Proteomic analyses indicated significant abundance changes in CM and renal mitochondria , but no differences in SkM. Taken together, these findings reveal the tissue-specific impact of chronic renal insufficiency on mitochondrial health.Chronic kidney disease (CKD) results in reduced kidney function, uremia, and accumulation of uremic metabolites. Mitochondrial alterations have been suggested to play a role in the disease pathology within various tissues. The purpose of this study was to perform a comprehensive bioenergetic and proteomic phenotyping of mitochondria from skeletal muscle (SkM), cardiac muscle (CM), and renal tissue from mice with CKD. The 5-month-old C57BL/6J male mice were fed a casein control or adenine-supplemented diet for 6 months. CKD was confirmed by blood urea nitrogen. A mitochondrial diagnostic workflow was employed to examine respiratory function, membrane and redox potential, reactive oxygen species production, and maximal activities of matrix dehydrogenases and electron transport system (ETS) protein complexes. Additionally, tandem-mass-tag-assisted proteomic analyses were performed to uncover possible differences in mitochondrial protein abundance. CKD negatively impacted mitochondrial energy transduction (all  Chronic kidney disease (CKD) stems from a progressive decline in renal function that results in elevated uremia and the accumulation of uremic metabolites which have deleterious effects on metabolism ,2,3. OveMitochondria are key regulators of energy homeostasis through oxidative phosphorylation (OXPHOS) as well as cellular redox homeostasis, calcium regulation, and apoptotic cellular signaling mechanisms, and have been identified as a potential target for uremic toxicity ,23,24. TThe goal of this study was to evaluate the organ-specific effects of CKD in mitochondrial health. To accomplish this goal, we performed comprehensive mitochondrial bioenergetic and proteomic phenotyping of mitochondrial isolated from skeletal muscle, cardiac muscle, and renal tissue from mice with and without CKD. Assessments included mitochondrial respiratory function, membrane potential, redox potential, reactive oxygen species production and electron leak, as well as enzymatic screening of matrix dehydrogenases and electron transport system protein complexes. Additionally, tandem-mass-tag-assisted proteomic analyses of mitochondrial isolates were performed to assess mitochondrial proteome abundance differences caused by CKD.C57BL/6J male mice (Stock #000664) were obtained from The Jackson Laboratory at 5 months of age . All rodents were housed in a temperature- (22 °C) and light-controlled (12 h light/12 h dark) room and maintained on standard chow diet  with free access to food and water prior to CKD induction. All animal experiments adhered to the Guide for the Care and Use of Laboratory Animals from the Institute for Laboratory Animal Research, National Research Council, Washington, D.C., National Academy Press, 1996, and any updates. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Florida.We utilized an established adenine diet model to induce CKD in mice ,30,31,32g for 10 min at 4 °C. Collected plasma was assessed using a commercial kit  according to manufacturer instructions.Blood urea nitrogen (BUN) levels were assessed in plasma collected prior to sacrifice as performed previously ,31. BrieRenal histology was performed using standard light microscopy as performed before . Brieflyg for 5 min to remove trypsin. Tissue pellets were then re-suspended in Buffer C (MOPS (50 mM), KCl (100 mM), EGTA (1 mM), MgSO4 (5 mM), bovine serum albumin ; pH = 7.1) and then homogenized via a glass-Teflon homogenizer (Wheaton) and subsequently centrifuged at 800× g for 10 min to pellet nuclei and other cellular components. The resulting supernatant was centrifuged at 10,000× g for 10 min to pellet mitochondria. All steps were performed at 4 °C or on ice. The mitochondrial pellet was gently washed to remove any damaged mitochondria and then re-suspended in Buffer B (MOPS (50 mM), KCl (100 mM), EGTA (1 mM), MgSO4 (5 mM); pH = 7.1) and protein concentration was determined using bicinchoninic acid protein assay .Skeletal muscle, cardiac muscle, and renal tissue were isolated as previously described ,24,33. BJO2) at 37 °C in Buffer Z , supplemented with either 5 mM creatine (conductance assay) or 20 mM creatine . In order to determine the integrity of mitochondrial outer membrane for each isolate, we performed respirometry using 10 mM pyruvate + 2 mM malate as substrates, followed by a bolus of ADP (16 mM) and cytochrome C (10 mM). Samples with a cytochrome C response greater than 15% were excluded from this study (1 sample was excluded). To better understand how mitochondria function under physiological energy demands, a creatine kinase (CK) clamp was used to leverage the enzymatic activity of CK, which couples the interconversion of ATP and ADP to that of phosphocreatine (PCr) and free creatine. This allows a titration of the extra mitochondrial ATP/ADP ratio and thus free energy of ATP hydrolysis (ΔGATP) across a range of energy demands [ATP can plotted against the corresponding JO2 creating a linear force-flow relationship, where the slope represents the conductance throughout the entire mitochondrial energy transduction system [High-resolution respirometry was measured using Oroboros Oxygraph-2k (O2K) measuring oxygen consumption /horseradish peroxidase (HRP) detection system (Ex/Em 530/590 nm) as described previously [2O2 via a H2O2 standard curve in identical substrate conditions as the experimental protocol. All experiments were performed at 37 °C in a 0.75 mL reaction volume. Buffer for all assays was identical to OXPHOS conductance experiments as well as the utilization of fuel sources .Mitochondrial Heviously ,24,34. F+ was carried out using a QuantaMaster Spectrofluormeter  [+ redox potential, NAD(P)+ auto fluorescence was measured kinetically via excitation/emission at 340/450 nm. All experiments were carried out at 37 °C in 0.75 mL of Buffer Z supplemented with 5 mM of creatine, 1 mM phosphocreatine (PCr), and 20 U/mL creatine kinase (CK). Initially, 75 µg of mitochondria was added to the assay buffer to determine a baseline measurement (0% reduction) followed by the addition of respiratory substrates  inducing state 2 respiration. Following state 2 respiration, adenosine triphosphate  was added, then sequential titrations of PCr  were added utilizing the CK clamp to titrate the extra mitochondrial ATP/ADP ratio. Last, 4 mM potassium cyanide (sigma#60178) was added to induce full reduction (100%) within the NAD(P)H/NAD(P)+ couple. The fluorescence of 340/450 nm was collected through the whole experiment allowing us to use the presence of mitochondria alone with no substrates as 0% reduction and the addition of cyanide as 100% reduction. The results were then expressed as percentage reduction using the formula: % reduction = (X − X0%red)/(X100%red − X0%red).Fluorescent determination of NAD(P)H/NAD(P)ay, USA) ,36. To mFluorescent determination of Δψ was performed using a QuantaMaster Spectrofluormeter  as performed previously ,36 by taJNAD(P)H) were measured fluorometrically using NAD(P)H auto fluorescence (Ex/Em = 340/450 nm) in a 96-well plate read kinetically on a BioTek Synergy 2 Multimode Microplate Reader as performed previously [The activity of several mitochondrial matrix dehydrogenases  at a protein concentration of 1 µg/µL. Dehydrogenase activity was measured fluorometrically using NAD(P)H auto fluorescence Ex/Em = 340/450 nm) in a 96-well plate read kinetically on a BioTek Synergy 2 Multimode Microplate Reader as performed previously [0/450 nm ATP synthase activity (complex V) was measured as previously described ,24. BrieThe enzyme activity of all electron transport system (ETS) complexes was determined spectrophotometrically as previously described ,38.g for 10 min at 4 °C and mitochondrial isolation medium was removed and mitochondria were resuspended in 150 µL of Chaps lysis buffer , and protease inhibitors . Samples were subjected to hand homogenization in glass homogenizers and three freeze/thaw cycles in liquid nitrogen and vortexed vigorously, followed by further disruption via sonication with a probe sonicator in three 5 s bursts (30% amplitude) on ice. The samples were then spun down at 16,000× g for 10 min at 4 °C. The supernatant was collected, and protein concentration was determined using a bicinchoninic acid protein assay  and volume was brought to 100 µL total per sample using 100 mM TEAB  with a total of 100 µg per sample. The samples were then reduced with the addition of 5 µL of 200 mM TCEP and incubated at 55 °C for 1 h. Following reduction, samples were alkylated via the addition of 5 µL of 375 mM iodoacetamide and incubated for 30 min at room temperature protected from light. Protein samples were then precipitated by adding 400 µL of methanol, vortexed to mix thoroughly, and centrifuged at 9000× g for 10 s at room temperature. A volume of 100 µL of chloroform was then added and vortexed followed by another 9000× g spin for 10 s. A volume of 300 µL of water  was added to the samples, vortexed vigorously and centrifuged for 1 min at 9000× g at room temperature. This step has 3 phases. The top layer (supernatant = mixture of water and methanol) is removed carefully and 300 µL of methanol is added and mixed vigorously and centrifuged at 9000× g for 2 min at room temperature. The supernatant was then removed, and remaining fluid was gently aspirated using a vacuum concentrator until the pellet was left a little moist. Precipitated proteins were then resuspended in 100 µL of 100 mM TEAB and 2.5 µg of trypsin  was added per 100 µg of protein and digested overnight at 37 °C. Following overnight protein digestion, samples were labeled using 11-plex tandem-mass-tag (TMT) reagents  and incubated for 1 h at room temperature. After incubation, the reaction was quenched with the addition of 8 µL of 5% hydroxylamine and incubated for 15 min. Labeled peptides were desalted with C18-solid phase extraction and dissolved in strong cation exchange (SCX) solvent A (25% (v/v) acetonitrile, 10 mM ammonium formate, and 0.1% (v/v) formic acid, pH 2.8). The peptides were fractionated using an Agilent HPLC 1260 with a polysulfoethyl A column . Peptides were eluted with a linear gradient of 0–20% solvent B (25% (v/v) acetonitrile and 500 mM ammonium formate, pH 6.8) over 50 min followed by ramping up to 100% solvent B in 5 min. The absorbance at 280 nm was monitored and a total of 16 fractions were collected. The fractions were lyophilized, desalted, and resuspended in LC solvent A (0.1% formic acid in 99.9% water (v/v)). A hybrid quadrupole Orbitrap (Q Exactive Plus) MS system  was used with high energy collision dissociation (HCD) in each MS and MS/MS cycle. The MS system was interfaced with an automated Easy-nLC 1000 system . Each sample fraction was loaded onto an Acclaim Pepmap 100 