Background: s/Aims: Sarcomatoid hepatocellular carcinoma (HCC) is a rare histological subtype of HCC characterized by extremely poor prognosis; however, its molecular characterization has not been elucidated. Methods: In this study, we conducted an integrated multiomics study of whole-exome sequencing, RNA-seq, spatial transcriptome, and immunohistochemical analyses of 28 paired sarcomatoid tumor components and conventional HCC components from 10 patients with sarcomatoid HCC, in order to identify frequently altered genes, infer the tumor subclonal architectures, track the genomic evolution, and delineate the transcriptional characteristics of sarcomatoid HCCs. Results: Our results showed that the sarcomatoid HCCs had poor prognosis. The sarcomatoid tumor components and the conventional HCC components were derived from common ancestors, mostly accessing similar mutational processes. Clonal phylogenies demonstrated branched tumor evolution during sarcomatoid HCC development and progression. TP53 mutation commonly occurred at tumor initiation, whereas ARID2 mutation often occurred later. Transcriptome analyses revealed the epithelial–mesenchymal transition (EMT) and hypoxic phenotype in sarcomatoid tumor components, which were confirmed by immunohistochemical staining. Moreover, we identified ARID2 mutations in 70% (7/10) of patients with sarcomatoid HCC but only 1–5% of patients with non-sarcomatoid HCC. Biofunctional investigations revealed that inactivating mutation of ARID2 contributes to HCC growth and metastasis and induces EMT in a hypoxic microenvironment. Conclusions We offer a comprehensive description of the molecular basis for sarcomatoid HCC, and identify genomic alteration (ARID2 mutation) together with the tumor microenvironment (hypoxic microenvironment), that may contribute to the formation of the sarcomatoid tumor component through EMT, leading to sarcomatoid HCC development and progression.

Background: s/Aims: Sarcomatoid hepatocellular carcinoma (HCC) is a rare histological subtype of HCC characterized by extremely poor prognosis; however, its molecular characterization has not been elucidated. Methods: In this study, we conducted an integrated multiomics study of whole-exome sequencing, RNA-seq, spatial transcriptome, and immunohistochemical analyses of 28 paired sarcomatoid tumor components and conventional HCC components from 10 patients with sarcomatoid HCC, in order to identify frequently altered genes, infer the tumor subclonal architectures, track the genomic evolution, and delineate the transcriptional characteristics of sarcomatoid HCCs. Results: Our results showed that the sarcomatoid HCCs had poor prognosis. The sarcomatoid tumor components and the conventional HCC components were derived from common ancestors, mostly accessing similar mutational processes. Clonal phylogenies demonstrated branched tumor evolution during sarcomatoid HCC development and progression. TP53 mutation commonly occurred at tumor initiation, whereas ARID2 mutation often occurred later. Transcriptome analyses revealed the epithelial–mesenchymal transition (EMT) and hypoxic phenotype in sarcomatoid tumor components, which were confirmed by immunohistochemical staining. Moreover, we identified ARID2 mutations in 70% (7/10) of patients with sarcomatoid HCC but only 1–5% of patients with non-sarcomatoid HCC. Biofunctional investigations revealed that inactivating mutation of ARID2 contributes to HCC growth and metastasis and induces EMT in a hypoxic microenvironment. Conclusions We offer a comprehensive description of the molecular basis for sarcomatoid HCC, and identify genomic alteration (ARID2 mutation) together with the tumor microenvironment (hypoxic microenvironment), that may contribute to the formation of the sarcomatoid tumor component through EMT, leading to sarcomatoid HCC development and progression.

Background: s/Aims: Sarcomatoid hepatocellular carcinoma (HCC) is a rare histological subtype of HCC characterized by extremely poor prognosis; however, its molecular characterization has not been elucidated. Methods: In this study, we conducted an integrated multiomics study of whole-exome sequencing, RNA-seq, spatial transcriptome, and immunohistochemical analyses of 28 paired sarcomatoid tumor components and conventional HCC components from 10 patients with sarcomatoid HCC, in order to identify frequently altered genes, infer the tumor subclonal architectures, track the genomic evolution, and delineate the transcriptional characteristics of sarcomatoid HCCs. Results: Our results showed that the sarcomatoid HCCs had poor prognosis. The sarcomatoid tumor components and the conventional HCC components were derived from common ancestors, mostly accessing similar mutational processes. Clonal phylogenies demonstrated branched tumor evolution during sarcomatoid HCC development and progression. TP53 mutation commonly occurred at tumor initiation, whereas ARID2 mutation often occurred later. Transcriptome analyses revealed the epithelial–mesenchymal transition (EMT) and hypoxic phenotype in sarcomatoid tumor components, which were confirmed by immunohistochemical staining. Moreover, we identified ARID2 mutations in 70% (7/10) of patients with sarcomatoid HCC but only 1–5% of patients with non-sarcomatoid HCC. Biofunctional investigations revealed that inactivating mutation of ARID2 contributes to HCC growth and metastasis and induces EMT in a hypoxic microenvironment. Conclusions We offer a comprehensive description of the molecular basis for sarcomatoid HCC, and identify genomic alteration (ARID2 mutation) together with the tumor microenvironment (hypoxic microenvironment), that may contribute to the formation of the sarcomatoid tumor component through EMT, leading to sarcomatoid HCC development and progression.

