Objective To observe the effect of pirfenidone on myocardial fibrosis in rats and inves-tigate its underlying mechanism.Methods Twenty-four SD rats were randomly divided into sham-operation group,model group,low-and high-dose pirfenidone groups,with 6 rats in each group.Rat model of myocardial fibrosis was established by injecting isoprenaline into the tail vein,while normal saline was given to the sham operation group.Pirfenidone of 150 and 300 mg/(kg·d)were infused gastrically to the rats of low-and high-dose pirfenidone groups after modeling.Mas-son staining was used to observe the severity of myocardial fibrosis,immunohistochemical assay was employed to detect the expression of Collagen-1,NOD-like receptor thermal protein domain associated protein 3(NLRP3)inflammasome,cysteinyl aspartate-specific protease-1(Caspase-1),and Western blotting was performed to detect the protein levels of Collogen-1 and atrial desmo-plakin D(gasdermin D,GSDMD).Results The model group showed obvious myocardial fibrosis,and elevated expression of Collogen-1,NLRP3,Caspase-1 and GSDMD when compared with the sham operation group(P＜0.05).Low-and high-dose pirfenidone treatment resulted in signifi-cantly reduced myocardial fibrosis and reduced expression of Collogen-1,NLRP3,Caspase-1 and GSDMD[(8.14±1.40)％,(6.56±0.75)％vs(22.15±2.57)％,P＜0.05;0.14±0.03 vs 0.33±0.05,0.42±0.13,P＜0.05;(10.34±1.40)％,(10.33±3.40)％vs(23.22±1.99)％,P＜0.05;(15.67±0.56)％,(17.33±0.78)％vs(22.87±1.92)％,P＜0.05;0.43±0.06,0.46±0.11 vs 0.65±0.03,P＜0.05].Conclusion Pirfenidone inhibits cardiomyocyte pyroptosis and attenuates myocar-dial fibrosis through the NLRP3/Caspase-1/GSDMD signaling axis.

Objective To observe the effect of pirfenidone on myocardial fibrosis in rats and inves-tigate its underlying mechanism.Methods Twenty-four SD rats were randomly divided into sham-operation group,model group,low-and high-dose pirfenidone groups,with 6 rats in each group.Rat model of myocardial fibrosis was established by injecting isoprenaline into the tail vein,while normal saline was given to the sham operation group.Pirfenidone of 150 and 300 mg/(kg·d)were infused gastrically to the rats of low-and high-dose pirfenidone groups after modeling.Mas-son staining was used to observe the severity of myocardial fibrosis,immunohistochemical assay was employed to detect the expression of Collagen-1,NOD-like receptor thermal protein domain associated protein 3(NLRP3)inflammasome,cysteinyl aspartate-specific protease-1(Caspase-1),and Western blotting was performed to detect the protein levels of Collogen-1 and atrial desmo-plakin D(gasdermin D,GSDMD).Results The model group showed obvious myocardial fibrosis,and elevated expression of Collogen-1,NLRP3,Caspase-1 and GSDMD when compared with the sham operation group(P＜0.05).Low-and high-dose pirfenidone treatment resulted in signifi-cantly reduced myocardial fibrosis and reduced expression of Collogen-1,NLRP3,Caspase-1 and GSDMD[(8.14±1.40)％,(6.56±0.75)％vs(22.15±2.57)％,P＜0.05;0.14±0.03 vs 0.33±0.05,0.42±0.13,P＜0.05;(10.34±1.40)％,(10.33±3.40)％vs(23.22±1.99)％,P＜0.05;(15.67±0.56)％,(17.33±0.78)％vs(22.87±1.92)％,P＜0.05;0.43±0.06,0.46±0.11 vs 0.65±0.03,P＜0.05].Conclusion Pirfenidone inhibits cardiomyocyte pyroptosis and attenuates myocar-dial fibrosis through the NLRP3/Caspase-1/GSDMD signaling axis.