pre-column  and separated on a PepMap RSLC analytical column  at a flow rate at 350 nL/min during a linear gradient from solvent A (0.1% formic acid (v/v)) to 30% solvent B (0.1% formic acid (v/v) and 99.9% acetonitrile (v/v)) for 95 min, to 98% solvent B for 15 min, and hold 98% solvent B for additional 30 min. Full MS scans were acquired in the Orbitrap mass analyzer over m/z 400–2000 range with resolution 70,000 at 200 m/z. The top ten most intense peaks with charge state ≥3 was fragmented in the HCD collision cell normalized collision energy of 28%, (the isolation window was 0.7 m/z). The maximum ion injection times for the survey scan and the MS/MS scans were 250 ms, respectively, and the ion target values were set to 3 × 106 and 1 × 106, respectively. Selected sequenced ions were dynamically excluded for 60 s. Proteomics data have been deposited to the jPOST repository (ID JPST000977) and Proteome Xchange (ID PXD021847).One hundred micrograms of isolated mitochondria were used per sample per tissue type (n = 3/group/tissue). Mitochondria were spun down at 10,000× 2 space and the average value from the combined samples was subtracted from each sample-specific measurement to normalize the relative measurements to the mean. For each comparison, protein abundances were analyzed for group average, standard deviation, two-tailed Student’s t-test , and a Benjamini–Hochberg [p-value.Raw MS/MS data files were processed against Uniprot Mus musculus reference proteome database using the Proteome Discoverer v2.4 . The following parameters were used: peptide tolerance at 10 ppm, tandem MS tolerance at ±0.02 Da, peptide charges of 2+ to 5+, trypsin as the enzyme, allowing one missed cleavage, TMT label (229.163 Da on-peptide N-term and K) and carbamidomethyl (C) as fixed modifications, oxidation (15.995 Da on M) as a variable modification. The false discovery rate (FDR) was calculated using Percolator algorithm in the Proteome Discoverer workflow based on the search results against a decoy database and was set at 1% FDR. For protein quantification, only MS/MS spectra that were unique to a particular protein and where the sum of the signal-to-noise ratios for all the peak pairs >9 was used for quantification. M2 reporter (TMT) intensities were summed together for each TMT channel, each channel’s sum was divided by the average of all channels’ sums, resulting in channel-specific loading control normalization factors to correct for any deviation from equal protein input in the 11-plex experiments. Reporter intensities were divided by the loading control normalization factors for each respective TMT channel. All loading control-normalized reporter intensities were converted to logHochberg  adjustedt-test. Pearson correlations were performed using two-tailed statistical testing. All statistical analysis was performed in GraphPad Prism (Version 8.4). In all cases, p < 0.05 was considered statistically significant. All data can be found at the following DOI: 10.6084/m9.figshare.16837924.Data are presented as the mean ± SD. Normality of data was assessed using the Shapiro–Wilk test. Data were first analyzed using two-tailed unpaired ATP = −15.24 and −14.72 Kcal/mol) the kidney exhibited a more reduced redox state (higher levels of NAD(P)H/NAD(P)+) compared to skeletal muscle and cardiac muscle mitochondria , the kidney was only significantly higher than cardiac muscle mitochondria , skeletal muscle had higher estimated electron leak than cardiac muscle mitochondria; however, there was no significant difference between skeletal and cardiac muscle H2O2 production  required for cellular tasks such as contraction  or production of ion gradients utilized for reabsorption or excretion of metabolites (kidneys). Given the diversity of cellular function, we first wanted to establish differences in mitochondrial energy transduction through these three highly metabolic tissues utilizing a rigorously developed mitochondrial energy transduction phenotyping platform ,35. Usinchondria D. Howeve tissues F. Intrigd levels G,H. At toduction G,H. Lastoduction I,J.p < 0.0001) and kidney weights  (p < 0.0001)  A–C compa < 0.05) D,F. Howe 0.0001) E. Notablp < 0.05) and cardiac muscle mitochondria   A,B. Not ductance C.ATP. Skeletal muscle mitochondria from CKD mice exhibited a more oxidized NAD(P)H/NAD(P)+ redox potential at all energy demands ; however, the Δψ was not significantly different between groups even though there appeared to be a leftward shift of the membrane potential curve when plotted against JO2 (reduced Δψ at each energy demand) . Although statistically insignificant, small changes in Δψ could implicate an impairment in the ability to pump protons into the inner membrane space via ETS enzymes.In parallel to respiratory function, we performed analysis of mitochondrial redox potential (NAD(P)H/NAD(P)+) and membrane potential (Δψ) across the range of ΔG demand) E–G. In s demand) D. Moreov demand) E–G, althATP values  in skeletal muscle, cardiac muscle, and renal mitochondria in severe CKD mice. Using the Amplex UltraRed combination with the CK clamp system, we were able to measure H2O2 production at each level of physiologically relevant energy demand and estimate electron leak by dividing the JH2O2 by the corresponding JO2 under identical conditions. Consistent with a previous report [2O2 production or electron leak (2O2 production at the lower (resting or near-resting) energy demands  and non-significant increases at the higher energy demands  . When analyzed as percentage electron leak at each energy demand, there were no statistically significant differences between control and CKD  production at numerous sites within the mitochondria. Intriguingly, oxidative stress has been postulated to play a significant role in the pathology and progression of CKD-induced cellular stress in skeletal, cardiac, and renal tissue ,15,19,20s report , skeletaron leak F–G undercal/mol) F. Interecal/mol) G. In sta and CKD G.p < 0.05), while other dehydrogenases demonstrated insignificant reductions in activity (p = 0.080), malic enzyme , and isocitrate dehydrogenase  (p < 0.01) (p = 0.051 vs. control)  displayed a significant reduction in the enzyme activity (activity A. These = 0.071) A. Consid < 0.01) B comparecontrol) C.p < 0.05 for both), and a non-significant reduction (~34%) in complex I activity (p = 0.068) (p < 0.05) (p < 0.05)  A. In con < 0.05) B. Renal  < 0.05) C. In con < 0.05) A,C and wTo determine if the functional changes observed in CKD mitochondrial were related to changes in protein abundance, we performed TMT-assisted proteomics analyses on isolated mitochondrial samples  were found to be significantly correlated with OXPHOS conductance (+), membrane potential (Δψ), enzyme activities, and oxidative stress were assessed. Skeletal muscle mitochondria demonstrated a more oxidized redox state and modest reductions in Δψ which were confirmed to be driven by impaired matrix dehydrogenase enzyme activities  and significant reductions in complex III and IV activities. Notably, these impairments in CKD skeletal muscle mitochondria did not cause increases in H2O2 production or electron leak. Cardiac muscle mitochondria displayed a similar oxidized redox potential and modest decrease Δψ, which were likely driven by the reduced activity of matrix dehydrogenase AKGDH and complex IV (cytochrome c oxidase). The large (75%) reduction in renal OXPHOS conductance, was paralleled by a hyper-reduced redox potential, a lower Δψ, which appear to have been caused by normal, or in some cases elevated, matrix dehydrogenase activity, and significant reductions in the activity of complexes II, III, and IV of the mitochondrial ETS. Additional proteomics profiling of all three mitochondrial proteomes uncovered tissue-specific changes in mitochondrial protein abundance caused by CKD.Altered mitochondrial function has been implicated to play a pathologic role in renal and cardiac fibrosis, left ventricular hypertrophy, inflammation, apoptosis, energy imbalance, muscle wasting, and reduced exercise capacity in the context of CKD. However, the tissue/organ-specific metabolic disturbances associated with CKD are not fully understood. In this study, a comprehensive mitochondrial phenotyping approach was applied to mice subjected to chronic adenine feeding or a control diet. Adenine-induced CKD was found to negatively impact mitochondrial OXPHOS in skeletal muscle (20% decrease), cardiac muscle (30% decrease), and the kidney (75% decrease). To identify potential sites of dysfunction, redox potential (NAD(P)H/NAD(P)Mitochondrial dysfunction has been postulated to play a role in the myopathic phenotype of CKD including muscle atrophy, weakness, and exercise intolerance. In the present, we confirm a previous report documenting muscle mitochondrial impairments in OXPHOS are driven by dehydrogenase enzymes and not robust changes in mitochondrial content or oxidative stress . Interes2O2 production at ‘resting’ levels of energy demand, while an estimation of electron leak was significantly increased at all ΔGATP levels tested. Oxidative stress has also been implicated to play a major role in pathological cardiac remodeling including cardiac hypertrophy, interstitial fibrosis, cardiac senescence, as well as metabolic aging [Heart disease is a common complication of CKD and approximately 30% of CKD patients suffer from heart failure . ATP turic aging ,22,47. Tic aging ; and in ic aging ,50. Alteic aging ,20,21,22ic aging . Taken tThe kidney has a high energy demand and renal mitochondrial function has been a consistently growing area of pre-clinical and clinical research across a wide range of renal pathologies, including CKD and AKI ,15,28. IAlthough this study employed a wide-ranging assessment of mitochondrial energetics in CKD across several tissues, there are some notable limitations. First, this study was performed only on fully mature male mice, as experiments with females were disrupted by the COVID-19 pandemic. There have been reports of sex differences in CKD-associated pathologies in C57BL6/J mice subjected to the adenine diet model ,56,57. BThis study utilized a wide-ranging array of functional analyses of mitochondria to uncover the biochemical nature of mitochondrial energetic impairment in skeletal muscle, cardiac muscle, and renal mitochondria in mice with CKD. Although each of the organs/tissues exhibit high levels of energy demand, each demonstrated unique functional characteristics and a differential impact of CKD on mitochondrial OXPHOS. Mitochondrial proteome analyses identify few abundance changes in skeletal muscle mitochondrial, whereas cardiac and renal mitochondrial displayed a modest number of differentially expressed proteins in CKD. These findings shed light on the underlying biochemical changes contributing to altered mitochondrial function and provide a platform for future interventional studies aimed to improve bioenergetics in the CKD milieu.


