutral balsam. The pathological changes in the tissues were observed under a light microscope and recorded.Liver tissue (30 μg) from each sample was used for protein analysis. The detection steps were as follows: 4 × loading buffer was added, the samples were placed in a boiling water bath for 10 min, centrifuged at 10,000 rpm for 10 min; 10% or 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was prepared according to the molecular weight of protein for electrophoretic separation of protein samples; the proteins on SDS-PAGE were transferred to a polyvinylidene fluoride (PVDF) membrane by electric transfer; after electric transfer, the PVDF was immersed in sealing solution containing 5% nonfat dry milk and incubated for 1 h at room temperature. Phosphate buffer solution-tween-20 (PBST) was used to wash the membrane three times for 5 min each time; the primary antibody of Akt (1: 1000), p-Akt (1: 4000), NF-κB p65 (1: 1000), IκB-α (1: 1000), IL-1β (1: 1000), IL-6 (1: 1000), TNF-α (1: 1000), E-cadherin (1: 1000) or vimentin (1: 1000) was added, respectively, then incubated overnight at 4°C. After washing 3 times with PBST for 20 min each time, horseradish peroxidase labeled secondary antibody solution  was added, and incubated at room temperature for 1 h. After washing three times with PBST for 10 min each time, the PVDF membrane was reacted with fresh enhanced chemiluminescence solution for 2 min, and quickly exposed in a dark room . The optp < 0.05 was considered statistically significant.Data are expressed as mean ± standard deviation (SD) (The water extract of ZDP was separated using the HPLC-MS system and its chromatographic fingerprinting was established. Comparing the retention time (Rt), ultraviolet and mass spectra with reference samples, the following five major components were identified: cosmosiin , luteolin 4′-O-glucopyranoside , quercimeritrin , luteolin  and apigenin  as shown in To investigate the therapeutic effects of ZDP on the pathological changes due to ALI, HE staining was performed. As shown in 4 significantly reduced the body mass of mice (p < 0.01). Compared with the model group, the intake of ZDP significantly slowed the decrease in mouse body mass (p < 0.01 or p < 0.05), indicating that ZDP could inhibit the decrease in body mass caused by CCl4. Compared with the normal group, the liver index of mice in the model group was significantly increased (p < 0.01). Compared with the model group, the liver index of mice in the bifendate-treated positive control group and three ZDP groups was significantly decreased (p < 0.01 or p < 0.05), but there was no statistically significant difference in the liver index among the ZDP dose groups (p > 0.05).Changes in body mass and liver index can reflect the degree of liver injury . As showp < 0.01). Compared with the model group, the serum AST and ALT activities in mice in the ZDP group treated with 2.5 or 1.25 g/kg and in the positive control (Bifendate-treated) group were significantly decreased (p < 0.01 or p < 0.05), and the activities of TBIL and ALP in the bifendate-treated group and all ZDP groups were also obviously decreased (p < 0.01). However, there was no significant difference in the above indices between the three ZDP groups (p > 0.05). These results show that ZDP repaired the damage to the liver cell membrane and organelles induced by CCl4, and liver function was significantly improved.The increase in ALT reflected the damage to hepatocyte membranes. The increase in AST indicated hepatocyte organelle damage. AST, ALT, TBIL and ALP are the most direct and important indicators of liver damage and show whether therapeutic agents have a protective effect against liver injury . As show4 increased, and the contents of SOD, CAT and GSH decreased (p < 0.01 or p < 0.05). Compared with the model group, the activity of SOD, the contents of CAT and GSH in the ZDP high dose group and bifendate-treated group were increased and the content of MDA was decreased (p < 0.01 or p < 0.05). The content of CAT in the ZDP low and medium dose groups was also increased significantly compared with the model group (p < 0.01). CCl4 increased the level of oxidative stress and reduced the antioxidant capacity of the liver (p < 0.01). These results show that ZDP can inhibit the degree of lipid peroxidation injury induced by CCl4 and improve the antioxidant capacity of mice.Oxidative stress induced by reactive oxygen species is the common pathophysiological basis in a variety of liver injuries. MDA, SOD, CAT and GSH can reflect the degree of lipid peroxidation and oxidative stress . As show4 were significantly elevated (p < 0.01). Compared with the model group, the level of TNF-α and IL-1β in liver tissue of mice in the medium and high dose ZDP groups and the bifendate-treated group decreased (p < 0.01).IL-1β and TNF-α are common indices used to detect the inflammatory response . As showp < 0.01) and the levels of NF-κB, IL-1β, TNF-α and IL-6 were elevated (p < 0.01 or p < 0.05) after CCl4 treatment. Compared with the model group, IκB protein expression level in both the ZDP high dose group and bifendate-treated group was increased (p < 0.01). However, there was no significant difference in the indices of Akt and IκB between the bifendate-treated group and the three ZDP groups (p > 0.05). Compared with the model group, protein expression levels of NF-κB, IL-1β, TNF-α and IL-6 in the ZDP high dose group and bifendate control group were obviously decreased (p < 0.01 or p < 0.05) as well as phosphorylated Akt (p-Akt) protein expression levels in both the ZDP high and medium dose groups. These results show that ZDP can inhibit the Akt-NF-κB pathway and its downstream inflammatory factors to relieve hepatic inflammatory activity in mice.The pathogenesis of ALI is closely related to oxidative stress and inflammation . During 4 administration, the E-cadherin protein expression levels were decreased (p < 0.05), but expression levels of vimentin were significantly increased (p < 0.01). Compared with the model group, E-cadherin protein expression in the ZDP high dose group and bifendate control group was higher (p < 0.01 or p < 0.05), and vimentin protein expression in the three ZDP groups also showed a downward trend. However, there was no significant difference in these three indices between the three ZDP groups and bifendate control group (p > 0.05). These results showed that ZDP had an inhibitory effect on EMT and promoted the repair of damaged hepatocytes.Acute liver disease is associated with ample re-modeling of liver parenchyma leading to functional impairment . Hepatoc4 in this study is a classic modeling method, as the symptoms, liver function indices and pathological changes are similar to human liver injury , CCl3-and CCl3O2- free radicals are generated in vivo. CCl3-binds freely with intracellular macromolecules, resulting in the destruction of hepatocyte membrane structure and function, and finally hepatocyte degeneration and necrosis (Zornia diphylla (L.) Pers. in CCl4-induced ALI in mice, and revealed that the main mechanisms underlying its efficacy were antioxidation, downregulation of the expression of pro-inflammatory signal factors and the promotion of liver tissue repair (The liver is the largest parenchymal organ in the human body, and its functions include catabolism, toxin removal, oxidation resistance and so on. However, the liver is easily damaged by various internal and external pathogenic factors. Liver disease is common and has been the focus of global medical attention and research, and ALI is the starting point of almost all liver diseases . Animal r injury . CCl4 isnecrosis . In thise repair . These f4.Following hepatocyte injury, the soluble enzymes AST and ALT present in the cytoplasm enter the blood, resulting in a significant increase in the levels of these enzymes in serum . This isin vivo. The higher the activity of SOD in vivo, the faster the free radical scavenging rate. When hepatocytes are attacked by free radicals, SOD can be reduced due to its excessive consumption. The reduction in its activity can lead to the accumulation of oxygen free radicals in the body, resulting in peroxidation of plasma membrane, damage to the membrane structure and function, and finally to the death of hepatocytes (in vivo, which can convert harmful substances in cells into harmless substances and eliminate them (4 liver injury in mice, the content of MDA increased significantly and the activities of SOD, GSH and CAT decreased significantly, indicating that the ability of hepatocytes to scavenge free radicals decreased. However, the content of MDA decreased significantly and the activities of SOD, GSH and CAT increased significantly in the liver tissue of mice in the ZDP groups, suggesting that ZDP has a certain antioxidant effect and can effectively improve the ability of cells to scavenge free radicals and reduce the damage to hepatocytes under free radical attack, which may be related to the antioxidant effect. These results were consistent with those of previous research (The pathogenesis of ALI is closely related to oxidative stress and the inflammatory response. Lipid peroxidation is an important mechanism of liver injury. SOD is the main antioxidant enzyme that scavenges free radicals and inhibits free radical reactions atocytes . MDA is atocytes . SOD canate them . In thisresearch .4, interleukin, as an important inflammatory mediator and immunomodulatory factor, activates inhibitory kappa B kinase, degrades IκB protein, dissociates NF-κB from IκB, and NF-κB then translocates from the cytoplasm to the nucleus and binds to the specific promoter, mediates the mutual expression of inflammatory cytokines within and between cells, multidirectionally regulates the inflammatory response and causes the cascade amplification effect of the inflammatory response (IL-1β, IL-6, TNF-α and other inflammatory factors play an important role in the inflammatory process of acute tissue injury . In ALI,response . In this4 can activate hepatic stellate cells, which can express vimentin in large quantities, and then start the process of liver fibrosis (4 administration, and decreased in the ZDP groups, but no statistically significant difference was observed. The reason for this may be that the time of ALI was relatively short and fibrosis had not yet occurred. Cell adhesion plays an important role in cell function. It is closely related to cell growth, differentiation, tissue repair, cell damage and stability of the internal environment. E-cadherin is the most common type of cadherin, which is expressed between epithelial cells (4 administration, the expression of E-cadherin in mouse liver decreased significantly, which was similar to that reported in other studies (4 and the expression of E-cadherin. It is speculated that ZDP may improve the adhesion between cells and promote the repair of injured hepatocytes by promoting the expression of E-cadherin protein.Vimentin, as a type III intermediate silk protein, anchors and supports organelles in the cytoplasm of mesenchymal cells. The expression of vimentin is up-regulated during EMT . CCl4 cafibrosis . Our resal cells . Althoug studies . In the Due to many common challenges existing in phytopharmacological research , and the4 in mice, improve the antioxidant capacity of liver cells, reduce the expression of inflammatory factors, promote liver tissue repair, and has a protective effect on CCl4-induced ALI in mice. Its protective effect may be related to inhibition of the activation of the inflammatory response-related Akt-NF-κB pathway.ZDP can effectively relieve the degree of ALI induced by CCl