AIM To establish a GC-MS method for the simultaneous content determination of sixteen pesticide residues in Lycii Fructus and perform safety assessment.METHODS The analysis was performed on DB-5MS chromatographic column(30 m×0.25 mm,0.25 μm)subjected to the programmed heating,with splitless injection of 1.0 μL dissolved sample at a flowing rate of 1.0 mL/min.Other parameters were as follows:injection port temperature of 250℃,electron impact ionization(EI),electron energy of 70 eV;ion source temperature of 230℃,multi-reaction monitoring mode,and collision gas.of high-purity N2.Pesticide residues with relatively high dietary risk were analyzed and discussed with regard to residue levels,dietary intake risk,risk ranking and cumulative exposure assessment.RESULTS Sixteen pesticides showed good linear relationships within their own ranges(r≥0.994 4),whose average recoveries were 70%-114%,with the RSDs of less than 2%.The highest average cyfluthrin residue of 0.999 2 mg/kg in Lycii Fructus of production regions and the highest average cypermethrin residue of 0.088 4 mg/kg in Lycii Fructus commodities were both detected.In Lycii Fructus of production regions with chronic hazard index(HI)value of 0.012 9 and acute HI value of 0.065 5 and their commodities with chronic HI of 0.001 2 and acute HI of 0.005 4,the pesticide residue of cypermethrin was the leading cause of chronic and acute dietary risk,and additionally,pyridaben within maximum residue limit(MRL)was the only detectectable highly toxic pesticide among the other most concerning pestcides of deltamethrin,pyridaben,chlorpyrifos,dichlorvos and methidathion.CONCLUSION There exist pesticide residues within MRL values in some samples of Lycii Fructus and the use of cypermethrin should be well-controlled.

A high performance liquid chromatography (HPLC) method utilizing correction factors was established for the quantitative detection of related substances in flumazenil. Separation was achieved using an Agilent Pursuit XRs C18 column (250 mm × 4.6 mm, 5 μm) with an isocratic elution of dilute phosphoric acid, methanol, and tetrahydrofuran as the mobile phases. Correction factors calculated from a standard curve method were applied to determine the impurity content. The quantification of impurities in flumazenil was conducted using both external standard and correction factor methods, followed by validation and comparison of the two. For the identification of degradation products, a forced degradation approach was employed to prepare a flumazenil degradation solution, and the resulting impurities were confirmed by LC-MS analysis. The separation of flumazenil and its impurities was found to be efficient. The limits of quantification for impurities A, B, D, and E were established at 0.169 9, 0.314 7, 0.143 9, and 0.270 8 ng, respectively, with the limits of detection at 0.055 8, 0.096 9, 0.048 8, and 0.089 0 ng. These impurities demonstrated a strong linear relationship across the concentration ranges of 0.034 9-7.847 0, 0.038 7-8.710 7, 0.034 6-7.794 1, and 0.032 4-7.292 8 µg·mL^-1, respectively (n = 7). The method achieved average recoveries between 98.25% and 99.42%, with an RSD of less than 2.0% (n = 9), indicating high accuracy. The external standard and correction factor methods were used to determine the related substances in flumazenil, and the results of the two methods were consistent. The established HPLC method is characterized by its high accuracy, sensitivity, and repeatability, and is suitable for determining related substances in flumazenil.