Therefore, we aimed to investigate the effects of JTXK granules on IR in skeletal muscle of high-fat diet-induced diabetic mice and C2C12 cells and analyze the underlying mechanisms. In the present study, we showed that JTXK granules attenuated body weight gain, reduced body fat mass, improved body lean mass, and enhanced muscle performance of diabetic mice. JTXK granules also improved glucose metabolism and skeletal muscle insulin sensitivity and partially reversed abnormal serum lipid levels, which might be related to the regulation of the AMPK/SIRT1/PGC-1α pathway, both in skeletal muscle tissue of diabetic mice and in C2C12 cells. Furthermore, drug-containing serum of JTXK granules was capable of enhancing glucose uptake and mitochondrial respiration in C2C12 cells, and AMPKα was proven to be closely involved in this process. Taken together, these results suggest that the JTXK granule ameliorates skeletal muscle IR through activation of the AMPK/SIRT1/PGC-1α signaling pathway, which offers a novel perspective of this formula to combat IR-related metabolic diseases.The Jiang Tang Xiao Ke (JTXK) granule is a classic Chinese herbal formula that has been put into clinical use in the treatment of type 2 diabetes mellitus for decades. However, whether its ability to ameliorate skeletal muscle insulin resistance (IR) is through modulation of the AMPK/SIRT1/PGC-1 Insulin resistance (IR) is a heterogeneous metabolic defect that primarily refers to the abnormality of insulin-mediated glucose disposal . IR is nα (PGC-1α). PGC-1α is known to increase gene expressions that are related to long-chain fatty acid oxidation, mitochondrial DNA replication, and cellular oxidative metabolism [+-dependent deacetylase activity, and it has been proven to interact with PGC-1α [α axis, mitochondrial synthesis and oxidative phosphorylation can be promoted, and high-fat diet- (HFD) induced obesity and IR thus can be retarded.Activation of adenosine monophosphate-activated protein kinase (AMPK) in response to insulin can boost the translocation of GLUT4 from the cytoplasm to the membrane and promote glucose uptake, transportation, and utilization . Besidestabolism . Meanwhih PGC-1α . By actiRehmannia glutinosa (Gaertn.) DC. (Dihuang), Panax ginseng C.A.Mey. (Renshen), Coptis chinensis Franch. (Huanglian), Salvia miltiorrhiza Bunge (Danshen), and so on, is a classic formula from traditional Chinese medicine. Our previous studies have shown that JTXK granules exhibited an overt therapeutic effect in the management of diabetes through the improvement of glucolipid metabolism, protection of islet ßeta-cells, and inhibition of oxidative stress and inflammatory reactions in various organs and tissues [α signaling pathway.The Jiang Tang Xiao Ke (JTXK) granule, composed of  tissues –18. Ther tissues , 20. Howα, SIRT1, PGC-1α, PPARα, and UCP3  were purchased from Abcam ; antibodies against CPT1 and Na,K-ATPase  were from Cell Signaling Technology ; and antibodies against GLUT4 and Lamin B1  were from Proteintech . All other materials are commercially available.JTXK granule was produced and quality-controlled as previously reported . The finMale 8-week-old C57BL6/J mice were purchased from Beijing Sibeifu Animal Technology Co., Ltd. . Mice were kept on an ad libitum basis and allowed free access to tap water and feed. All the mice were housed in the animal laboratory with a barrier environment in Beijing University of Chinese Medicine (certification number SCXK (Jing) 2016-0002) with a temperature of 25°C and humidity of 55 ± 5%. The protocols were approved by the Animal Care Committee of Beijing University of Chinese Medicine .n = 8) and HFD-fed group (n = 32). Mice in the normal group were fed the standard chow diet , while their counterparts received HFD feeding, of which 45% of calories are from fat  during the entire experiment. The diabetic model induction process lasted 12 weeks. After 12 weeks of feeding, mice whose body weight gain was 20% heavier than the average weight of the normal group with fasting blood glucose higher than 7.0 mmol/L were considered diabetic mice [After one week of adaptive feeding, mice were randomly divided into the normal group  was performed after the mice were fasted for 8 h from 7:00 a.m. to 3:00 p.m. Blood glucose levels were assessed before and 30, 60, 90, and 120 min after intragastric administration of glucose (2 g/kg) to the mice. For the insulin tolerance test (ITT), mice were firstly fasted for 5 h from 8:00 a.m. to 1:00 p.m., then were injected intraperitoneally with insulin (0.5 U/kg). Blood glucose levels were measured before and 30, 60, 90, and 120 min after injection.A treadmill test was used to evaluate exercise capacity and endurance. Before the formal test, mice in each group received a 6-day treadmill adaption training, which was to set the speed to 10 m/min and the running platform to 5° and allow mice to run for 5 min. When the formal test was performed, set the treadmill to 5° and the speed to 20 m/min [Serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were determined using the commercial assay kits from Nanjing Jiancheng Bioengineering Institute . All the assays were conducted in accordance with the manufacturer's instructions. Absorbance values of these measurements were detected by the automatic biochemical analyzer.μm sections and stained with hematoxylin and eosin according to the set routine. The optical microscope  was used to observe the histopathological changes in muscular tissue.The skeletal muscles were exposed, and then the gastrocnemius muscles from the hind limb were collected. The skeletal muscle tissue was fixed in a 4% neutral formaldehyde buffer for 24 h and then embedded in paraffin. Next, the embedded paraffin blocks were cut into 4 Skeletal tissue was immediately rinsed in PBS after removing from the mice. The tissue was diced into 2 mm cubes and fixed with 4% glutaraldehyde and 0.2% tannic acid in PBS overnight. Samples were washed with PBS and postfixed in 1% osmium tetroxide for 2 h. Samples were then dehydrated through a graded series of acetone and embedded in SPI-Pon 812 resin  24 h at 36°C, 48°C, and 60°C, respectively. Embedded tissue was cut into 70 nm sections and stained with uranyl acetate (30 min), followed by 0.2% lead citrate (30 min). Images were photographed under an electron microscope .2. Cells were passaged every 2-3 days. When cells grew to 80% confluence, the medium was replaced by high-glucose (25 mM) DMEM containing 2% horse serum to induce differentiation. Differentiation media were replenished every other day by removing 75% of the cell medium, followed by adding the fresh medium. Then, the differentiated C2C12 myotubes were treated with DMEM containing 0.4 mmol/L of palmitic acid and 1% FBS for 24 h to induce IR. For the treatment, the C2C12 myotube cells were incubated in the medium containing DCS of JTXK or control serum for 48 h.C2C12 cells were cultured in DMEM containing 10% fetal bovine serum (FBS). All the cells were cultured at 37°C with 95% humidity and 5% COThe drug-containing serum (DCS) of JTXK was obtained following our previous procedures . Specifiα knockdown cell line was accomplished by Genloci Biotechnologies, Inc. . The sequence of the siRNA oligo specific for AMPKα (Gene ID: 105787) was GTTGGATTTCCGTAGTATTAT. Cells were suspended in a sterile tube, centrifuged at 1000 rpm for 4 min, and added with DPBS. And then, 30 μg of pEGFP-N1 plasmid with siRNA was added to the system and delivered to the cells by electroporation at 600 V . The transfected cells were cultured in DMEM for 48 h and then transferred into a medium containing 2 μg/mL Puro. Then, limiting dilution analysis was applied to screen out positive clones and RT-PCR was conducted to confirm the transfection rate.Generation of the AMPKμL of the supernatant or different concentrations of the glucose standard solution were added into a 195 μL working solution. The absorbance was determined at 570 nm after incubation for 20 min at 37°C. The glucose consumption was calculated by the glucose concentration of blank wells subtracted from that of cell-plated wells.The glucose oxidase method was used to examine the glucose levels in the culture medium of the C2C12 cells. According to the manufacturer's instructions, 5 μM oligomycin, 5 μM carbonyl cyanide-(trifluoromethoxy)phenylhydrazone, and 0.5 μM rotenone/antimycin A. The mitochondrial respiratory parameters, such as basal respiration, ATP production, maximum respiration, and spare respiratory capacity, were calculated.Mitochondrial function was evaluated by measuring the oxygen consumption rate on the Seahorse XF Extracellular Flux Analyzer  according to the manufacturer's protocol. The C2C12 cells were treated with the JTXK drug-containing serum or control vehicle in XF24 analyzer microplates. 48 h later, the medium was changed to an XF basic medium supplemented with 11 mM glucose, 4 mM glutamine, and 2 mM pyruvate. Then, the cells were incubated with the sequential addition of 1 α, CPT1, PGC-1α, PPARα, SIRT1, UCP3, and GLUT4  at 4°C overnight. On the following day, the membrane was incubated for 1.5 h with the horseradish peroxidase- (HRP-) conjugated secondary antibody (1 : 5000 diluted) at room temperature. Then, the membranes were incubated with high-sensitivity ECL luminous liquid , and images were captured with Azure Biosystems, Inc., USA. Densitometry analyses of immunoblots were performed with the ImageJ software package.Proteins were extracted from skeletal muscle tissue and C2C12 cells with a precool RIPA lysis buffer containing 1% PMSF. After centrifugation, the total protein was extracted and the concentration was measured according to the BCA kit. Equal amounts of total protein per sample were subjected via SDS-PAGE separation gel and concentration gel and transferred to polyvinylidene fluoride (PVDF) membranes using a wet blotting system . Membranes were blocked with 5% skim milk dissolved in Tris-buffered saline and incubated with diluted AMPKΔΔCT-).Total RNA was isolated from skeletal muscle and C2C12 cells with the TRIzol reagent according to the manufacturer's instructions. cDNA was synthesized using a RevertAid First Strand cDNA Synthesis Kit . RT-PCR was performed on a StepOne™ RT-PCR System  using the SYBR Green Master Mix . The amplification conditions were set as follows: predenaturation at 95°C for 10 min, followed by 40 cycles of amplification . For each sample, PCR reactions were run in triplicate. And for each group, there were six samples. The gene-specific primers are shown in P < 0.05 was considered statistically significant. All the figures and statistical analyses were generated on GraphPad Prism 7 .All data were presented as mean ± standard deviation (SD). Datasets with more than two groups to be compared were analyzed using one-way analysis of variance (ANOVA) and Tukey's post hoc multiple comparison test. For statistical analyses of groups comparing two variables, two-way ANOVA was conducted, followed by Tukey's post hoc multiple comparison test. P < 0.05). Meanwhile, in comparison with DM mice, metformin and JTXK granules both improved the body lean mass of diabetic mice (P < 0.05). Moreover, according to results from the treadmill test, a higher body fat rate and less body lean mass accompanied impaired skeletal muscle performance, while treatment with metformin and JTXK granules prevented this alteration (P < 0.05). Next, we performed H&E staining and electron microscopy of skeletal muscle tissue . The area under the curve (AUC) among groups was 14.28 ± 4.17 (NC), 27.57 ± 8.19 (DM), 14.57 ± 2.45 (Met), and 14.53 ± 3.96 (JTXK), which indicated that treatment with metformin and JTXK granules improved glucose tolerance significantly (P < 0.05). Consistent with OGTT, results from serum glucose also revealed that diabetic mice exhibited apparent hyperglycemia, while metformin and JTXK granules could improve glucose metabolism (P < 0.05). In addition, long-time HFD feeding also leads to reduced insulin sensitivity. The results of ITT showed that the blood glucose level of diabetic mice was still higher than that of the other mice 30, 60, 90, and 120 min after insulin injection intraperitoneally. And AUC of ITT in the DM group (700.90 ± 61.85) was evidently increased when compared with that in the NC group (403.50 ± 28.43). Notably, metformin and JTXK granule treatment decreased blood glucose levels at 30, 60, 90, and 120 min and decreased AUC of ITT significantly . Moreover, treatment with metformin and JTXK granules also attenuated the HFD-induced hyperinsulinemia and abnormal HOMA-IR index markedly . As for blood lipid profiles, long-time HFD feeding caused dyslipidemia in diabetic mice, represented by the ascended FFA, TC, TG, and LDL-C level and descended HDL-C level . Aside from HDL-C, metformin and JTXK granule treatment markedly attenuated these alterations . Compared with diabetic mice, JTXK mice also exhibited a higher level of serum HDL-C (P < 0.05). In brief, JTXK granule treatment improved glucose metabolism and insulin sensitivity and partly reversed abnormal serum lipid levels.To investigate the effect of JTXK granules on glucose tolerance and insulin sensitivity, OGTT and ITT were conducted after 8 weeks of treatment. Blood samples were collected for detection of serum glucose, insulin levels, and lipid contents. Compared with normal mice, mice in the DM group showed evidently impaired glucose tolerance with obvious hyperglycemia before and after oral administration of glucose , CPT1, and UCP3 in skeletal muscle of diabetic mice were evidently lower than that of normal mice (P < 0.05). After 8 weeks of intervention with metformin or JTXK granules, expressions of those proteins in these two groups were significantly increased in comparison with that in diabetic mice (P < 0.05). Diabetic mice also exhibited reduction of GLUT4 membrane translocation, while treatment with metformin and JTXK granules increased GLUT4 translocation (P < 0.05). As shown in Figures α, SIRT1, and UCP3 in skeletal muscle of diabetic mice were lower than that of normal mice (P < 0.05), while treatment with metformin or JTXK granules markedly increased these gene expressions by approximately 2-fold (P < 0.05). Compared with the DM group, metformin or JTXK granules also increased PGC-1α, PPARα, and SIRT1 expression significantly (P < 0.05). The above results indicated it is possible that JTXK granules ameliorated skeletal muscle IR through modulation of the AMPK/SIRT1/PGC-1α pathway.To explore the underlying mechanism of JTXK granules in ameliorating skeletal muscle IR of diabetic mice, we analyzed the expression of important proteins and genes in the AMPK/SIRT1/PGC-1P > 0.05). Next, we induced C2C12 cell insulin resistance by exposing them to 0.4 M palmitic acid for 24 h. According to the results, IR C2C12 cells exhibited impaired glucose uptake capacity, while treatment with metformin or DCS for 24 or 48 h significantly improved the glucose consumption of IR C2C12 cells . In the in vivo experiment, obvious alteration in the AMPK/SIRT1/PGC-1α pathway was observed, so we assumed that JTXK probably acts through this signaling pathway. Therefore, we next analyzed the glucose uptake ability in the AMPKα knockdown C2C12 cell line (A-KD group in the figure). As shown in Figures P < 0.05).To further verify the effect of JTXK granules through in vitro experiment, we analyzed the glucose uptake ability and mitochondrial respiration in C2C12 cells after intervening with DCS of JTXK or metformin. As shown in Figures P < 0.05). For the AMPKα knockdown C2C12 cell line (A-KD group), compared with C2C12 cells, the basal respiration, ATP-linked respiration, maximum respiration, and spare respiratory capacity of C2C12 cells were significantly decreased (P < 0.05). As expected, the impaired mitochondrial respiration of AMPKα knockdown C2C12 cells was restored by intervention with DCS (P < 0.05). Furthermore, the effect of DCS on A-KD C2C12 cells was weaker than that on C2C12 cells, indicating that the effect of DCS on improving mitochondrial energy metabolism of C2C12 cells is closely related to the regulation of AMPKα. Thus, we concluded that AMPKα was closely involved in the effect of JTXK on enhancing glucose uptake and mitochondrial respiration in C2C12 cells.As AMPK is the pivot regulator in energy metabolism, we further investigated the effects of JTXK on mitochondrial energy metabolism . Compareα signaling in C2C12 cells. As shown in Figures α, SIRT1, PGC-1α, PPARα, UCP3, and CPT1 (P < 0.05). Consistent with results of western blotting, DCS of JTXK increased AMPKα, SIRT1, PGC-1α, PPARα, UCP3, and CPT1 mRNA expressions by 1.87-, 2.09-, 2.17-, 2.03-, 2.19-, and 2.62-fold  The JTXK granule was capable of enhancing glucose uptake and mitochondrial respiration in C2C12 cells, and AMPKα was proven to be closely involved in this process.The present study demonstrated the following. (1) JTXK granules attenuated body weight gain, reduced body fat mass, improved body lean mass, and enhanced muscle performance of diabetic mice. (2) JTXK granules improved glucose metabolism and insulin sensitivity and partly reversed abnormal serum lipid levels. (3) JTXK granules increased the gene as well as protein expression of important molecules in the AMPK/SIRT1/PGC-1Rehmannia glutinosa (Gaertn.) DC. (Dihuang), Panax ginseng C.A.Mey. (Renshen), Salvia miltiorrhiza Bunge (Danshen), Cornus officinalis Siebold & Zucc. (Shanzhuyu), Coptis chinensis Franch. (Huanglian), Pueraria montana var. lobata (Gegen), and so on. To better understand the essential and substantial basis of this formula, the fingerprint technique was applied to illustrate the main ingredients (Fig α expression and increasing AMPK phosphorylation [α signaling concerning mitochondrial respiration.In the last decades, significant investigation breakthroughs have been witnessed in the study of traditional Chinese medicine. Among these, herbal medicine stands in a critical position in traditional Chinese medicine practice. The JTXK granule is an effective herbal formula derived from rich clinical practice and exhibits a satisfying therapeutic effect in the management of IR-related metabolic disorders, including obesity, prediabetes, and overt diabetes. This formula is composed of ents Fig . Accordirylation . Here, wWestern diet and sedentary lifestyle have made metabolic disorders such as diabetes, obesity, and metabolic syndrome become an important disease spectrum threatening human health. Among metabolic disorders, IR is a common phenomenon as well as the main pathological factor. Therefore, investigating effective drug candidates for IR has been a hot topic in this research field. In the present study, we chose metformin, the first-line drug for diabetes, as the positive control to compare the effect of JTXK granules on skeletal muscle IR. In line with our previous results, JTXK granules exhibited a favorable effect on attenuating body weight gain and reducing food intake. In addition, JTXK granules also helped to regulate body composition, ameliorate pathological changes in skeletal muscle, and enhance muscle performance. In the skeletal muscle of diabetic mice, we also observed lipid droplets and swelling mitochondria, and JTXK granule intervention obviously relieved these changes. Studies have shown that compared with insulin-sensitive individuals, the mitochondrial DNA copy number in leukocytes of obese people was 6.9-fold lower, and the number of mitochondria was positively correlated with glucose utilization and oxidation while negatively correlated with BMI and serum FFA levels , 23. MorAlthough the exact mechanism that leads to the development of IR in skeletal muscle is not yet fully understood, accumulation of glucose and FFA have been shown to play a primary role , 26. In α, PGC-1α, SIRT1, PPARα, UCP3, and CPT1. JTXK granules also enhanced GLUT4 membrane translocation, which is considered a primary mediator of glucose removal from the blood circulation as well as an essential regulator of whole-body glucose homeostasis [α emerged as a key transcriptional coactivator in understanding how nuclear regulatory signals are linked to the biogenesis of mitochondria, antioxidant defense, and inflammatory response in skeletal muscle [α, while its overexpression prevents HFD-induced reduction of mitochondrial respiration in hepatocytes and promotes exercise-induced autophagy in skeletal muscle [α by deacetylation [α/PPARs might offer a novel strategy in the treatment of mitochondrial-related diseases [α. There may be an internal relationship between them, which plays a significant role in energy metabolism. After AMPK activation, it acts on the downstream SIRT1 by affecting the NAD+ level, which enhances mitochondrial oxidative phosphorylation capacity and ATP production through the SIRT1/PGC-1α signaling pathway [α network is a critical energy-sensing signaling pathway and that AMPK activation will induce the concurrent deacetylation and phosphorylation of its downstream targets and relieve the susceptibility to IR-associated metabolic disorders [α knockdown C2C12 cells exhibited apparent impaired mitochondrial respiration capacity, indicating the essential position of AMPKα in mitochondrial metabolism. JTXK granules restored mitochondrial respiration capacity in IR C2C12 cells but were less effective on A-KD C2C12 cells, which proved that AMPKα regulation was closely involved in the therapeutic mechanisms of JTXK granules. Taken together, our findings provide us a novel image that the JTXK granule ameliorates skeletal muscle IR through activation of AMPKα and modulation of the AMPK/SIRT1/PGC-1α signaling network.In the present study, we claimed that JTXK granules enhanced expression levels of proteins and genes related to mitochondrial energy metabolism, including AMPKeostasis . PGC-1α l muscle . Accumull muscle , 32. As tylation . Besidesdiseases . AMPK an pathway . So it iisorders . It reveisorders –39. To tα signaling pathway (Fig In sum, we demonstrate that JTXK granules could improve glucose and lipid metabolism and ameliorate skeletal muscle IR, which are associated with activation of the AMPK/SIRT1/PGC-1hway Fig . Our res