Hengshun aromatic vinegar (HSAV), produced by typical solid-state or liquid-state fermentation techniques, is consumed worldwide as a food condiment. HSAV shows multiple bioactivities, but its activity in type 2 diabetes mellitus (T2DM) and possible mechanisms have not been reported. In this study, the effects of HSAV against T2DM were evaluated in insulin-induced HepG2 cells and high-fat diet (HFD) and streptozotocin (STZ) induced T2DM rats. Then, the mechanisms of HSAV against T2DM were explored by Real-time PCR, Western blot, immunofluorescence assays, siRNA transfection and gene overexpression experiments. Results indicated that HSAV significantly improved glucose consumption and reduced triglycerides (TG) contents in metabolic disordered HepG2 cells. Meanwhile, HSAV obviously alleviated general status, liver and kidney functions of T2DM rats, and decreased hyperglycemia and hyperlipidemia, improved insulin resistance, and reduced lipid accumulation in liver. Mechanism studies indicated that HSAV markedly down-regulated the expression of proliferator-activated receptor γ coactivator-1α (PGC-1α), then regulated peroxisome proliferators-activated receptor α (PPAR-α)/protein kinase B (AKT) signal pathway mediated gluconeogenesis and glycogen synthesis. Meanwhile, HSAV significantly up-regulated proliferator-activated receptor γ coactivator-1β (PGC-1β), and subsequently decreased sterol regulatory element binding protein-1c (SREBP-1c) pathway mediated lipogenesis. In conclusion, HSAV showed potent anti-T2DM activity in ameliorating dysfunction of glycolipid metabolism through regulating PGC-1α/PGC-1β pathway, which has a certain application prospect as an effective diet supplement for T2DM therapy in the future. Type 2 diabetes mellitus (T2DM) is characterized by chronic hyperglycemia and relative deficiency of insulin, resulting in disorders of lipid, glucose and protein metabolisms . In 2015The pathogenesis of T2DM is very complicated, and there are many factors affecting its occurrence, including environmental and genetic factors . Among tThere are many kinds of drugs for the treatment of T2DM at present, but some undesirable adverse effects often occur after administrating, including hypoglycemia, fluid retention, osteoporosis and heart failure . Hence, Vinegar contains a variety of nutrients, and has been used as acidic condiment more than three thousand years around the world . The maiHSAV was produced by Jiangsu Hengshun Vinegar Industry Co., Ltd. , and was diluted to the suitable concentrations with purified water. Glibenclamide was purchased from MedChemExpress  and was dissolved in the normal saline containing 10% DMSO, 40% PEG300 and 5% Tween-80 for rat administration.2 and 95% O2 at 37°C.Human hepatocellular carcinoma HepG2 cells were purchased from Shanghai Institute of Biochemistry and Cell Biology , and cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum  in humidified air containing 5% CO4 cells/well overnight, and different concentrations of HSAV  were added and incubated for 12, 24, and 36 h, respectively. The cell viabilities were evaluated by CCK-8 assay (n = 6). To select a suitable condition for model development, HepG2 cells were administrated with different concentrations  of insulin  for 12, 24 and 36 h, and then the cell viabilities were detected (n = 6).To select the safe concentrations of HSAV, the cytotoxicity of HSAV was evaluated in HepG2 cells. Cells were incubated in 96-well at a density of 5 × 104 cells/well overnight. The culture medium was changed to DMEM containing 0.5, 1.0 or 1.5% HSAV , respectively. After 24 h incubation, culture medium of cells was changed to DMEM containing 10–6 mol/L insulin. After another 24 h incubation, cell viability, glucose consumption and TG content were detected according to the manufacturer’s instructions (n = 6).HepG2 cells were incubated in 96-well at a density of 5 × 10Male Sprague Dawley (SD) rats (weighing 180–220 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. (SCXK (Liao) 2015–0,001, Benxi, China). Rats were housed with a 12:12 h of light/dark cycle at an ambient temperature of 22–25°C for 7 days. Animal experiments were approved by the Animal Care and Use Committee of Dalian Medical University (SYXK (Liao) 2013–0,006), and all experimental procedures were conducted in accordance with the People's Republic of China regulations on the use and care of laboratory animals. In this study, T2DM model of rats was established by high-fat diet  and a single low-dose intraperitoneal injection of streptozotocin . Initially, rats were randomly divided into two groups: HFD group and normal diet group. Rats in HFD group were fed HFD for 4 weeks, and then given an intraperitoneal injection of a single dose of STZ (40 mg/kg)  to induce T2DM . Rats inThe body weight of rats was recorded weekly during the experiment. The food intake and water intake were recorded in the last week before the rats were sacrificed.The weights of liver and renal were weighed and the liver and renal indexes were calculated, respectively. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE), urea nitrogen (BUN), triglyceride (TG), total cholesterol (TC), free fatty acid (FFA), low density lipoprotein (LDL), and high-density lipoprotein (HDL) were measured by kits according to the manufacturer’s instructions .via trapezoidal method using GraphPad Prism 5.0 software  , serum insulin (INS)  and glycosylated hemoglobin  were measured by kits according to protocols. In the last week of administration, oral glucose tolerance test (OGTT) was performed. Rats were fasted for 6 h and orally administrated with 2 g/kg glucose. At 0, 30, 60, 90 and 120 min after glucose administrated, blood glucose level was measured . Then, t States) . In addi States) . The forLiver and kidney tissues were buried in 4% polyformaldehyde solution for 24 h. Then, formalin-fixed liver and kidney tissues were embedded in paraffin. Then, liver and kidney sections with 5 μm thick were stained with hematoxylin-eosin (H&E). Furthermore, the liver sections were also stained with periodic acid-schiff stain (PAS) to observe hepatic glycogen. The frozen liver tissue slices were stained with Oil Red O to evaluate lipid droplet accumulation. Images were acquired by a light Nikon Eclipse TE2000-U microscope  with 200× magnification.The paraffin sections of liver were dewaxed and incubated with anti-PGC-1α antibodies in a humidified box at 4°C overnight. On the next day, the sections were incubated with alexa fluorescein-labeled secondary antibody out of light for 1 h at 37°C. Then, the sections were stained with 4′,6-diamidino-2-phenylindole  for 10 min. After washed with PBS, the images were obtained by a TE 2000U fluorescence microscopy (Nikon) with 200× magnification.Total proteins of liver tissues and HepG2 cells were extracted by cold lysis buffer  containing 1 mmol/L phenylmethyl sulfonyl fluoride. Then bicinchonininc acid (BCA) protein assay kit  was used to detect the protein concentration. Proteins were dispersed through SDS-PAGE (8–12%) and then transferred to PVDF membranes . After blocking nonspecific binding sites with 5% dried skim milk, the membranes were individually incubated with the primary antibodies for 4°C overnight. Then, specific antibody binding was detected by horseradish peroxidase-conjugated secondary antibody for 2 h incubation at room temperature. Protein expression was detected by enhanced chemiluminescence (ECL) method with a Bio-Spectrum Gel Imaging System . The relative expression of target proteins was normalized to that of β-actin.® RT kit . Real-time PCR experiment was carried out on CFX96 PCR system . β-actin was used to normalize mRNA levels, and the relative mRNA levels were calculated using the 2−△△Ct method.Total RNA were extracted using TransZolTM, and reverse-transcribed into cDNA according to the instructions of the Prime ScriptHepG2 cells were incubated in 6-well plates for 24 h and then transfected with PGC-1α or empty plasmid  using lipofectamine 2000  for 6 h. After discarding the medium, the cells were added fresh medium containing 10% fetal bovine serum and incubate for 24 h. Then, the cells were pretreated with HSAV for 24 h and exposed to insulin for an additional 24 h. The cells were then collected for the subsequent experiments.HepG2 cells were incubated in 6-well plates for 24 h and then transfected with PGC-1β siRNA (Genepharma) or empty plasmid (NC) using lipofectamine2000 for 6 h. After discarding the medium, the cells were added fresh medium containing 10% fetal bovine serum and incubate for 24 h. Then, the cells were pretreated with HSAV for 24 h and exposed to insulin for an additional 24 h. The cells were collected for subsequent experiments.Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 5.0 software. Significant differences among multiple groups were analyzed by one-way ANOVA followed by Newman–Keuls test. An unpaired t-test was carried out in the comparison two different groups. The results were considered to be statistically significant at p < 0.05. The data and statistical analysis were in accordance with standard recommendations for pharmacological experiment design and analysis.in vitro. In order to select the suitable concentration for modeling, HepG2 cells were treated with different concentrations of insulin, and the results showed that the glucose consumption of cells was markedly decreased when the cells were incubated with 10–6 mol/L insulin for 24 h (p < 0.01)  . Then, tp < 0.01) (p < 0.01). These results demonstrated that HSAV ameliorated general state of rats with T2DM.The body weights of rats in each group are as shown in  < 0.01) , but thep < 0.01, ). The serum levels of ALT and AST of rats in model group were obviously higher than those of control group (p < 0.01). Administration of HSAV (middle and high dose) or glibenclamide declined the levels of ALT and AST compared with model group . The serum BUN level of rats in HSAV or glibenclamide group was lower than that of model group , while administration of HSAV or glibenclamide increased liver glycogen content , and HSAV or glibenclamide reduced OGTT-AUC as shown in p < 0.05). Furthermore, we also found that rats in middle dose of HSAV showed the best glucose tolerance in OGTT.In OGTT, blood glucose level in model group was much higher than control group after given glucose, while it was lower in HSAV or glibenclamide group than model group . Accordip < 0.01). After 4 weeks of treatment, HSAV and glibenclamide markedly reduced HOMA-IR value compared with model group (p < 0.05). Moreover, ISI remarkably declined in model group compared with control group (p < 0.01), but it was raised by HSAV or glibenclamide treatment , but administration of HSAV or glibenclamide markedly decreased those indicators compared with model group , while HSAV obviously reversed the abnormal expression of PGC-1β  and increased the mRNA level of PGC-1β (p < 0.01) comepared with model group in vitro , but it was upregulated by HSAV treatment  went down in model group, which was up-regulated in HSAV group in comparison with model group  as decreased in model group compared with control group (p-GSK-3β) significantly declined in model group (p < 0.01), while HSAV reversed its expression. Meanwhile, the phosphorylation levels of forkhead transcription factors 1 (p-FoxO1) was declined (p < 0.01), and the expression levels of phosphoenolpyruvate carboxykinase (PEPCK) was accordingly increased in model group  and down-regulated PEPCK expression compared with model group , and there was no significant change after HSAV treatment  and the expression level of SREBP-1c was obviously increased in HepG2 cells transfected with PGC-1β siRNA (p < 0.01). As a consequence, the mRNA levels of ACC, SCD1 and FAS were significantly upregulated by PGC-1β siRNA transfection compared with NC group (p < 0.01, HepG2 cells were transfected with PGC-1β siRNA to further determine the effect of HSAV on PGC-1β pathway. As shown in T2DM is a chronic disorder, which can cause a variety of complications including cardiovascular disease, renal and urogenital disorder . As we aHSAV, one of the most famous vinegar in China, contains various physicochemical and bioactive compositions including organic acids, amino acids and phenolic compounds . It has in vitro and in vivo. In insulin-induced HepG2 cells, HSAV significantly increased the glucose comsumption, and reduced TG content. In T2DM rats, HSAV obviously improved the general states, liver and kidney function of rats, and ameliorated the histopathological changes in liver and renal tissues. Moreover, HSAV significantly decreased levels of blood glucose, INS and GHb, increased liver glycogen content, and improved insulin sensitivity and glucose tolerance of T2DM rats. In lipid metabolism, HSAV remarkably reduced lipid level in serum and lipid accumulation in the liver. Strikingly, HSAV evidently alleviated dysfunctions of glucose and lipid metabolism, and showed an excellent anti-T2DM effect.The pharmacological effects of HSAV were evaluated both via interacting with diverse transcription factors involved in cellular energy metabolism. By regulating those transcription factors, PGC-1α acts as a molecular switch in multiple cellular processes, including mitochondrial biogenesis and respiration, gluconeogenesis and glucose transport, glycogenolysis (in vitro and in vivo.PGC-1α is a transcriptional coactivator expressed as multiple, alternatively spliced variants transcribed from different promoters. PGC-1α is not directly bound to DNA but is recruited to the template enolysis . Studiesenolysis . Reversienolysis . Hence, p-GSK-3β and p-FoxO1 were up-regulated, and PEPCK was down-regulated by HSAV, which elucidated that HSAV remodeled glycogen synthesis and gluconeogenesis in T2DM by regulating PGC-1α signal pathway.PGC-1α binds numerous transcription factors involved in regulating lipid and glucose metabolism after activation. PPAR-α is one of such transcription factor that can partially mediate the downstream signaling effects . PPAR-α PGC-1β is involved in the development of various diseases, including liver cancer, diabetic cardiomyopathy, and hyperlipidemia . PGC-1β It has been reported that there is a certain correlation between the activity compounds of aromatic vinegar and its antioxidant activity . AromatiHSAV showed a potent effect against T2DM, and regulates PGC-1α and PGC-1β mediate glycolipid metabolism pathway, thereby improving glycogen synthesis, reducing gluconeogenesis and lipid synthesis. This study provided a new perspective and evidence for the application of HSAV as diet supplement in T2DM patients and susceptible population.


The β-carbolines, mainly including harman and norharman, are a group of naturally occurring, plant-derived alkaloids, and are also considered as nonpolar heterocyclic aromatic amines. Sesame seed oils contain a high level of β-carbolines (harman and norharman). In China, sesame seed oil blends are one of the most popular types of vegetable oils blends, which can be used as cooking oils or frying oils. Thus, it is meaningful to investigate the degradation of β-carbolines (harman and norharman) in sesame seed oil blends as frying oils during heating. In this work, the loss of harman and norharman in different types of sesame seed oil blends have been investigated. The results showed that the degradation of harman and norharman were dependent both on the type of oil blends, heating temperature and time. Harman and norharman were more degraded during heating  in oleic acid-rich oil blends compared to polyunsaturated acid-rich oil blends. Mechanistic investigation suggested that the reduction in harman and norharman in oil blends during heating was mainly due to the oxidative degradation reaction between β-carbolines and lipid oxidation products. Therefore, the contents of β-carbolines (harman and norharman) in sesame seed oil blends when used as frying oils and heated can be decreased with prolonged cooking time. H-pyridoindole (norharman)  and 9rharman) , are bio,4-bindolHarman and norharman can be found in many processed foods, including cookies, maize, soy and coffee ,9. CoffeVegetable oil blends are produced by blending two or more kinds of vegetable oil in a certain proportion, which can meet the requirements of nutrition, oxidative stability or flavor. Due to the flavor of sesame seed oils, vegetable oil blends containing sesame seed oil (sesame seed oil blends) is one of the most popular oil blends in China ,19. ThouIn China, there are many kinds of commercial vegetable oil blends containing sesame seed oil (sesame seed oil blends), which can be used as cooking oil to meet the requirements of nutrition and flavor. Furthermore, the contents of sesame seed oil in different oil blends range from 1–10% . TherefoNotably, no heterocyclic aromatic amines (including harman and norharman) were detected in the used vegetable oil samples, including SBO, SFO, HOSFO and HOPNO . Indeed,The four sesame seed oil blends  containing β-carbolines (78.88 ± 0.60 μg/kg harman and 88.90 ± 0.49 μg/kg norharman) were heated at two cooking temperatures (150 °C and 180 °C) . Harman The contents of harman and norharman in the sunflower seed oil blend (SFO&SSO) were decreased to 71% and 33%, respectively, when heating at 150 °C for 3 h C,D. WhenThe contents of harman and norharman in the high-oleic-acid sunflower seed oil blend (HOSFO&SSO) were decreased to 65% and 25%, respectively, when heating at 150 °C for 3 h A,B. WhenMoreover, harman and norharman in the high-oleic-acid peanut oil blend (HOPNO&SSO) were decreased to 62% and 25%, respectively, when heating at 150 °C for 3 h C,D. HarmIt was found that the level of decrease in harman in different oil blends at 150 °C occurred in the following order: HOSFO&SSO ≈ HOPNO&SSO > SFO&SSO > SBO&SSO. Additionally, the decrease in level of norharman in different oil blends at 150 °C followed the same order: HOSFO&SSO ≈ HOPNO&SSO > SFO&SSO > SBO&SSO. At 180 °C, the decrease in level of harman in the oil blends followed the following order: HOSFO&SSO ≈ HOPNO&SSO > SFO&SSO > SBO&SSO. At 180 °C, the decrease in level of norharman in the oil blends followed the following order: HOSFO&SSO > HOPNO&SSO ≈ SFO&SSO > SBO&SSO. Based on the above results, it was concluded that both harman and norharman were more strongly decomposed in monounsaturated fatty acid-type oil blends  than in polyunsaturated fatty acid-type oil blends  during heating under aerobic conditions.The fatty acid composition of the four oil blends were summarized in A previous report has studied the stability of HAAs in pure water during heating . ResultsIn general, harman and norharman showed more stability than other HAAs  . TherefoThen, the changes in acid value (AV), peroxide value (POV), anisidine value (p-AnV) and total oxidation value (TV) of the four oil blends during heating at 150 °C and 180 °C under aerobic conditions are summarized in The changes in AV in the four oil blends  indeed increased slightly with increasing heating time at 150 °C or 180 °C, and the final AV in the four different oil blends after heating at 180 °C were slightly higher than that at 150 °C. Peroxide value (POV) and anisidine value (p-AnV) were important indicators to measure the initial hydroperoxide concentration and secondary oxidation products concentration, respectively. During heating at higher temperature, the unstable lipid hydroperoxide underlie decomposition . Thus, tFinally, the degradation mechanism of β-carbolines (harman and norharman) during heating under aerobic conditions was discussed. The thermal decomposition pathway of harman and norharman during heating in edible oil media (under aerobic conditions) could be ruled out based on the control experiments . In factn-hexane were of HPLC grade, and other chemicals were of analytical reagent grade. Oasis MCX solid-phase extraction cartridges  were purchased from Waters . The water used was Wahaha purified water purchased from a local supermarket. The standards AαC , MeAαC , Trp-P-1 , Trp-P-2 , DMIP , Glu-P-2 , MeIQ , MeIQx , IQ , PhIP , 4,8-DiMeIQx , 7,8-DiMeIQx  were purchased from Toronto Research Chemicals . Harman , norharman  and 4,7,8-TriMeIQx  were purchased from Alta scientific .Soybean oil, sunflower seed oil, high-oleic-acid sunflower seed oil, high-oleic-acid peanut oil and sesame seed oil were purchased from a local supermarket in China. Acetonitrile (HPLC-grade) was purchased from Thermo Fisher Scientific . Ammonium hydroxide was obtained from Kemiou Chemical Reagent Co. Ltd. . Hydrochloric acid (HPLC grade) was obtained from Kemiou Chemical Reagent Co. Ltd. . Methyl alcohol, acetic acid and One type of vegetable oil (360 g), such as soybean oil (SBO), sunflower seed oil (SFO), high-oleic-acid sunflower seed oil (HOSFO) and high-oleic-acid peanut oil (HOPNO), was blended with sesame seed oil  to afford four kinds of sesame seed oil blends . Based on this method, the contents of β-carbolines (harman and norharman) in different blended oils remain at the same level, which is suitable for our investigation.Considering the common practical oil frying temperatures (150–180 °C), the four kinds of sesame seed oil blends  were heated at 150 °C and 180 °C, respectively, for 0.5 h, 1 h, 1.5h, 2 h, 2.5 h and 3 h in an open glass container . In the control group, 20 g blended oil (90% high-oleic-acid sunflower seed oil blended with 10% sesame seed oil) was heated for 1 h, 2 h and 3 h under the protection of nitrogen at 150 °C and 180 °C in a pressure reaction tube, respectively.The fatty acid composition was determined by conversion of oil to fatty acid methyl esters, prepared following the standard IUPAC method 2.301. Fatty acid compositions of oil blends were analyzed by Agilent Technologies 6890 N gas chromatography (GC) equipped with a 30.0 m × 250 µm × 0.25 µm BPX-70 capillary column and detected using a flame ionization detector (FID). Then, 1 µL of sample was injected under the following conditions: the nitrogen flow rate was 1.0 mL/min, the oven was programmed from the set temperature of 170 to 210 °C at 2 °C/min, the split ratio was 50:1, the GC injection temperature was 250 °C, and the detector temperature was 300 °C.Oil sample (1 g) was added with 10 μL 5mg/L 4,7,8-TriMeIQx  and combined with 10 mL acetonitrile (containing 1% acetic acid) in a 50 mL centrifuge tube, after which the mixture was shaken for 5 min, followed by ultrasonic extraction for 10 min, and shaken vigorously for 1 min before being centrifuged at 10,000 rpm (−4 °C) for 10 min. The supernatant liquid was collected into a 50 mL centrifuge tube. The above extraction operations with acetonitrile were repeated twice. All extracts were collected together.v/v). All extracts were transferred to the MCX column for enrichment and purification. Then, it was washed with 10 mL water, 10 mL methanol, and 10 mL methanol/ammonia/water  mixed solution. Finally, 10 mL methanol/ammonia  solution was used for elution. The eluent was collected and evaporated to dryness under nitrogen. The residue was dissolved in 10 mL 5% formic acid/acetonitrile  mixture and filtered through a 0.45 μm microporous filter for LC/MS analysis.A solid phase extraction column cartridge—Oasis MCX (150 mg/6 mL)—was flushed in advance with 10 mL of methanol and 10 mL 0.1mol/L HCl-methanol mixed solution . Multiple reaction monitoring (MRM) conditions were automatically optimized. The capillary voltage was 5.5 kV and the ion source temperature was 550 °C. The MRM parameters for 14 HAAs and internal standard are summarized in p < 0.05.All experiments were carried out in triplicate, the mean and standard deviation (SD) for each of the determination were calculated and reported. Figure preparation was performed using Origin Pro software . The difference between groups were tested by ANOVA and Duncan’s multiple range tests. Means were compared and they were considered significant when In this work, the loss of β-carbolines (harman and norharman) in different types of edible oils (sesame seed oil blends) has been investigated. The results showed that the degradation of harman and norharman were dependent both on the type of oil blends and heating temperature and time. Harman and norharman were more degraded during heating  in the oil blends rich in oleic acid compared to the oil blends rich in polyunsaturated acids. Mechanism investigation suggested that the reduction in harman and norharman in oil blends during heating was mainly due to the oxidative degradation of β-carbolines (harman and norharman) under aerobic conditions. Thus, the contents of harman and norharman in vegetable oils as frying oils during heating can be decreased rapidly with prolonged cooking time. Furthermore, oil frying provides a simple food processing method for the reduction of heterocyclic aromatic amine  risk in our diet.


Pancreatic cancer stem cells (PCSCs) play a key role in the aggressiveness of pancreatic ductal adenocarcinomas (PDAC); however, little is known about their signaling and metabolic pathways. Here we show that PCSCs have specific and common proteome and lipidome modulations. PCSCs displayed downregulation of lactate dehydrogenase A chain, and upregulation of trifunctional enzyme subunit alpha. The upregulated proteins of PCSCs are mainly involved in fatty acid (FA) elongation and biosynthesis of unsaturated FAs. Accordingly, lipidomics reveals an increase in long and very long-chain unsaturated FAs, which are products of fatty acid elongase-5 predicted as a key gene. Moreover, lipidomics showed the induction in PCSCs of molecular species of cardiolipin with mixed incorporation of 16:0, 18:1, and 18:2 acyl chains. Our data indicate a crucial role of FA elongation and alteration in cardiolipin acyl chain composition in PCSCs, representing attractive therapeutic targets in PDAC. It represents the eleventh most common cancer worldwide3 and the seventh leading cause of cancer-related deaths in the world, being on track to become the second most common cause of cancer-related deaths by 20301. It is the most lethal cancer with a 5-year survival rate of less than 9%4. PDAC is defined as an intractable malignancy for several reasons, but mainly due to lack of early diagnosis and effective treatments. Increasing evidence suggests that drug resistance and metastatic capability of PDAC are mainly influenced by the presence of highly plastic stem cells within the tumour, known as pancreatic cancer stem cells (PCSCs). PCSCs, described for the first time in 20075, represent a small (less than 1%) sub-population of undifferentiated quiescent cells6 characterized by self-renewal, unique plasticity and metabolism, and capacity to organize the tumour bulk producing a hierarchy of differentiated cells7. Currently, the origin of cancer stem cells (CSCs) is still unknown. Two interrelated models have been proposed to explain the heterogeneity, chemoresistance, and aggressiveness of pancreatic cancer8. Firstly, the hierarchical model that proposes CSCs as “entities” driving tumour formation, metastasis, chemoresistance, and relapse. Secondly, the stochastic model that describes CSCs as “states”, i.e. they can originate from all cancer cells because of accumulated mutations or epigenetic changes. PCSCs are characterized by aberrant expression of the embryonic stem cell transcription factors, including Oct3/4, Nanog, and Sox210, as well as of epithelial-to-mesenchymal transition markers, including CDH1 and Zeb16. Recently, by proteomic and metabolomic profiling, our group reported that PCSCs obtained from Panc-1 PDAC cell line rely on fatty acid and mevalonate pathways for their survival11. However, therapies targeting cancer-associated biosynthetic pathways have not given satisfactory results on overall patient survival12, since these do not target either the plastic PCSC sub-population or the transient cells that replenish the PCSC pool8.Pancreatic cancer comprises different types of neoplasia, among which the most common is the infiltrating neoplasm named pancreatic ductal adenocarcinoma (PDAC). PDAC arises in exocrine glands of the pancreas13 few metabolomics14 and proteomics17 studies have been published, while no lipidomic analysis has as yet been carried out on PCSCs. Further elucidation of the signaling pathways that regulate PCSC growth, survival, and metabolomic plasticity is needed to detect therapeutic targets and to explore novel therapeutic approaches against pancreatic cancer18. Therefore, in the current study proteomic and lipidomic analyses were integrated to investigate the signaling and metabolic dysfunctions implicated in the pathophysiology of PCSCs obtained from parental (P) PDAC cell lines. Protein and lipid profiles of PCSCs derived from four PDAC cell lines harbouring different genetic backgrounds19, e.g. PaCa3 (p16-met), PaCa44 , MiaPaCa2 , and PC1J  were analysed. To our knowledge, the current work presents the first proteome and lipidome investigation of PaCa3, PaCa44, MiaPaCa2, and PC1J CSCs obtained from relative PDAC cell lines. The results shown here support the potential of a multiomic approach and contribute to the understanding of PDAC biology revealing mitochondrial cardiolipin remodelling, as well as enhanced fatty acid elongation and phosphoinositol phosphatase pathways, as promising targets in PCSCs.Despite the importance of a multi-omics characterization of PCSC, in-depth characterization is still deficient. In addition to our previous findings20, the morphological differences between the four PCSCs and P cell lines  were initially investigated. Different growth patterns between P and PCSCs were observed , cell cycle (CYCLIN B1) and Wnt signaling  were evaluated. Figure OCT3/4, SOX2 and NANOG genes were upregulated in PaCa3, PaCa44, and MiaPaca2 CSCs compared to their respective P cell lines. However, no significant (p < 0.05) mRNA level differences of stemness genes were detected for PC1J CSCs. Therefore, protein expression of CSC markers was evaluated by immunoblotting in the four P cells and PCSCs. The results show that Oct3/4 was not detectable in either PCSCs or P cells (data not shown); whilst Sox2 and Nanog were induced in PaCa3, PaCa44, and PC1J CSCs. Moreover, not observable levels were established in MiaPaCa2 for both P and CSC cell lines  genes was also investigated  of upregulated proteins in PCSCs related to lipid metabolism, including FA elongation and biosynthesis of unsaturated FAs, are reported in Fig. Possible dysregulated signaling and metabolic pathways of PCSCs were investigated by performing proteomics and lipidomics analyses. Comparing the quantitative proteome