Objective To observe the improvement effect and mechanism of salvianolic acid B on renal fibrosis mice.Methods Thirty-six BALB/c mice were randomly divided into sham operation group,model group,salvianolic acid B low-,medium-and high-dose groups and VX765(caspase-1 inhibitor)group,with 6 mice in each group.Salvianolic acid B low-,medium-and high-dose groups were intragastrically administered with corresponding doses of 50,100,200 mg-kg-1-d-1 salvianolic acid B normal saline suspension,respectively,VX765 group was intragastrically administered with 50 mg·kg-1·d-1 VX765 normal saline suspension,and the sham operation group and the model group were intragastrically administered with the same volume of normal saline,once a day.After seven days,except for the sham operation group,the mice in other groups were treated with unilateral renal ischemia-reperfusion injury combined with contralateral nephrectomy to establish an acute kidney injury-induced renal fibrosis model.After modeling,each group continued to be administered with the corresponding volume of drugs for 21 days.After the administration,the pathological changes of renal tissue were observed by hematoxylin-eosin(HE)staining and Masson staining.The serum creatinine(SCr)was detected by sarcosine oxidase method.The urea nitrogen(BUN)was detected by urease method.The level of serum neutrophil gelatinase-associated lipocalin(NGAL)was detected by enzyme-linked immunosorbent assay(ELISA).The expression of fibrosis-related proteins fibrosis-related proteins α-smooth muscle actin(α-SMA),vimentin and transforming growth factor-β(TGF-β)and pyroptosis-related proteins cleaved cysteine aspartate proteolytic enzyme 1(cl-caspase-1),pro-caspase-1(pro-caspase-1),NOD-like receptor hot protein domain-related protein 3(NLRP3),cleaved interleukin-1β(cl-IL-1β),interleukin-1β(IL-1β),GSDMD-N,GSDMD in renal tissue was detected by Western Blot.Results Compared with the sham operation group,the serum levels of SCr,BUN and NGAL in the model group were increased(P＜0.05 or P＜0.01).HE and Masson staining showed inflammatory cell infiltration and a large amount of collagen deposition in the renal interstitium,and the expression levels of fibrosis-related proteins α-SMA,vimentin and TGF-β were increased(P＜0.01).The expression levels of pyroptosis-related proteins cl-caspase-1/pro-caspase-1,NLRP3,cl-IL-1β/IL-1β,GSDMD-N/GSDMD were also increased(P＜0.05 or P＜0.01).Compared with the model group,the levels of serum SCr,BUN and NGAL in the high-dose salvianolic acid B group were significantly decreased(P＜0.05 or P＜0.01),the infiltration of renal interstitial inflammatory cells and collagen deposition were reduced,and the expression levels of fibrosis proteins α-SMA,vimentin,TGF-β and pyroptosis-related proteins cl-caspase-1/pro-caspase-1,NLRP3,cl-IL-1β/IL-1β,GSDMD-N/GSDMD were decreased(P＜0.05 or P＜0.01).There was no significant difference in the above indexes between the high-dose salvianolic acid B group and the VX765 group(P＞0.05).Conclusion Salvianolic acid B may alleviate renal fibrosis in mice by inhibiting pyroptosis mediated by NLRP3/caspase-1/GSDMD pathway.

AIM To establish an HPLC method for the simultaneous content determination of gallic acid,catechin,epicatechin,rhein 8-O-β-D-glucoside,chlorogenic acid,chrysophanol 8-O-β-D-glucoside,emodin-8-O-β-D-glucoside,emodin-3-methyl ether-8-O-β-D-glucoside,aloe emodin,rhein,cyperotundone,luteolin,α-cyperone,emodin,chrysophanol and emodin-3-methyl ether in Binglang Sixiao Pills.METHODS The analysis was performed on a 30℃thermostatic Kromasil C18 column(250 mm×4.6 mm,5 μm),with the mobile phase comprising of methanol-0.1%formic acid flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelength was set at 260 nm.Subsequently,cluster analysis and principal component analysis were made.RESULTS Fifteen constituents showed good linear relationships within their own ranges(r＞0.999 0),whose average recoveries were 96.00%-101.00%%with the RSDs of 0.50%-1.50%.Various batches of samples were clustered into two categories,and four principal components demonstrated the accumulative variance contribution rate of 90.004%.CONCLUSION This simple,accurate and reproducible method can be used for the quality control of Binglang Sixiao Pills.

Background: Intrahepatic cholangiocarcinoma (ICC) is a highly desmoplastic tumor with poor prognosis even after curative resection. We investigated the associations between the composition of the ICC stroma and immune cell infiltration and aimed to develop a stromal-immune signature to predict prognosis in surgically treated ICC. Patients and methods: We recruited 359 ICC patients and performed immunohistochemistry to detect α-smooth muscle actin (α-SMA), CD3, CD4, CD8, Foxp3, CD68, and CD66b. Aniline was used to stain collagen deposition. Survival analyses were performed to detect prognostic values of these markers. Recursive partitioning for a discrete-time survival tree was applied to define a stromal-immune signature with distinct prognostic value. We delineated an integrated stromal-immune signature based on immune cell subpopulations and stromal composition to distinguish subgroups with different recurrence-free survival (RFS) and overall survival (OS) time. Results: We defined four major patterns of ICC stroma composition according to the distributions of α-SMA and collagen: dormant (α-SMAlow/collagenhigh), fibrogenic (α-SMAhigh/collagenhigh), inert (α-SMAlow/collagenlow), and fibrolytic (α-SMAhigh/collagenlow). The stroma types were characterized by distinct patterns of infiltration by immune cells. We divided patients into six classes. Class I, characterized by high CD8 expression and dormant stroma, displayed the longest RFS and OS, whereas Class VI, characterized by low CD8 expression and high CD66b expression, displayed the shortest RFS and OS. The integrated stromal-immune signature was consolidated in a validation cohort. Conclusion We developed and validated a stromal-immune signature to predict prognosis in surgically treated ICC. These findings provide new insights into the stromal-immune response to ICC.