Our results showed that serum USP25 concentration was higher in AP patients than in healthy controls and was positively correlated with disease severity. AP patients’ serum USP25 levels after treatment were significantly lower than that at the onset of AP. Moreover, USP25 expression was upregulated in cerulein-induced AP in mice, while USP25 deficiency attenuates AP and AP-related multiple organ injury. In vivo and in vitro studies showed that USP25 exacerbates AP by promoting the release of pro-inflammatory factors and destroying tight junctions of the pancreas. We showed that USP25 aggravates AP and AP-related multiple organ injury by activating the signal transducer and activator of transcription 3 (STAT3) pathway. Targeting the action of USP25 may present a potential therapeutic option for treating AP.Ubiquitin-specific protease 25 (USP25) plays an important role in inflammation and immunity. However, the role of USP25 in acute pancreatitis (AP) is still unclear. To evaluate the role of USP25 in AP, we conducted research on clinical AP patients, USP25 Acute pancreatitis (AP) is characterized by autodigestion, edema, hemorrhage, and even necrosis of the pancreatic tissue after activation of pancreatin. Inflammation of the pancreas with or without organ failure can be observed during the course of pancreatitis. AP can be classified according to severity into mild acute pancreatitis (MAP), moderate severe acute pancreatitis (MSAP), and severe acute pancreatitis (SAP) . ApproxiThe intercellular spaces between pancreatic epithelial cells are usually closed by tight junctions. These connections are assembled from the components of adjacent cells that form a gasket-like band that surrounds the surface of each cell at the intersection of the apical and basolateral regions. This seal restricts the movement of molecules between the epithelial cavity and the interstitium according to molecule charge and size. Structurally, tight junctions are related to the actin-based cytoskeleton . SeveralDeubiquitinating enzymes (DUBs) inversely regulate protein ubiquitination by dissociating ubiquitin chain molecules and maintaining the dynamic balance of cell ubiquitin. DUBs play an important role in the regulation of protein degradation and ubiquitin recovery, DNA repair pathways, and cell signal regulation . DUBs caUbiquitin-specific protease 25 (USP25) belongs to the cysteine protease family of DUBs, and is involved in the regulation of inflammation and immunity. Long et al. found that USP25 can promote the stability of histone acetyltransferase HBO1 in bacterial infections, thereby enhancing HBO1-mediated transcription of inflammatory genes and promoting inflammation . USP25 iHere, we investigated whether USP25 affects inflammation in patients with AP. We found that serum USP25 concentrations were higher in AP patients than in healthy controls and were positively correlated with disease severity in AP patients. We demonstrated that USP25 plays a critical role in mediating AP development by activating the STAT3 pathway.the Atlanta classification of acute pancreatitis (2012 revision) . The diagnosis of AP was based on evision) . Patient−/− mice were kindly provided by Dr. Chen Dong , and C57BL/6 wild-type (WT) mice were purchased from the Vital River Laboratory . All experimental mice were housed in a specific pathogen-free animal facility at the Animal Experiment Center at Renmin Hospital of Wuhan University.Animal protocols were approved by the Animal Ethics Committee of Wuhan University for animal experiments (No. WDRY2018-K033). C57BL/6 USP25WT and USP25−/− mice. C57BL/6 USP25WT and USP25−/− male mice  were randomly divided into four groups: USP25WT + normal saline (NS), USP25WT + cerulein, USP25−/− + NS, and USP25−/− + cerulein. We administered intraperitoneal injections of cerulein  (50 μg/kg body weight) seven times at an interval of 1 h to induce AP in mice  and mouse pancreatic acinar cells (MPAC) were treated with 10-8 M cerulein, and the control group was treated with the same amount of phosphate buffered saline (PBS).To induce AP in vitro study, the cells AR42J and MPAC were incubated with 10 μM stattic for 2 h, followed by the addition of PBS or cerulein.Stattic  was administered to mice at a dose of 1 mg/kg/day per mouse for 7 days to inhibit STAT3 phosphorylation, and then mice were injected intraperitoneally with NS or cerulein. In the In the colivelin treatment study, the cells AR42J and MPAC were incubated with 100 fM colivelin, followed by the addition of cerulein.g at 4°C for 10 min, and the serum was stored at −80°C before examination. Serum AMY, LIPA, ALT, AST, Cr, and BUN were measured using a fully automated chemical analyzer . Serum IL-6, TNF-α, and IL-1β levels were examined using ELISA kits  according to the manufacturer’s instructions. All samples were analyzed three times.The blood samples were centrifuged at 3,000 × The pancreases, lungs, livers, kidneys, and small intestines obtained from the experimental mice were fixed in 4% buffered formalin for 24 h and then embedded in paraffin. Tissue sections (4 μm) were stained with hematoxylin and eosin (H&E). The histopathological changes were analyzed under an optical microscope by two expert pathologists who were blinded to the experimental method.The pathological changes in pancreatic tissue were evaluated according to the criteria proposed by Schmidt et al., including edema, inflammation, perivascular infiltration, hemorrhage, fat necrosis, and acinar necrosis . Lung inAfter deparaffinization, hydration, antigen retrieval, and serum block, pancreatic sections were incubated overnight with the following primary antibodies at 4°C: CD45  and USP25 . Goat anti-rabbit horseradish peroxidase , or goat anti-mouse horseradish peroxidase  was added to the slices.Y705 antibody  at 4°C. After washing with PBS, CY3-labeled goat anti-rabbit antibody  was added, and the nuclei were stained with 4,6-diamino-2-phenylindole . To evaluate the expression level of ZO-1 and p-STAT3, ImageJ  was used to measure the fluorescence intensity of ZO-1 and p-STAT3, and the fluorescence intensity was quantified under a high-power field.After the paraffin sections of the pancreas were deparaffinized, washed, and blocked, ZO-1 antibody  was added to it and incubated at 4°C. MPAC were incubated with p-STAT3A total protein extraction kit (Beyotime Bio-technology) was used to extract total protein from different groups, and protein concentration was determined using the BCA kit . From each group, a 20 μg protein sample was electrophoresed on a 10% SDS polyacrylamide gel and then transferred to a polyvinylidene fluoride membrane. After blocking the protein with a protein-free rapid blocking buffer , polyvinylidene fluoride membranes were incubated for 12 h at 4°C with the following antibodies: USP25 antibody , STAT3 antibodies , p-STAT3 (Tyr705) antibody , ZO-1 antibody , and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody .Mouse primary acinar cells were cultured as described below. Briefly, pancreas were isolated from 8-week-old male USP25 KO and WT C57BL/6J mice and minced for 5 min in Hank’s.2.Balanced salt solution  + 10 mmol/L of HEPES  and 0.1% bovine serum albumin . After washing, pancreatic segments were cultured in 10 ml collagenasa IA solution  at 37°C for 20-30 min. Then the solution containing collagenase was removed and replaced with Dulbecco’s modified Eagle medium  supplemented with 10% fetal bovine serum , 1% Penicillin and streptomycin , 25 ng/ml recombinant mouse epidermal growth factor  and 0.25 mg/ml trypsin inhibitor at 37°C, 95% humidity and 5% COAR42J cells were purchased from the American Type Culture Collection . The cells were cultured in a 5% CO2 humidified incubator at 37°C in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum , 1% Penicillin and streptomycin .Usp25 gene (siRNA Usp25) and non-targeting siRNAs (NC) were purchased from GenePharma Genomics Co. Ltd. . Cells were cultured with Lipofectamine 6000  and transfected with siRNA according to the manufacturer’s instructions. After the cells were incubated with siRNA for 72 h, cell protein was extracted and transfection efficiency was verified by WB.Small interfering RNA (siRNA) targeting the p < 0.05.Statistical analyses were performed using the GraphPad Prism software version 8.0. Data are expressed as mean ± SD. Comparisons between the two groups were performed using the unpaired Student’s t-test for normally distributed parameters and the Wilcoxon rank-sum test for non-normally distributed parameters. The Pearson correlation analysis was used to test correlations, and one-way analysis of variance (ANOVA) combined with the Bonferroni’s post hoc test was used for multi-group testing. Statistical significance was defined at p < 0.05). The levels of serum USP25 protein in patients and controls from ELISA are shown in p < 0.05). In addition, as shown in We collected clinical data from 79 patients diagnosed with AP  and 53 healthy controls, and serum USP25, IL-6, TNF-α, and IL-1β levels