levels between PCSCs and P cells revealed a total of 121, 186, 212, and 235 proteins differentially expressed in PaCa3, PaCa44, MiaPaCa2, and PC1J CSCs, respectively (Supplementary Table p < 0.05) accumulation of FAs in Paca44, MiaPaca2, and PC1J CSCs  of the fold-change of PCSCs/P ratio showed that all the cell lines cluster away from each other Fig. a indicatSCs Fig. b. AlthouSCs Fig. c.Figure LC–MS results also showed an increase of some neutral lipids in PCSCs, therefore cells were imaged using confocal microscopy to evaluate lipid droplets (LDs) properties Fig. d. A trenSYNJ1, SYNJ2, SACM1L, MTMR1, MTMR2, MTMR3, MTMR4, MTMR6, MTMR7, MTMR8, MTMR9, MTMR14, and PTEN) encode for phosphoinositide lipid phosphatase proteins, and one for the fatty acid elongase-5 (ELOVL5).The lipidomics analysis performed by BioPAN revealed active pathways and related genes for each PCSCs Table . BioPAN As concerning the substrates and the products of phosphoinositide lipid phosphatases, a statistically significant increase (at 95% CI) in the levels of phosphatidylinositol (PI) in PaCa3, MiaPaCa2 and PC1J CSCs was observed Fig. b. In addELOVL5, are also included in Table Based on the proteomics results, FA elongation and biosynthesis of unsaturated FAs emerged as key pathways for PCSCs, therefore the role of the elongases and desaturases was also investigated in the FA metabolism  dependency26. LDHA was found downregulated in all the PCSCs, which is consistent with the OXPHOS as energy source in PCSCs rather than glycolysis27. Moreover, LDHA phosphorylation of tyrosine in position 10 was also evaluated by immunoblotting, as p-LDHA promotes the activity of LDHA and the Warburg effect28, demonstrating a downregulation of p-LDHA in PCSCs.Our comprehensive analysis of protein modulation in PCSCs and P cells portrays specific and common proteome changes for the four PCSCs, most of which are related to dysregulated lipid-metabolism pathways. Interestingly, among these latter are the biosynthesis and metabolism of cholesterol, the main product of the already reported mevalonate pathwayPTEN (among many other phosphoinositide lipid phosphatases) and ELOVL5. Pten has a dual-specificity phosphatase activity: protein and lipid dephosphorylation. As a lipid phosphatase, preferentially dephosphorylates phosphoinositide substrates, removing the phosphate from phosphatidylinositol 3,4,5-trisphosphate (PIP3), PIP2, PIP and inositol 1,3,4,5-tetrakisphosphate (I4P)30. In contrast, Elovl5 plays a role in the addition of two carbon units to the carboxyl ends of fatty acyl CoA substrates, acting on monounsaturated palmitoleoyl-CoA (C16:1)31 and linoleoyl-CoA (C18:2), and participating in the production of very long chain FAs 20:3, 20:4, 22:4 and 22:533.To further dissect the metabolic pathways, a comprehensive analysis of lipid modulations in PCSCs and P cells was performed. The findings suggest that PCSCs are characterized by induction of long chain fatty acids and accumulation of lipid droplets. Phosphoinositide signaling and fatty acid elongation come off as specifically noteworthy pathways in PCSCs. Accordingly, some main genes involved in these pathways were detected: 34 and comprises two diacylglycerol phosphate residues connected by a glycerol backbone and four fatty acyl chains. CL is derived from the catalytic activity of Cardiolipin synthase (CRLS) on two units of phosphatidylglycerol (PG), but activity of the phospholipase A2 produces monolysocardiolipin, which is the substrate for HADHA. Therefore, fatty acyl chain combinations in CL give a wide variety of mature CL species. On the one hand HADHA catalyses the last three steps of mitochondrial β-oxidation of FAs, when is accompanied by the β subunit (HADB)35. On the other hand, HADHA plays a role in the acylation of monolysocardiolipin to form CL37. Thus, further analysis of the CL lipid molecular species linked with CL remodelling by HADHA were performed. Our lipidomic analysis indicated, for MiaPaCa2 and PC1J CSCs, increased levels of CL molecular species showing incorporation of oleoyl-CoA (C18:1), linoleoyl-CoA (C18:2), and palmitoyl-CoA (C16:0). However, not direct correlation can be suggested with CL results and HADHA upregulation.Interestingly, lipid biosynthetic pathway analysis did not show genes in reaction pathways involving Cardiolipin (CL) as no increased levels were detected; despite the fact that proteomics showed an upregulation of HADHA in all the four PCSCs, and a significant enrichment of “acyl chain remodelling of CL” pathway for CSCs derived from MiaPaca2 and PC1J cells. CL is a major mitochondrial membrane glycerophospholipid subclass39. These protein complexes can be linked with oxidative stress modulation by reduction of superoxide levels, proton pump homeostasis, and energy metabolism40. Therefore, CL plays a critical role in oxidative phosphorylation (OXPHOS). During this process, many protons are transported across the mitochondrial membrane, causing a pH shift. In this context, CL molecular species function as a proton trap within the membranes of this organelle41. Mechanistically, H+ ions interact with PO4− groups of CL to neutralize its negative charge and to reduce the size of the hydrate coat around the polar head42. The neutralized CL acquires a reverse molecular shape, a non-bilayer packing of CL results, and this leads to inverted micelles accumulating near highly-protonated regions of membranes. These non-bilayer structures favour the oligomerization of the F0 sector of the ATP synthase complex and act as a proton trap to shuttle protons to the ATP synthase complex leading to enhanced energy production43. Very little recently published research have shown a prominent role for CL alterations in cancer stem cells. Interestingly, it has been shown that inhibition of mitochondrial phospholipid production, such as CL, reduces stemness and increases differentiation of acute myeloid leukaemia cells45. To the best of our knowledge, this is the first time that the changes in CL acyl-chain composition have been demonstrated for a solid tumour like PDAC as well as its PCSCs.It has been reported that changes in CL molecular species composition has an enormous impact on the structural integrity of the inner membrane of mitochondria and on the enzymatic activity of all mitochondrial respiratory complexesIn conclusion, a proteomic and lipidomic analysis of pancreatic parental cancer cells and cancer stem cells established major differences in the signaling and lipid metabolic pathways between these two cell types. In summary, the data obtained in this multiomics study showed that alteration of cardiolipin acyl-chain composition, fatty acid elongation, and phosphoinositide signaling are specific pathways of PCSCs and potential new targets in novel therapeutic strategies for pancreatic cancer.2 for some passages. As previously described by Dalla Pozza et al.46, PCSCs were obtained by growing adherent cells in “CSC medium”  for 2 weeks. “CSC medium” was refreshed twice a week with new medium solution and maintained at 37 °C with 5% CO2. PCSCs were passed through a cell strainer (> 40 μm) to separate cell spheres, followed by examination under a light microscope  at 20 × and 40 × magnifications, prior to spheres collection. The cell numbers and viabilities of P cells and PCSCs were determined with the trypan blue exclusion test. Cells with viability higher than 85% were pelleted, frozen with liquid nitrogen, and stored at − 80 °C for further analyses.The human PDAC cell lines PaCa3, PaCa44, MiaPaCa2, and PC1J, called parental (P) cells, were obtained from American Type Culture Collection (ATCC). These cells were grown in RPMI 1640 media supplemented with 10% foetal bovine serum, 2 mM glutamine, and 50 µg/ml gentamicin sulphate . Cells were maintained at 37 °C with 5% CO6 cells  using TRIzol Reagent (Life Technologies) according to the manufacturer's instructions. The extracted RNA was quantified by NanoDrop One (Thermo Fisher Scientific) and checked for integrity loading on 1.5% agarose gel. 1 μg of RNA was reverse transcribed using first-strand cDNA synthesis. The real-time PCR reaction was performed according to the protocol of the SYBR‑Green detection chemistry with GoTaq qPCR Master Mix (Promega) on a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). The sequence of the primers used in this experiment are provided in Supplementary Table −ΔΔCT method.Total RNA was extracted from 1 × 1047. Briefly, cells were collected , washed and lysed in 1X PBS with protease inhibitors cocktail and 0.1% SDS. Acetone was used for protein precipitation/denaturation followed by resuspension in 100 mM NH4HCO3. Protein content was measured by Bicinchoninic Acid Protein Assay. Thirty µg of protein extract was subjected to reduction (with dithiothreitol) and alkylation (with iodoacetamide), prior to tryptic digestion at 37 °C overnight.Identification and quantification of proteome modulation of P cells and CSCs of the four cell lines were performed as previously reported by Brandi et al.48, performed by using a micro-LC system  interfaced with a 5600 + TripleTOF mass spectrometer . Samples were subjected first to data-dependent acquisition (DDA) analysis to generate the SWATH-MS spectral library, and then to cyclic data independent analysis (DIA), based on a 25-Da window, using three technical replicates of each sample. The MS data were acquired by Analyst TF v.1.7 (AB SCIEX), while PeakView v.1.2.0.3, Protein Pilot v.4.2 (AB SCIEX) and Mascot v.2.4 (Matrix Science) programs were used to generate the peak-list. The database search was performed using the UniProt/Swissprot . Samples were input in the Protein Pilot software v. 4.2 , with the following parameters: cysteine alkylation, digestion by trypsin, no special factors and FDR at 1% were used for database search with Protein Pilot, while for Mascot search the following parameters were used: trypsin as digestion enzyme, 2 missed cleavages, search tolerance of 50 ppm for the peptide mass tolerance and 0.1 Da for the MS/MS tolerance. The charges of the peptides to search for were set to 2 +, 3 + and 4 +, and the search was set on monoisotopic mass. The instrument was set to ESI-QUAD-TOF and the following modifications were specified for the search: carbamidomethyl cysteines as fixed modification and oxidized methionine as variable modification. FDR was fixed at 1%. Peak lists generated by both Protein Pilot and Mascot were compared to UniProt/Swissprot  database. The obtained files from the DDA acquisitions were used for the library generation using a FDR