Aim To explore the effect of astragaloside IV (AS-IV) on cell proliferation and collagen expression in cardiac fibroblasts (CFs) of rats induced with angiotensin II (Ang II) and its mechanism. Methods CFs were pretreated with short-chain acyl-CoA dehydrogenase (SCAD) siRNA1186 for 12 h and then co-treated with Ang TJ and AS-IV for 36 h. The expressions of SCAD, α-SMA, collagen I and collagen III in CFs were detected by Western blot. mRNA expression levels of SCAD, a-SMA, collagen I and collagen III in CFs were detected by quantitative real-time PCR. The SCAD enzymatic activity, the content of ATP, hydroxyproline and free fatty acid were measured by detection kits. Results The expression of α-SMA, collagen I and collagen III were up-regulated (all P < 0. 01) in CFs induced by Ang II compared with the control cells, and the expression and enzymatic activity of SCAD significantly decreased (P < 0. 01, P< 0. 05). The content of ATP decreased (P < 0.01), and the content of hydroxyproline and free fatty acids increased (all P < 0.01). Compared with Ang II group, SCAD expression and enzymatic activity, and ATP content were significantly increased (all P < 0.01) in Ang II + AS-TV group, but the content of hydroxyproline and free fatty acids, and the expression of α-SMA, collagen I and collagen III significantly decreased (all P < 0.01). However, compared with the Ang II + NC group, there was no significant difference in all indices in the Ang II + SiRNA1186 + AS-TV group. The protective effect of AS-TV on Ang II -induced cell proliferation and collagen expression in CFs was eliminated by the interference of SCAD SiRNA1186. Conclusions AS-IV may inhibit Ang II-induced cell proliferation and collagen expression in CFs by activating SCAD.

Objective: To examine the association of greenness exposure with waist circumference (WC) and central obesity in older adults in China. Methods: Based on the cross-sectional data from the Chinese Longitudinal Healthy Longevity Survey in 2017-2018, 14 056 participants aged 65 years and over were included. Demographic characteristics, lifestyle, WC, and other information were collected through a questionnaire and physical examination. Based on the satellite monitoring data of moderate-resolution imaging spectroradiometer (MODIS) provided by NASA, the annual mean of normalized difference vegetation index (NDVI) within a radius of 1 000 meters was obtained as the measurement value of greenness exposure. Multivariate linear regression model, multivariate logistic regression model, and restricted cubic splines (RCS) model were used to analyze the association and dose-response relationship between greenness exposure and WC and central obesity in older adults in China. Results: A total of 14 056 participants were enrolled with a median age of 84.0 years [IQR: 75.0-94.0 years]. About 45.0% (6 330) of them were male and 48.6% (5 853) were illiterate. There were 10 964 (78.0%) participants from rural. The mean of WC was (84.4±10.8) cm. Central obesity accounted for 60.2% (8 465), and the NDVI range was (-0.06, 0.78). After adjusting for confounding factors, the multivariate linear regression model showed that the change value of WC in the urban group [β (95%CI):-0.49 (-0.93, -0.06)] was smaller than that in the rural [-0.78 (-0.98, -0.58)] for every 0.1 unit increase in NDVI (P_interaction=0.022). Compared with the Q₁ group in NDVI, WC of Q₂ and Q₃ groups in rural decreased, and the β (95%CI) values were-1.74 (-2.5, -0.98) and-2.78 (-3.55, -2.00), respectively. The multivariate logistic regression model showed that after adjusting for confounding factors, the risk of central obesity decreased for urban and rural older adults with an increase of 0.1 unit in NDVI, and the OR (95%CI) values were 0.87 (0.80, 0.95) and 0.86 (0.82, 0.89), respectively (P_interaction=0.284). Compared with the Q₁ group in NDVI, the risk of central obesity in the Q₂ and Q₃ groups in rural was lower, and the OR (95%CI) values were 0.68 (0.58, 0.80) and 0.57 (0.49, 0.68), respectively. The results of the multivariate regression model with RCS showed that there was a non-linear association of NDVI with WC (P_nonlinear=0.006) and central obesity (P_nonlinear=0.025). Conclusion: Greenness exposure is negatively associated with WC and central obesity in older adults in China.