in AP patients and healthy controls were detected by ELISA. As shown in p < 0.05), LIPA , ALT , Cr , BUN , CRP , IL-6 , TNF-α , and IL-1β  levels were positively related with the USP25 level. We followed up with 16 AP patients to compare the serum USP25 level before and after treatment (p < 0.05).In the correlation analysis between serum markers, and USP25 , AMY  (p < 0.05) /mouse weight (g) was calculated, and the mouse pancreas, lung, liver, kidney, and small intestine were examined using H&E staining. The results showed that the relative weight of the pancreas of AP mice was significantly higher than that of the control group ( < 0.05) . As show < 0.05) . The his < 0.05) .p < 0.05).To further verify the difference in inflammation between wild-type mice treated with saline and cerulein, we stained the tissues for a marker of inflammation (CD45) by IHC  and quanp < 0.05; WB, p < 0.05) in the pancreas of AP mice were significantly higher than those of the control group.The expression levels of USP25 in mouse pancreas were examined by IHC and WB. The results are shown in p < 0.05), LIPA (p < 0.05), ALT (p < 0.05), AST (p < 0.05), Cr (p < 0.05), and BUN (p < 0.05) of AP mice were significantly higher than those of the control group (p < 0.05), TNF-α (p < 0.05), and IL-1β (p < 0.05) were significantly higher in AP mice than in the control group  mice. The DNA identification results of the experimental mice are shown in WT mice, USP25 deficiency had a protective effect on cerulein-induced AP mice, which showed a lower degree of pancreatic inflammation and multiple organ injury. The relative weight of the pancreas of mice in the USP25−/− group was significantly lower than that of the USP25WT group . The lungs of USP25−/−AP mice showed less bleeding and interstitial and alveolar edema, inflammation, and cell infiltration than that of the USP25WT AP mice (p < 0.05). In addition, hepatocellular edema, cytoplasmic vacuolation, necrosis, loss of intercellular boundaries and inflammatory cell infiltration in the livers of the USP25−/−AP mice decreased significantly (p < 0.05). Furthermore, dilated renal tubules, disordered tubular structure and inflammatory cell infiltration around the renal tubules were rarely observed in the kidneys of the USP25−/−AP mice (p < 0.05). Moreover, the villi tip and lamina propria were rarely peeled off or fell off in pieces in the small intestine of the USP25−/−AP mice (p < 0.05).To study the contribution of USP25 in the pathogenesis of AP, we examined cerulein-induced AP in USP25 knockout (USP25WT group . The damp < 0.05), LIPA (p < 0.05), ALT (p < 0.05), AST (p < 0.05), BUN (p < 0.05), and Cr (p < 0.05) of the USP25−/−AP group were significantly lower than those of the USP25WT AP group (p < 0.05), TNF-α (p < 0.05), and IL-1β (p < 0.05) in the USP25−/−AP group were also significantly lower than those in the USP25WT AP group.Consistent with the above mentioned histology, the levels of serum AMY   . In addiAP group .−/− + cerulein was significantly lower than that in the NC + cerulein group or USP25WT + cerulein, while the ZO-1 protein level in siRNA + cerulein group or USP25−/− + cerulein was significantly higher than in NC + cerulein group or USP25WT + cerulein. Similarly, as shown in −/− AP mice was significantly lower than that in USP25WT AP group, but ZO-1 in USP25−/− AP mice was significantly higher than in USP25WT AP mice. Expression levels p-STAT3 in MPAC and ZO-1 levels in pancreatic tissues respectively measured using IF were consistent with the results of the WB  (p < 0.05), lung (p < 0.05), liver (p < 0.05), kidney (p < 0.05), and small intestine (p < 0.05). In addition, the levels of AMY (p < 0.05) and LIPA (p < 0.05) in the AP group with stattic were significantly lower than those in the AP group without stattic  activation inhibitor stattic to interfere with STAT3 phosphorylation in mice and the cells AR42J and MPAC. The results showed that the relative weight of the pancreas in AP group with stattic was significantly lower than that in AP group without stattic ( < 0.05) . H&E sta < 0.05) , 7 showe05 activa stattic .p < 0.05), p-STAT3 (p < 0.05) of AP group with stattic were significantly reduced compared to those in AP group without stattic. In contrast, the expression of ZO-1 protein in AP group with stattic was significantly higher (p < 0.05) than in AP group without stattic. We used the IF method to examine the expression level of ZO-1 in the pancreatic tissue (p < 0.05), which was consistent with the results of WB , TNF-α (p < 0.05), and IL-1β (p < 0.05) in the AP group with stattic were significantly reduced, suggesting that inflammation of pancreas in AP group was reduced , while the expression of ZO-1 decreased significantly (p < 0.05), which suggest that activation of STAT3 by colivelin counteracted the protective effect of USP25 deficiency on AP in AR42J, and MPAC.To further verify that USP25 regulates STAT3, the STAT3 agonist colivelin was added to AR42J and MPAC, and then cerulein was used to induce AP in the cells. As shown in Therefore, USP25 aggravates AP-and AP-related multiple organ injury by destroying tight junctions through activating the STAT3 pathway.Patients with AP can experience systemic damage caused by a systemic inflammatory response, which manifests as multiple organ injuries. The proportion of AP patients with organ failure is 8–20% . In tertInflammation is a local and systemic pathological reaction in AP. During the development of AP, pancreatic acinar cells release digestive enzymes, inflammatory cytokines, and pro-inflammatory mediators into the blood circulation, including IL-6, TNF-α, and IL-1β . Pro-infIn this study, it was observed that serum AMY, LIPA, etc., and pro-inflammatory cytokines  increased after cerulein injection, and the pathological damage to the pancreas and other organs  were aggravated. These results were consistent with the results of previous studies , indicatUsp25 gene . In addition, signals triggered by type I IFN transduction induce the expression and protein synthesis of IFN regulatory factor 7 (IRF7). IRF7 binds to the USP25 promoter to activate the transcription of the p25 gene . USP25 c−/− mice .STAT3 activation plays an important role in the development of AP. Research by Li et al. found that when mouse AP was caused by intraductal hypertension, STAT3 phosphorylation was activated, serum AMY, IL-6, TNF-α, and IL-1β levels were all significantly increased, and the tight junction of the pancreas disappeared (the expression levels of ZO-1 and occludin decreased) . In addiUSP25, as a member of the deubiquitination family, can effectively reduces protein degradation by the proteasome through reversibly removing the ubiquitin chain. USP25 may regulate the inflammatory response in AP through multiple signaling pathways. Nino et al. found that USP25 restrained the degradation of the EGFR by assisting in the association of the E3 ubiquitin ligase c-Cbl with EGFR, thereby modulating the amplitude of ubiquitylation on the receptor. . PreviouOur current work demonstrates the role of USP25 in AP and AP-related multiple organ injuries. First, USP25 is highly expressed in patients with AP and is positively correlated with the degree of inflammation in AP and AP-related multiple organ injury. Second, we confirmed that USP25 deficiency can alleviate cerulein-induced AP and AP-related multiple organ injury. Last, our results show that USP25 promotes the release of pro-inflammatory factors and destroys the tight junction of the pancreas by activating the STAT3 pathway, thereby aggravating AP. Therefore, our conclusion emphasizes the role of USP25 in exacerbating AP-and AP-related multiple organ injuries. Targeting the action of USP25 may be a potential therapeutic option for treating AP.


Background: Chemotherapy-induced peripheral neuropathy (CIPN) negatively impacts cancer survivors’ quality of life and is challenging to treat with existing drugs for neuropathic pain. TNF-α is known to potentiate TRPV1 activity, which contributes to CIPN. Here, we assessed the role of TMI-1, a TNF-α-converting enzyme inhibitor, in paclitaxel (PAC)-induced neurotoxicity in dorsal root ganglion (DRG) cells.Materials and Methods: Immortalized DRG neuronal 50B11 cells were cultured and treated with PAC or PAC with TMI-1 following neuronal differentiation. Cell viability, analysis of neurite growth, immunofluorescence, calcium flow cytometry, western blotting, quantitative RT-PCR, and cytokine quantitation by ELISA were performed to determine the role of TMI-1 in neurotoxicity in neuronal cells.Results: PAC administration decreased the length of neurites and upregulated the expression of TRPV1 in 50B11 cells. TMI-1 administration showed a protective effect by suppressing inflammatory signaling, and secretion of TNF-α.Conclusion: TMI-1 partially protects against paclitaxel-induced neurotoxicity by reversing the upregulation of TRPV1 and decreasing levels of inflammatory cytokines, including TNF-α, IL-1β, and IL-6 in neuronal cells. Paclitaxel (PAC) is one of the most widely used chemotherapeutic agents against several types of cancers, including ovarian, breast, esophageal, lung, and other solid cancers . HoweverThe underlying mechanism of paclitaxel-induced peripheral neuropathy (PIPN) is not fully understood, but neuroinflammation and increased ion channel activity have been well identified as possible mechanisms in previous studies . PAC incTNF-α is synthesized as a transmembrane protein and released into the soluble form by proteolytic cleavage of a disintegrin and metalloprotease 17 (ADAM17), called the TNF-α-converting enzyme (TACE). TMI-1 is a TACE inhibitor that prevents the secretion of soluble TNF-α. It shows tumor-selective cytotoxic action against breast cancer and other neoplasms . It alsoIn the present study, we evaluated the protective effect of TMI-1 on paclitaxel-induced neurotoxicity in differentiated 50B11 cells, which are immortalized DRG neuronal cell lines derived from rats on embryonic day 14.5 . In addid-glucose , 0.2% l-glutamine, and 1% antimycotic antibiotics . The cells were plated for 24 h before differentiation and experimentation, and the exhausted medium was replaced by a serum-free medium. For neuronal growth of the 50B11 cells, 75 μM forskolin  was used, which was dissolved in DMSO: ethanol = 1: 1 solution. Cells were treated with PAC  or PAC with TMI-1 . The final