threshold of 1%. Protein quantification was performed by PeakView v.2.0 and MarkerView v.1.2. (AB SCIEX) programs by extracting from SWATH files ten peptides per protein with the highest MS1 intensity, and ten transitions per peptide. Peptides with FDR lower than 1.0% were exported, and up- and downregulated proteins were selected using p value < 0.05 and fold change > 1.5.Tryptic peptides were analysed by label-free LC–MS/MS as previously49. Briefly, frozen cell pellet lipids were extracted using the Folch method with Chloroform/Methanol/Water (2:1:1 ratio). Lipids were dried using a SpeedVac  and re-suspended in Chloroform/Methanol (1:1), prior to injection into a Shimadzu Prominence 20-AD system . Chromatographic separation was achieved using a Waters Acquity UPLC C4  column . The column was kept a 45 °C and 7 μl of samples were eluted using a mobile phase composed of solvent A (water) and B (acetonitrile), each containing 0.025% formic acid. The gradient started at 45% B for 5 min, then increased to 90% B for 5 min, and 100% B was reached after an additional 10 min and held for 7 min before re-equilibration at 45% B for 5 min. The flow rate was maintained at 100 μl/min. Accurate mass (with an error below 5 ppm) was acquired on an Orbitrap Elite mass spectrometer . Source parameters for positive polarity were: capillary temperature 275 °C; source heater temperature 200 °C; sheath gas 10 AU; aux gas 5 AU; sweep gas 5 AU. Source voltage was 3.8 kV. Full scan spectra in the range of m/z 340–1500 were acquired at a target resolution of 240,000 (FWHM at m/z 400). Manual inspection of the results was carried out using Xcalibur and further processed using Lipid Data Analyzer (LDA) 2.7.0_2019 software50.Lipids extraction and analysis was performed as previously outlinedTargeted analysis of lysolipids, Cer, and dhCer, was performed using a QTRAP 6500 LC–MS/MS System (AB SCIEX) operating in MRM mode. Quantification of multiple species of ceramides was carried out by the integration of the peak area as normalized against the peak area of the non-endogenous odd-chain ceramide C17:0. C17 was present at a known concentration and served as the internal standard (IS). Collision energy (CE) was optimized previously.Thirtyfive lipid subclasses were identified, including alkenyl-acylphosphatidylcholine (P-PC), alkenyl-acylphosphatidylethanolamine (P-PE), alkyl-acylglycerol (O-DG), alkyl-acylphosphatidylcholine (O-PC), alkyl-acylphosphatidylethanolamine (O-PE), alkyl-triacylglycerol (O-TG), cardiolipin (CL), ceramides (Cer), cholesterol (CH), cholesterol ester (CE), diacylglycerol (DG), dihydroceramides (dhCer), dihydrosphingomyelin (dhSM), free fatty acids (FA), phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM), sphingosine (SG), triacylglycerol (TG), lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylglycerol (LPG), lyso phosphatidylinositol (LPI), phosphatidylserine (LPS), alkenyl-lysophosphatidylcholine (P-LPC), alkenyl-lysophosphatidic acid (P-LPA), alkyl-lysophosphatidylethanolamine (O-LPE), alkyl-lysolysophosphatidylcholine (O-LPC), phosphatidylinositolmonophosphate (PIP), phosphatidylinositoldiphosphate (PIP2), phosphatidylinositoltriphosphate (PIP3).51 (https://www.R-project.org) with in-house built scripts. Statistical comparison between the P cells and PCSCs was performed using the paired t-test (p < 0.005), principal component analysis (PCA), and log2 ratio transformation of PCSCs versus parental lipid levels.Lipid relative quantitation levels were calculated using the R-studio (v3.2.4) software52. Briefly, to characterize the function of proteins, gene ontology (GO) annotation, KEGG and Reactome pathways enrichment analyses were performed using STRING v.11.0 (http://string-db.org)53. The differentially expressed proteins were analysed for candidate functions and pathways enrichment, setting Homo sapiens as taxonomy, p < 0.05 and gene count > 2 as cut-off point.Bioinformatics analysis of proteomic data was performed as previously describedhttps://lipidmaps.org/biopan/)54. BioPAN provides Z-scores (Z > 1.645 at p < 0.05) using substrate and product lipid levels and integrate a list of genes, which could be involved in the activation or suppression of enzymes catalysing lipid metabolic pathways.Lipid profile levels obtained from the four PCSCs and P cells were loaded into an open access tool BioPAN, on LIPID MAPS Lipidomics Gateway /Tween-20 and incubated with primary antibodies diluted as reported in Supplementary Table Western Blot analysis was performed as previously described2) for lipid droplets (LDs) staining. Supernatant was removed and, after washing with PBS, cells were fixed using 8% formaldehyde, for 10 min at room temperature. After fixation and washing, cells were incubated with Oil Red O (Bio-Optica) for 20 min at room temperature. Thus, cells were washed again, treated with 0.1% Triton X-100 and 1% bovine serum albumin for 15 min at room temperature, and stained with DAPI  for 30 min for nuclei visualization. Samples were scanned by a confocal microscope (Leica TCS SP5 AOBS) using 416 nm and 545 nm lasers under a 40 × oil objective. The images were analysed using Leica Application Suite X 3.7.0.20979 software. Imaris X64 9.1.0 software to generate z- and three-dimensional projections for the analysis of lipid LDs shape and volume. Twenty images per sample were recorded and LDs were counted manually using the ImageJ multipoint tool after background correction. Detected LDs were normalized per cell nucleus. Statistical analysis (t-test) was performed to identify significant changes.CSCs and P cells were grown on a L-lysine coverslip inside a 24-well plate and incubated at 37 °C  according to the manufacturer's instructions. Briefly, cells were lysed in 1% Triton X-100 in chloroform (w/v) and centrifuged at 13,000 × g for 10 min to remove insoluble debris. The organic phase was collected, dried on 50 °C dry bath for 20 min, fatty acid assay buffer was added and then dissolved by extensive vortex mixing. After that, absorbances for individual wells were read at 570 nm using a microplate reader, and values reported as μM FFA/million cells.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.


Veratrilla baillonii Franch (V. baillonii) has been applied in the intervention and treatment a diverse range of diseases, including diabetes. In this study, we revealed that water extracts of V. baillonii (WVBF) can ameliorate liver injury and insulin resistance in T2DM rat model. To elucidate the anti-diabetic mechanisms of WVBF, we performed liver transcriptome analysis that displayed WVBF treatment significantly suppressed many gene expressions involved in insulin resistance. Furthermore, functional experiments showed that WVBF treatment reduced the pathological damages of liver and pancreas, which may be regulated by Foxo1, Sirt1, G6pc, c-Met, Irs1, Akt1, Pik3r1. These results indicated that WVBF improves diabetic liver injury and insulin resistance in diabetic rats. Therefore, this study demonstrated WVBF could be used as a promising therapeutic agent for intervention and treatment of diabetes.Type 2 diabetes mellitus (T2DM) is a complex and polygenic disorder with diverse complications.  As evidsistance . In type of T2DM . An accu of T2DM . Inflamm of T2DM . Hypergl of T2DM  and indu of T2DM . Accumul of T2DM .Swertia corymbosa, a plant in the family Gentianaceae and used in traditional medicine to treat diabetes in Indian , a member of Gentianaceae family, is characterized by anti-toxic and liver protection properties (V. baillonii (WVBF) has significant protective effects on oxidative stress-induced liver injury, diabetic liver injury, and drug induced liver toxicity  in vitro . Furtherin vitro . HoweverTRIzol was obtained from Tiangen Biochemical Technology Co., Ltd. ; Streptozotocin was obtained from Saiguo Biotechnology Co., Ltd. ; Blood glucose test paper and blood glucometer purchased from Sinocare Biosensor co. ; Rat Insulin ELISA kit was obtained from Biyuntian Biological Technology Co., Ltd.n = 8), group two rats were administrated intragastrically with Metformin Hydrochloride 150 mg/kg (n = 8) once a day for 6 weeks; group three and four rats were administrated intragastrically with WVBF 12.5 mg/kg (n = 8) and 25 mg/kg (n = 8) once a day for 6 weeks, respectively. The NC group and the MC group were orally given 0.9% normal saline according to their body weight. The PC group, WVBF 12.5 mg/kg group, and WVBF 25 mg/kg group were weighed according to their body weight, dissolved with 0.9% normal saline and administered orally.Sprague-Dawley (SD) rats, male, 8 weeks old , weighin50 mg/kg  positive25 mg/kg  (n = 8) Three liver samples were collected from NC, MC, WVBF 12.5 mg/kg and 25 mg/kg each group. After overnight fasting for 12 h, liver tissues were collected. rinsed with diethyl pyrocarbonate (DEPC) water prepared with phosphate buffer saline (PBS), frozen in liquid nitrogen for 30 s, transferred to enzyme-free EP tube and frozen at -80°C. Total RNA was isolated using TRIzol reagent  according to the manufacturer’s protocol. The concentration, quality and integrity were determined using a Nanodrop spectrophotometer  and an Agilent bioanalyzer 2100 .p < 0.05 for the four groups. A total of 12 cDNA libraries were generated by the TruSeq RNA sample preparation kit . Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. The established sequencing library was sequenced with Illumina Hiseq novaseq 6000 by whbioacme. Differential expression analysis of RNA-seq data was performed as described in previous study  Remove the connector sequence (adapter); 2) Remove reads with low quality  alkali base exceeding 40% of the length proportion of the read; 3) Remove reads with base number of “n” base exceeding 5; 4) Start from the 5 “end of reads (i.e. the beginning of reads), filter the sliding window quality, and cut off the sliding window (sliding window size 4) whose average base quality value is lower than the threshold (20); 5) Remove the filtered reads whose length is less than 36. FeatureCounts version 1.5.0 was used to obtain the fragments per kilobase of exon per million fragments mapped (FPKM) of all annotated genes. DEGs were identified using DESeq2 with the default parameters at us study . Venn diFBG was monitored at 0, 1, 2, 3, 4, 5 and 6 weeks after-treatments of diabetic and normal rats. Blood was collected from the tail of the animals after 12–16 h overnight fasting. The tail tip was sanitized with alcohol and pricked and a drop of blood was used for blood glucose measurement using a glucometer . FBG was presented by a broken line graph of weekly blood glucose dynamic changes.OGTT was performed in rats as described previously in our lab after 6 weeks of WVBF treatment . BrieflyInsulin resistance index, HOMA-IR, was assessed using fasting glucose ( mmol/L) × fasting insulin ( mμ/L)/22.5. HOMA-β cell function index, HOMA-β, was assessed using 20×fasting insulin ( mμ/L)/fasting glucose (mmol/L)-3.5.Liver and pancreas tissues were removed, fixed with 10% neutral buffered formalin over-night, embedded in paraffin, and sectioned at 5 μm. Next, H&E staining was were performed as previously described in detail in our lab .ß-actin served as the endogenous housekeeping gene to normalize the expression of the RNA sample levels. Relative gene expression levels were calculated using the 2−ΔΔCT method.Total RNA was extracted from rat liver tissues with TRIZOL reagent . The concentration and purity of different RNA samples were evaluated using a spectrophotometer .The cDNA was synthesized by reverse transcriptase . Real-time qPCR was performed using the Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific (China) Co., Ltd.). The primers of real-time qPCR were used to amplify transcripts for c-Met, Irs1, Akt1, Pik3r1, Foxo1, Sirt1 and G6pc . ß-actinp < 0.05. Data are presented as means ± SEM. *p < 0.05, **p < 0.01, two-tailed Student’s t-test  test comparison post hoc test for normal distribution data. The significance level was fixed at s t-test ; one-ways t-test .To explore the effects of WVBF on liver injury and insulin resistance in diabetic rats, a desirable T2DM model that induced by HFD and STZ was proposed and its metabolic features were measured. As shown in th week  index that reflects insulin resistance. The FINS level of MC group was significantly lower than that in NC group in the 6th week . WVBF  were detected . We idenTo validate the WVBF effects on ameliorating insulin resistance by aforementioned analysis, we also chose certain key molecules linking insulin signaling and glycolysis/gluconeogenesis, such as Pikr1, Akt1, Foxo1 and Sirt1 . ResultsAccumulating studies demonstrate that many Chinese herbal medicines have beneficial effects on the treatment of metabolic disorders including diabetes . Herein,ß cell failure , which modulate glucose-stimulated insulin secretion through their ability to reverse otherwise irreversible glycolytic steps  were administered orally in different groups of mice in the presence or absence of WVBF and the protective effect of WVBF on diabetic liver injury were evaluated . Moreove glucose . Pancreaic steps . In thisTo validate the WVBF effects on ameliorating diabetic liver injury, this study demonstrated that WVBF treatment alleviated liver and pancreas islet injury by mediating the insulin signaling cascade , which orchestrated the normal secretion of insulin to control blood glucose . c-Met iet al. found that FoxO1-dependent decrease of IRS1 induced the inactivation of Akt signaling and alleviation of insulin resistance (PI3K-Akt signaling is the various mechanistic links in metabolic disorders, including diabetes . PIK3R1,sistance . Foxo1-Ssistance . SIRT1 hsistance . Sirt1 usistance . Varioussistance . Moreovesistance . Interesin vivo, and improve insulin resistance in rats. WVBF treatment exhibited the protective effects on liver injury in diabetic rats, which restored the gene expression of insulin resistance and insulin signaling cascade. These findings provide a valuable therapy option for the well-known WVBF, which could be used as a promising therapeutic agent in treating diabetes and its complications.In conclusion, this present study uncovered that WVBF could alleviate HFD-STZ -induced diabetic liver injury


Cardiac remodeling in rheumatic mitral stenosis (MS) is complex and incompletely understood. The objective of this study was to evaluate cardiac structural and functional changes in a cohort of patients with rheumatic MS using cardiovascular magnetic resonance (CMR).This retrospective study included 40 patients with rheumatic MS, consisting of 19 patients from India, 15 patients from China, and 6 patients from Mexico (median (interquartile range (IQR)) age: 45 years (34–55); 75% women). Twenty patients were included in the control group. CMR variables pertaining to morphology and function were collected. Late gadolinium enhancement (LGE) sequences were acquired for tissue characterization. Statistical analyses were performed using the Kruskal–Wallis test and the chi-square test.p < 0.001), lower right ventricular (RV) ejection fraction (44% (40%–52%) vs 64% (59%–67%), p < 0.001), higher RV end-diastolic volume (72 (58–87) mL/m2 vs 59 (49–69) mL/m2, p = 0.003), larger left atrial volume (87 (67–108) mL/m2 vs 29 (22–34) mL/m2, p < 0.001), and right atrial areas (20 (16–23) cm2 vs 13 (12–16) cm2, p < 0.001). LGE was prevalent in patients with rheumatic MS (82%), and was commonly located at the RV insertion sites. Furthermore, the patient cohorts from India, China, and Mexico were heterogeneous in terms of baseline characteristics and cardiac remodeling.Compared to the control group, patients with MS had lower left ventricular (LV) ejection fraction (51% (42%–55%) vs 60% (57%–65%), Our findings demonstrated that biventricular dysfunction, right and left atrial remodeling, and LGE at the RV insertion sites are underappreciated in contemporary rheumatic MS. Further studies are needed to elucidate the prognostic implications of these findings.The online version contains supplementary material available at 10.1186/s12968-022-00853-5. Rheumatic heart disease is the most common cause of mitral stenosis (MS), and remains a major cause of valve disease globally, occurring predominantly in developing countries –4. ProgrWhile echocardiography is typically the noninvasive modality of choice in the evaluation of patients with rheumatic MS due to its availability and low cost, cardiovascular magnetic resonance (CMR) imaging is a promising tool to provide more detailed characterization of morphologic and functional changes that occur in response to MS. CMR has the unique ability to precisely quantify chamber size and function due to its high spatial resolution, and can also provide tissue characterization through late gadolinium enhancement (LGE) imaging and parametric mapping. Prior studies have shown that CMR can improve the evaluation and prognostication of patients with other valve diseases, such as aortic stenosis, mitral regurgitation, and tricuspid regurgitation, through more accurate assessment of cardiac remodeling and tissue characterization –11. HoweIn this study, we assembled a cohort of patients with rheumatic MS who underwent CMR at one of three endemic countries on two continents, and characterized the cardiac structural and functional changes in this population.We studied consecutive patients with symptomatic rheumatic MS referred for CMR in China between 2013 and 2019 , India between 2018 and 2020 , and Mexico between 2012 and 2020  (n = 76). Patients in India underwent CMR as part of a part of a study protocol, patients in Mexico underwent CMR as part of either a study protocol or preoperative evaluation, and patients in China underwent CMR as part of a preoperative evaluation. We excluded patients with prior balloon valvuloplasty, prior cardiovascular procedures, prior myocardial infarction, or greater than moderate concomitant valvular disease as assessed by echocardiography. The control group was derived from a cohort in India that underwent CMR for the evaluation of coronary artery disease (CAD). These patients did not have any abnormalities on rest imaging or stress perfusion imaging, did not have any known cardiovascular conditions, and did not have a history of prior percutaneous coronary intervention or coronary artery bypass graft surgery. This study received approval from each hospital’s respective Institutional Review Board, and patients gave written informed consent for the study.The following CMR scanners were utilized in this study: (1) India: 1.5 T , (2) China: 3 T , and (3) Mexico: 1.5 T . At all three sites, after acquiring localizers, electrocardiogram gated standard balanced steady-state free precession cine images with 8 to 14 short-axis and 3 long-axis planes  were obtained. The parameters were as follows: (1) India: Repetition time (TR)/Echo time (TE) 4.4–5.4/1.1–1.2 ms; slice thickness: 8 mm; flip angle: 66–67 degrees; in-plane spatial resolution: 1.4–1.7 × 1.7–2.3 mm; (2) China: TR/TE, 2.8 /1.2 ms; slice thickness: 8 mm; flip angle: 38 degrees; in-plane spatial resolution: 1.6 × 2.0 mm; (3) Mexico: TR/TE 3.7–3.9/1.8–1.9 ms; slice thickness: 8 mm; flip angle: 60 degrees; in-plane spatial resolution: 1.6–1.7 × 1.6–1.7 mm. Ten to 20 minutes after the administration of a gadolinium contrast agent, segmented two-dimensional LGE images were acquired using routine inversion recovery sequences with the following parameters: (1) India: TE 1.1 ms; slice thickness: 8 mm; flip angle: 40 degrees; field of view: 274 × 319 mm; in-plane spatial resolution: 1.2 × 2.1 mm; (2) China: TE 1.2 ms; slice thickness: 8 mm; flip angle: 40 degrees; field of view: 225 × 300 mm; in-plane spatial resolution: 2.1 × 1.6 mm; (3) Mexico: TE 3.0 ms; slice thickness: 10 mm; flip angle: 25 degrees; field of view: 360–460 × 360–460 mm; in-plane spatial resolution: 1.3–1.6 × 1.6–2.1 mm. All studies included in the analysis were judged to be of good quality. CMR studies from India and Mexico were analyzed at the coordinating center  by two investigators. CMR studies from China were analyzed locally due to export restrictions, but LGE images were virtually reviewed together with the coordinating center. Measurements of biventricular volumes and function, LA volume (using biplane area-length method) and emptying fraction, right atrial (RA) maximal area, and mitral valve area (MVA) by planimetry were performed, and inter-observer and intra-observer variability analyses were conducted. Papillary muscles were included in the LV cavity, not in the LV mass. Native T1 measurements and quantification of extracellular volume fraction (ECV) were performed on a portion of CMR studies from India (n = 18) and China (n = 9). Wall motion abnormalities were assessed by reviewing cine images, and LGE images were analyzed for the presence and location of LGE. CMR studies from India and Mexico were analyzed using suiteHEART . CMR studies from China were analyzed on Qmass  at the time of the CMR were obtained from the medical record. N-terminal pro brain natriuretic (NTpro-BNP) levels were available for the cohort