concentrations of PAC and TMI-1 were 1, 10, and 100 ng/ml and 0.04, 0.4, and 4 ng/ml , respectively.The 50B11 cells, a friendly gift from Dr. Ahmet Höke, were used as a model of DRG neurons and cultured in neurobasal medium  supplemented with 10% FBS , 2% B27 supplement , 11 mM 4 cells in a 96-well plate. The cells stabilized and adhered to the wall, the serum-free media was changed, and the cells were treated with PAC  and TMI-1 (0–40 ng/ml) for 24, 48, and 72 h. The assay was performed in triplicate, and the results were expressed as absorbance at 450 nm. The fold-number of living cells could be determined by comparing these results with those of the untreated control.Cell viability was determined using the EZ-CYTOX cell viability, proliferation, and cytotoxicity assay kit  according to the manufacturer’s instructions. Rat-immortalized DRG neuronal cells were cultured and seeded at 4 × 10Frontiers cells were seeded in 24-well plates to measure neurite growth for 24 h and imaged using a Nikon E300 microscope. The wells were photographed in five randomly selected fields at ×100 magnification for analysis. Images were analyzed using the ImageJ software.Cells were seeded on coverslips in six-well plates coated with collagen and allowed to attach. After attachment, the cells were washed with washing buffer (PBS with 0.1% Tween 20), fixed with 2% paraformaldehyde, permeabilized with 0.01% Triton X-100, and blocked with blocking buffer (PBS with 10% FBS) at room temperature. The coverslips were incubated with primary antibodies [TRPV1  and βIII tubulin ] overnight at 4°C, washed three times, and incubated with secondary antibodies [Alexa 488  and Alexa 647 ] at room temperature. Cells were mounted in Prolong Gold antifade reagent with DAPI  and imaged using a laser-scanning microscope .For flow cytometry, the cells were incubated for 24 h, differentiated with FSK, and treated with PAC and PAC with TMI-1. Intracellular calcium levels were detected using the EZCell Calcium Detection Kit  following the manufacturer’s protocol. The cells were resuspended in PBS and the fluorescence intensity of the calcium indicator in cells was measured by flow cytometry ; a minimum of 10,000 events were counted for each sample. For calcium imaging, cells were seeded in six-well plates, loaded with calcium indicators , and washed with PBS. The stained cells were observed under a confocal microscope (green).2, 0.1 mM EDTA, 10 mM KCl, and protease inhibitor), incubated on ice, and centrifuged, and the cytosolic protein was then separated. Pellets were washed entirely with PBS, suspended in ice-cold saline buffer , incubated on ice, sonicated, and centrifuged, and used to separate the nuclear protein.For whole protein quantification, the control and treated cells were scraped off the plates in the presence of ice-cold lysis buffer  with phosphatase inhibitor cocktail III . For nuclear protein quantification, the cells were lysed in hypotonic buffer . Blots were incubated with the corresponding primary antibodies  and subsequently with the secondary antibody [HRP-goat anti-rabbit IgG ]. Immunoreactive bands were visualized using an enhanced chemiluminescence peroxidase substrate solution . The relative densities of the bands were normalized and quantified with β-actin using the ImageJ software.Total RNA was isolated using TRIzol reagent  according to the manufacturer’s protocol. The concentration of total RNA was determined using a Nanodrop spectrophotometer . RNA was reverse transcribed into cDNA using Maxime RT PreMix . Quantitative PCR was performed using Brilliant III SYBR Green QPCR kit  on an Applied Biosystems Real-Time PCR system . The Ct values were determined for each product and normalized against the Ct value of the reference gene (GAPDH). The values were calculated using ΔΔCT, where Ct is the cycle threshold. The primer sequences were as follows: IL-1β sense CACCTCTCAAGCAGAGCACAG, antisense GGGTTCCATGGTGAAGTCAAC; IL-6 sense TCCTACCCCAACTTCCAATGCTC, antisense TTGGATGGTCTTGGTCCTTAGCC; TNF-α sense AAATGGGCTCCCTCTCATCAGTTC, antisense TCTGCTTGGTGGTTTGCTACG AC; GAPDH sense GTATTGGGCGCCTGGTCACC, and antisense CGCTCCTGGAAGATGGTGATGG.To quantify TNF-α secretion, sandwich ELISA was performed according to the manufacturer’s instructions. First, TNF-α was measured in the culture medium using the Rat TNF-α Pre-coated ELISA Kit . Next, cells were analyzed using a 450 nm ELISA microplate reader . The cells were quantified in the culture medium and a standard curve was used.® Statistical Software for Windows, version 18.11.6 . After testing for normal distribution, data were presented as the median and interquartile range (IQR) and analyzed using the Mann-Whitney U test or Kruskal-Wallis test. If the differences between the groups were significant, the Conover tests were performed for post hoc multiple comparisons. Finally, the Jonckheere-Terpstra trend test was performed to demonstrate the dose-response relationship [i.e.  and TMI-1 (0–40 ng/ml) were added to the rat-immortalized DRG neuronal cell lines and incubated for 24, 48, and 72 h. Paclitaxel at concentrations up to 100 ng/ml did not affect cell viability (data not shown). TMI-1 at concentrations up to 4 ng/ml was ineffective in modulating cell viability except for at 72 h (data not shown). The final concentrations of paclitaxel that cause neurotoxicity but do not affect cell viability were determined to be 1, 10, and 100 ng/ml. In addition, the concentrations of TMI-1 were optimized to 0.04, 0.4, and 4 ng/ml.p = 0.0090 by Mann-Whitney U test).We evaluated the effect of forskolin on the morphological differentiation of 50B11 cells using low magnification images. Compared to 50B11 cells cultured without any mitogenic agents, the cells treated with 75 µM forskolin showed increased neurite length per cell .Morphological changes were observed 24 h after treatment of 50B11 cells with paclitaxel (100 ng/ml) and paclitaxel with TMI-1 (4 ng/ml). In the immunofluorescence analysis, the cells treated with paclitaxel showed degeneration as blebbing of axons, and some recovery of axonal injury was observed after administration of TMI-1 . Comparep = 0.005372 by Kruskal–Wallis test; From FSK to P100, p = 0.000108, From P100 to P100 + T4, p = 0.01052 by Jonckheere-Terpstra trend test). In immunofluorescence assays . Fluorescence microscopy images showed that intracellular calcium increased in cells treated with paclitaxel and decreased in those treated with TMI-1 with a dose-dependent trend. However, the quantified intracellular calcium fluorescence intensity was not statistically significant .The increased concentration of intracellular calcium level was shown by flow cytometry in cells treated with 1, 10, 100 ng/ml paclitaxel, showing a dose-dependent trend. After treatment with TMI-1 , the concentration of intracellular calcium decreased in a dose-dependent manner (p < 0.05) (p < 0.05). Treatment with 4 ng/ml TMI-1 decreased the levels of PKC, PI3K, and NF-κB . The low dose (0.04 ng/ml) of TMI-1 did not show a statistically significant decrease in all phospho-MAPK levels  and a significant decrease after treatment with 4 ng/ml TMI-1 (p < 0.05).The measurement of signaling protein levels, including PKC, PI3K, nuclear NF-κB, and phospho-MAPK , is shown in  < 0.05) . The proK levels . Neitherp = 0.004502 by Kruskal–Wallis test; From FSK to P100, p = 0.00692, From P100 to P100 + T4, p = 0.00012 by Jonckheere-Terpstra trend test). The expression of proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, was measured by quantitative PCR .ELISA was used to estimate the level of TNF-α secretion under different treatment conditions. Secretion of TNF-α was significantly increased in 50B11 cells treated with 100 ng/ml paclitaxel and decreased in the cells treated with 0.04, 0.4, and 4 ng/ml TMI-1, showing dose-dependent trend , but TMI-1 reversed these effects. Therefore, these results demonstrate that TMI-1 counteracts the neurotoxic effect of paclitaxel by contributing to neuroinflammation, which is one of the mechanisms of PIPN.The present study was performed to demonstrate the protective effect of TMI-1 against paclitaxel-induced neurotoxicity. Paclitaxel induced axonal degeneration, which was identified by axonal blebbing and shortening of the length of neurites, and TMI-1 attenuated these changes in DRG neuronal cells. In line with previous research results that demonstrate that paclitaxel induces an increase in TRPV1 expression , the incPaclitaxel induces peripheral neuropathy through multiple mechanisms: 1) arrest of the cell cycle by stabilizing the microtubules, 2) mitochondrial dysfunction, 3) sensitization of ion channels, including voltage-gated sodium channels and TRPV1, and 4) neuronal damage and neuroinflammatory processes . The halvia TLR4  . TMI-1 inhibits the enzymatic activity of ADAM17 and MMPs by binding zinc at the catalytic site . A previ of MMPs . TMI-1 w of MMPs . Howeverin vitro environment with immortalized cells, and it is not clear whether TMI-1 has the same neuroprotective effect in vivo. Thus, further in vivo clinical studies, such as on rodent pain models of PIPN, should be performed to evaluate the protective effect of TMI-1. Second, our results were attributed to short-term treatment with paclitaxel and TMI-1. A longer duration of treatment may be suitable for the CIPN model because CIPN is a chronic and progressive disease. Third, there seem to be no studies on TMI-1 associated with CIPN. Although this study suggests the possibility of TMI-1 as a therapeutic target for PIPN via attenuation of TRPV1 overexpression, further studies on the mechanisms underlying the neuroprotective effect of TMI-1 and on the clinical association between TMI-1 and CIPN are needed. Previous studies have reported that TMI-1 is effective and beneficial in treating arthritis and neoplasm via inhibition of ADAM17/MMPs. Considering the cytotoxicity on tumor cells, TMI-1 may be used as an ideal therapeutic agent for both chemotherapy and CIPN.There are several limitations to the present study. First, this study was limited to an In conclusion, TMI-1 has a neuroprotective effect on differentiated DRG cells treated with paclitaxel. It reduced the secretion of soluble TNF-α and subsequently decreased the expression of TRPV1. These results suggest that the inhibition of ADAM17 may be beneficial for the treatment of paclitaxel-induced peripheral neuropathy.