from India. MVA, as determined by pressure half-time (PHT), tricuspid regurgitation peak velocity for determination of pulmonary artery systolic pressure and other concomitant valve diseases were extracted from the transthoracic echocardiogram report from the study performed closest to the time of the CMR. Severe MS was defined as MVA ≤ 1.5 cmNon-normally distributed continuous variables are shown as median (interquartile range) and analyzed using the Kruskal–Wallis test. Categorical variables are shown as total counts with percentages and analyzed using the chi-square test. All statistical analyses were performed with JMP Pro .p = 0.03). The control group was derived from a cohort in India that underwent CMR for the evaluation of CAD, and patients in the control group had higher rates of CAD, diabetes mellitus, and hypertension. The MS cohort reported a higher burden of symptoms, including dyspnea on exertion, palpitations, and angina. Echocardiography data for the calculation of MVA by PHT was available for the majority of the overall cohort . Within the context of this limitation, most patients in the MS cohort were classified as severe MS, as indicated by the median MVA by PHT of 0.85 cm2 (IQR: 0.73 to 1.10 cm2) and median MVA by CMR planimetry of 1.17 cm2 (IQR: 0.89 to 1.56 cm2). The patients in the MS cohort had the following breakdown of coexistent mitral regurgitation: none (12/40 [30%]), trace (7/40 [17.5%]), mild (11/40 [27.5%]), mild-moderate (1/40 [2.5%]), and moderate (9/40 [22.5%]).The final study cohort consisted of 40 patients, including 19 patients from India, 15 patients from China, and 6 patients from Mexico . Patients in China had higher rates of atrial fibrillation, and patients in Mexico had higher rates of hypertension. Furthermore, there was a higher burden of dyspnea on exertion in the India and Mexico groups, and a higher burden of angina in the China and Mexico groups. There were no significant differences in MVA by PHT or CMR planimetry between the three groups.Characteristics of the MS cohort, stratified by country, are shown in Table The CMR findings in the MS cohort and the control group are shown in Table p = 0.001), and RV ejection fraction (RVEF) (44% (IQR: 40% to 52%) vs 64% (IQR: 59% to 67%), p < 0.001). RV volumes were greater in the MS cohort (end-diastolic volume index (RVEDVI) of 72 (IQR: 58 to 87) mL/m2 vs 59 (IQR: 49 to 69) mL/m2, p = 0.003), and there was no difference in LV end-diastolic index (LVEDVI) between the two groups. LV global longitudinal strain (GLS) was worse (less negative) in the MS cohort (− 16% (IQR: − 18% to − 14%) vs − 20% (IQR: − 22% to − 18%), p < 0.001). Pulmonary artery systolic pressures (PASP) by transthoracic echocardiography were mildly to moderately elevated in the MS cohort. In the MS cohort, there was a weak negative correlation between PASP and RVEF  and PASP and RV stroke volume (SV)/end systolic volume (RVESV) , but no correlation was observed between PASP and RV diastolic or systolic volumes.Compared to the control group, the MS patient cohort had significantly lower LV ejection fraction (LVEF) (51% (IQR: 42% to 55%) vs 60% (IQR: 57% to 65%), 2 vs 29 (IQR: 22 to 34) mL/m2, p < 0.001), and LA emptying fraction was significantly lower in the MS group (20% (IQR: 13% to 30%) vs 61% (IQR: 51% to 65%), p < 0.001). RA size was larger in the MS group (20 (IQR: 16 to 23) cm2 vs 13 (IQR: 12 to 16) cm2, p < 0.001). There were no significant correlations between MVA by echocardiography, biatrial or biventricular size/function, or PASP  mL/mp < 0.001). In the MS group, the two patterns of LGE observed were 1) enhancement at the inferior RV insertion site (79%) and 2) enhancement at the superior RV insertion site (13%). There were no patients with subepicardial or subendocardial LGE. One patient in the control group exhibited midmyocardial enhancement at the inferior RV insertion site. There were no patients with wall motion abnormalities in either group. One patient in Mexico underwent endomyocardial biopsy at the RV insertion site that revealed myocardial fibrosis . The China and India cohorts had a mildly reduced median LVEF of 48%, while the Mexico group had a normal median LVEF of 63%. RV function was reduced across all three groups, and biventricular volumes were similar in the three cohorts. There was also no difference in LV GLS between the India and Mexico cohorts.The CMR findings in the MS cohort, stratified by country, are shown in Table 2) compared to the India (80 (IQR: 59 to 98) mL/m2) and Mexico (80 (IQR: 68 to 98) mL/m2) cohorts, but LA emptying fraction was similar in all groups. RA maximal areas were similar across the three cohorts.Indexed LA volumes were significantly greater in the China cohort (median: 109 (IQR: 90 to 153) mL/mp = 0.43). Patients with LVEF ≤ 50% had similar ECV when compared to patients with LVEF ≥ 50% (ECV: 28.5% (IQR: 25.6% to 30.2%) vs 28.0% (IQR: 23.5% to 29.7%), p = 0.41).The prevalence of LGE was similar across all three MS cohorts. There was no difference in the frequency of midmyocardial enhancement at the inferior RV insertion point, but midmyocardial LGE at the superior RV insertion point was more commonly observed in the China group. In the India cohort, the median native T1 time (at 1.5 T) (n = 18) was 990 ms (IQR: 964 to 1029 ms). In the China cohort, the median native T1 time (at 3 T) (n = 9) was 1275 ms (IQR: 1236 to 1340 ms). The median ECV in both cohorts was 28.3% (IQR: 24.5% to 29.9%), and there was no difference in ECV between the India and China groups (27.3% (IQR: 23.3% to 30.4%) vs 28.9% (IQR: 28.1% to 29.4%), p < 0.001) and RVEDVI , and significant negative correlations with RVEF , RV SV/ESV , LVEF , and LA emptying fraction   Table .Table 4COverall, 22/38 (58%) patients with MS ultimately underwent a mitral valvuloplasty or valve replacement . The characteristics of patients, stratified by whether a mitral valve procedure was performed, are described in Additional file In this study of a cohort of patients from China, India, and Mexico with rheumatic MS who underwent CMR imaging, we assessed detailed cardiac structural and functional changes that occurred in response to MS. We found that biventricular and biatrial remodeling was common in this population. We also identified a high prevalence of nonischemic fibrosis at the RV insertion points in the overall cohort, but no significant elevation in native T1 or ECV. In addition, we found notable differences in cardiac structure and function between patients from the three countries.As MS progresses and becomes more severe, the resistance to blood flow through the narrowed mitral valve orifice results in LA pressure elevation leading to LA enlargement and functional decline, pulmonary hypertension, and ultimately RV dilation and dysfunction . AccordiInterestingly, in the overall cohort, LV function was more reduced among MS patients, as assessed by LVEF and GLS. Although the LV was traditionally thought to be spared from adverse hemodynamic consequences due to MS, LV dysfunction in patients with MS has been recognized , 17.The not be a significant cause of subsequent myocardial dysfunction in MS patients. Given the burden of RV dilation and dysfunction in this cohort, it is possible that LV dysfunction is a result of abnormal septal motion and altered RV-LV interactions. In addition, biventricular dysfunction could be a consequence of a tachycardia-mediated cardiomyopathy in the setting of atrial fibrillation. Additional work is required to understand the mechanisms and prognostic implications of LV dysfunction and LGE in this cohort. The indexed LV volumes were at the lower limits of normal, possibly due to reduced preload or reduced filling, but this requires additional study [Our CMR study provides the opportunity to better understand the mechanisms related to LV dysfunction in MS. We found LVEDVs were similar in the MS and control groups, and we did not identify segmental wall motion abnormalities in the MS cohort. LGE imaging showed evidence of inferior and superior RV insertion site fibrosis, which has not been shown previously, and was likely due to RV remodeling in the setting of pulmonary artery hypertension –26. Impoal study .There were several key differences between the China, India, and Mexico cohorts. The India cohort was younger than the China and Mexico cohorts, suggesting that advanced valvular disease and subsequent heart failure symptoms developed more rapidly in this cohort. Prior studies have noted that patients in endemic regions tend to present with more rapidly progressive valve disease, possibly due to recurrent bouts of carditis . FurtherThis study should be interpreted in the context of its strengths and limitations. Strengths include the careful selection of patients with rheumatic MS without concomitant valve disease and cardiovascular conditions, who underwent CMR across three countries. We were able to identify a sizable cohort of patients meeting our stringent criteria, despite the limited access and availability of CMR in these countries. In addition, tissue characterization was performed in the majority of CMR studies.Our study has a number of limitations. Only patients undergoing preoperative CMR were included which reflect a selection bias. Despite this limitation, these patients represent a cohort of patients with symptomatic rheumatic MS. Native T1 mapping was not available on all scans. T2 mapping was not performed, and acute inflammation and edema could not be assessed. However, in general, MS is a late finding in patients with rheumatic heart disease, and patients are typically decades past the acute inflammatory carditis/valvulitis stage. Finally, outcome data are not available and therefore associations of imaging parameters with clinical outcomes could not be performed.In this multicenter international study of patients with symptomatic rheumatic MS, we found that biventricular and biatrial remodeling, including reduced biventricular function and nonischemic fibrosis at the RV insertion sites. In addition, the patient cohorts from India, China, and Mexico were heterogeneous in terms of baseline characteristics and ventricular remodeling, possibly due to differences in disease progression and access to care. These findings further our understanding of the cardiac structural and functional changes in rheumatic MS, and demonstrate the value of CMR imaging in the evaluation of this population. Further studies are needed to understand the prognostic implications of these findings.Additional file 1. Additional tables.