Cyclophilin A (CyPA) is widely expressed by all prokaryotic and eukaryotic cells. Upon activation, CyPA can be released into the extracellular space to engage in a variety of functions, such as interaction with the CD147 receptor, that contribute to the pathogenesis of cardiovascular diseases. CyPA was recently found to undergo acetylation at K82 and K125, two lysine residues conserved in most species, and these modifications are required for secretion of CyPA in response to cell activation in vascular smooth muscle cells. Herein we addressed whether acetylation at these sites is also required for the release of CyPA from platelets based on the potential for local delivery of CyPA that may exacerbate cardiovascular disease events. Western blot analyses confirmed the presence of CyPA in human and mouse platelets. Thrombin stimulation resulted in CyPA release from platelets; however, no acetylation was observed—neither in cell lysates nor in supernatants of both untreated and activated platelets, nor after immunoprecipitation of CyPA from platelets. Shotgun proteomics detected two CyPA peptide precursors in the recombinant protein, acetylated at K28, but again, no acetylation was found in CyPA derived from resting or stimulated platelets. Our findings suggest that acetylation of CyPA is not a major protein modification in platelets and that CyPA acetylation is not required for its secretion from platelets. Cyclophilin A (CyPA) is the best-known member of the cyclophilin family and is expressed by all known prokaryotic and eukaryotic cells, accounting for ~0.4% of the total cytosolic protein fraction . CyPA waExtracellular CyPA has been involved in the pathophysiology of different cardiovascular diseases, including cardiovascular remodeling after myocardial ischemia, cardiac hypertrophy, abdominal aortic aneurysm formation, atherosclerosis, and vascular remodeling . These ePlatelets are anucleated cells derived from both bone marrow and lung megakaryocytes  with a v2+ mobilization from intracellular stores 2+, describing acetylation of K28, was the only promising candidate identified with two spectra, a mass deviation of +0.81 and +0.41 ppm and Mascot ion scores of 59 and 41 B. The otFinally, we analyzed CyPA secretion from thrombin-activated human platelets. After stimulation, the released proteins were separated from platelets by centrifugation. The purity of the fraction was confirmed by the absence of platelet membrane-specific protein GPαIIb . WesternThe data are in stark contrast to other vascular cell types, e.g., VSMCs studied by Soe et al. that showed an acetylation-dependent release of CyPA into the medium . StimulaSeveral papers describe acetylation of CyPA in human cells and platelets ,26,28,29An acetylated band around 14 kDa was also seen sporadically in human platelet lysates, and after incubation with p300. However, no CyPA band was detected at the same size as a confirmation in platelet lysates. Interestingly, very low amounts of N-terminally truncated form of the protein (8–14 kDa) were identified in cultured mouse glial cells, which, in contrast to full-length CyPA (16 kDa), induces a distinct pattern of cytokine release in cultured microglia or astroglia [2+ mobilization from intracellular stores [Upon platelet activation, platelets rapidly release their granule content. Alpha granules release their contents into the open canicular system (OCS) at the platelet surface, while dense granules fuse directly with the membrane to exocytose activation mediators, e.g., coagulation proteins, cytokines, and proinflammatory molecules . CyPA war stores , integrir stores , in lineQuantification of acetylated versus non-acetylated CyPA in Jurkat and Hela cells revealed only 38–50% of intracellular CyPA to be acetylated . Lysine Acetylsalicylic acid (ASA) is known to irreversibly acetylate COX in platelets at serine residues within the active site. ASA has also been described to acetylate a lysine in CyPA in human colon cancer cell lines, so that in principle ASA may also acetylate CyPA in platelets ,36,37. HIn VSMCs, acetylated CyPA can be found after 4 h of Ang II treatment, and release of CyPA is noted after 16 h of stimulation. In our control experiments, we also detected CyPA acetylation after Ang II treatment in HASMCs and observed secretion of CyPA into the cell supernatant . CyPA isOur data suggest an acetylation-independent release of the protein from activated platelets. Neither increased CyPA acetylation in supernatants of platelets nor in the platelet lysate was found after stimulation. Our findings thus describe low levels of acetylation of CyPA in platelets and, in contrast to VSMC, no CyPA acetylation at K82 or K125, indicating that CyPA acetylation at these sites is not prerequisite for its rapid secretion from platelets.g for 15 min to obtain platelet-rich plasma, and platelets were subsequently collected by centrifugation at 400× g for 7 min and resuspended in Tyrode’s buffer , at a density of 1 × 109 cells/mL.Anonymized residual waste of citrated blood was obtained from healthy donors, participating in a study performed at the University of Würzburg . At no time point, any backtracking to the donors  was possible. Blood was centrifuged at 330× g for 10 min to obtain platelet-rich plasma. Platelets were subsequently collected by centrifugation at 720× g for 6 min and resuspended in Tyrode’s buffer at a density of 1× 109 cells/mL.To obtain a sufficient number of platelets for protein analyses, blood from 4 C57BL/6 mice (8–12 weeks old), randomly selected from different cages and healthy by visual inspection, was collected. Mice were housed within the Zentrum für Experimentelle Molekulare Medizin (ZEMM) at the University of Würzburg, in standard cages containing hardwood chip bedding and enriched with mouse homes and cellulose tissue. Mice had ad libitum access to water and standard chow. Mice were maintained on a 12 h (hr) light/12 h dark cycle, at a temperature of 20–24 °C with 45–62% humidity. Mice that had not been subject to any sort of treatment and were sacrificed for scientific purposes did not require ethical approval by local authorities. Mice were euthanized by an overdose of isoflurane anesthesia (5% concentration) until one minute after breathing stops, and subsequent exsanguination by the punctuation of the heart with a syringe, preloaded with 100 µL citrate buffer. Pooled blood was diluted 1:1 with Tyrode’s buffer and centrifuged at 300× 8 platelets/mL was used. Following activation, platelets were removed by 1000× g centrifugation. The purity of the supernatant was confirmed by the absence of platelet membrane-specific GPαIIb protein.Washed platelets (5 × 108 cells/mL) were stimulated with 0.5 U/mL thrombin for 5 min. Cells were harvested and lysed in 500 µL in IP buffer  substituted with Complete Mini ) and 10 µM EX527 acetylase inhibitor , known to increase CyPA acetylation in HeLa cells . Cell lyReactions (20 µL) containing 50 nM recombinant human CyPA , 1.2 mM acetyl-CoA , and 1 µg p300 catalytic domain  in HAT buffer  were incubated at 30 °C for 30 min.The cell lysate was resolved by SDS polyacrylamide gel electrophoreses. Proteins were transferred onto nitrocellulose and blocked subsequently with 5% milk in Tris-buffered saline/0.1% Tween20 (TBS/T) for 1 h. Next, blots were incubated overnight at 4 °C with appropriate antibodies: rabbit polyclonal acK antibody ; rabbit polyclonal CyPA ; p-P38 ; β-Actin ; GPαIIb .After washing 3 times with TBS/T, membranes were probed with secondary horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse antibody , and rabbit HRP protein A , and visualized using enhanced chemiluminescence reagent . Chemiluminescence images were taken using the Amersham Imager 600 . Uncropped Western Blot images are provided as 2. HASMCs were used between passages 3 and 6 and growth arrested with Dulbecco’s modified Eagle medium/nutrient mixture F-12 (DMEM/F-12) containing 0.3% FBS overnight before the medium was changed to serum-free DMEM/F-12 one hour before the experiment. Cells were stimulated with Ang II (300 nM) for 2 and 24 h. The supernatant was collected, complemented with 10 µM EX527 and processed for immunoprecipitation similar to platelet supernatants. Cell lysates were washed with PBS and subjected to immunoprecipitation and Western blot as described for platelets.Human primary aortic smooth muscle cells  were purchased from American Type Culture Collection . Cells were cultured in vascular basal media (ATCC PCS-100-030) supplemented with vascular smooth muscle growth kit (ATCC PCS-100-042), containing 10% FCS, 10 U/mL penicillin, and 10 μg/mL streptomycin, at 37 °C under 5% COSilver staining was performed using the PierceTM Silver Stain for Mass Spectrometry Kit  according to the manufacturer‘s instructions. Gels were illuminated by a lightbox, and protein bands were excised with a clean scalpel and placed in 0.5 mL microcentrifuge tubes for in-gel trypsin digestion and mass spectrometry analysis.Cutout gel bands were washed alternately 3× with 50 mM ammonium bicarbonate (ABC), pH 7.8, and 25 mM ABC, pH 7.8/50% acetonitrile (ACN). Gel bands were dried, digested for 14 h with 125 ng Trypsin Gold ) in 50 mM ABC, and subjected to nanoLC–MS/MS. For peptide recovery, gel bands were incubated twice in 20 µL 0.1% TFA at 37 °C for 15 min followed by 25 µL 0.1% TFA/60% ACN. The resulting peptide fractions were combined, dried, and redissolved in 0.1% TFA for LC–MS.m/z 400), followed by up to 10 low-resolution MS/MS of the most intense precursor ions (top N) using collision-induced dissociation. AGC target values were set to 106 and 105 for MS and MS/MS, respectively, and the maximum injection time was set to 100 ms. Singly charged ions and ions of unassigned charge were excluded from fragmentation, and fragmented precursors were excluded from refragmentation for 20 s (dynamic exclusion). The lock mass at m/z = 371.1012 was used as an internal calibrant.Liquid chromatography–mass spectrometry (LC–MS) was conducted using a U3000 RSLCnano ProFlow system online-coupled to an LTQ Orbitrap Velos Pro for data-dependent acquisition (DDA) or to a Q Exactive HF mass spectrometer  for parallel reaction monitoring (PRM). For DDA, samples were loaded in 0.1% TFA at a flow rate of 30 µL/min. After 5 min, the pre-column was switched in line with the main column , and peptides were separated using a 90 min binary acetonitrile gradient ranging from 2% to 32% ACN in presence of 0.1% formic acid at 60 °C and a flow rate of 250 nL/min. The LTQ Orbitrap Velos Pro was operated in data-dependent acquisition mode, starting with a survey scan with a resolution of 60,000 (@ m/z), with maximum ion injection times of 128 and 256 ms for MS and MS/MS, respectively. AGC target values were set to 3 × 106 and 20 precursors were included using a normalized collision energy of 27. The lock mass at m/z = 445.1200 was used as an internal calibrant. Data have been uploaded to ProteomeXchange [For targeted LC–MS, we used a 45 min binary gradient and a Q Exactive HF operated in parallel reaction monitoring mode (PRM). The resolution was set to 120,000 and 60,000 (at 200 eXchange  (PXD0272