Objective To study the role of microRNA(miR)-483-3p in reducing myocardial fibrosis in rats,and explore the relationship between its mechanism and autophagy.Methods A total of 24 male SD rats were randomly divided into sham operation group,model group,blank transfec-tion group and high expression group,with 6 rats in each group.The blank transfection group and the high-expression group were pretreated with a single injection of adeno-associated virus(AAV)-blank transfection and AAV-miR-483-3p(5×1011 vg)in the tail vein,respectively.In 14 d later,the sham group was injected with 2.5 ml/(kg·d)normal saline for 14 d,and rat model of myocardial fibrosis was established by 2 mg/ml isoproterenol[2.5 ml/(kg·d)]injection through tail vein for 14 consecutive days.Myocardial pathological damage,severity of myocardial fibrosis,and expression levels of collagen-Ⅰ,microtubule-associated protein light chain 3(LC3),autoph-agy-related protein 5(Atg5)and autophagy degradation substrate(P62)in cardiomyocytes were evaluated and measured.Results Compared with the sham operation group,the model group had obviously larger myocardial fibrosis area,higher positive expression of Collagen-Ⅰ,and increased protein levels of Atg5 and LC3-Ⅱ/LC3-Ⅰ,and decreased expression level of P62 protein(P＜0.05).The myocardial fibrosis area,positive expression of Collagen-Ⅰ,the expression levels of Atg5 and LC3-Ⅱ/LC3-Ⅰ protein[(13.64±1.51)％vs(27.47±1.55)％,(13.48±3.07)％vs(30.91±2.45)％,0.98±0.17 vs 1.24±0.28,0.66±0.05 vs 1.26±0.09,P＜0.05]were significant-ly decreased,and the expression level of P62 was notably increased(0.91±0.11 vs 0.74±0.06,P＜0.05)in the high expression group than the model group.Conclusion MiR-483-3p attenuates myocardial fibrosis in rats,and the mechanism may be related to the inhibition of cardiomyocyte autophagy.

Objective To explore the therapeutic effect of bicyclol(BIC)on rat model of myocardial fibrosis and its possible mechanism.Methods Twenty-four SPF male SD rats were randomly di-vided into sham group,model group,low-and high-dose groups,with 6 rats in each group.Except for the sham group,all other groups were injected with 5 mg/(kg·d)isoproterenol by tail vein to establish myocardial fibrosis model,and the low-and high-dose groups were administered by ga-vage with 100 and 200 mg/(kg·d)BIC,respectively for 14 consecutive days.HE staining and Masson staining were used respectively to observe the severity of myocardial injury and fibrosis.Western blot assay was employed to detect the protein expression of Collagen Ⅰ,Collagen Ⅲ,al-pha smooth muscle actin(α-SMA),methyltransferase-like protein 3(METTL3),α-ketoglutarate-dependent dioxygenase AlkB homolog 5(ALKBH5)and YTH domain family protein 1(YTHDF1)in rat myocardium.Results Compared with the sham group,myocardial cell necrosis and myocardial fibrosis were significantly more serious in the model group.Low-and high-dose BIC treatment reduced myocardial cell rupture and necrosis and myocardial fibrosis when com-pared with the model group.The expression levels of Collagen Ⅰ,Collagen Ⅲ,α-SMA,METTL3 and YTHDF1(P＜0.05)were significantly higher,and that of ALKBH5(0.58±0.02 vs 0.88±0.07,P＜0.05)was notably lower in the myocardial tissues of the model group than the sham group.While,both doses of BIC treatment significantly reversed above changes in protein levels(P＜0.05).Conclusion BIC can effectively alleviate myocardial structural damage and interstitial collagen deposition in rats with isoproterenol-induced myocardial fibrosis,and its mechanism may be related to m6A methylation modification.

Single-cell transcriptome sequencing (scRNA-seq) can resolve the expression characteristics of cells in tissues with single-cell precision, enabling researchers to quantify cellular heterogeneity within populations with higher resolution, revealing potentially heterogeneous cell populations and the dynamics of complex tissues. However, the presence of a large number of technical zeros in scRNA-seq data will have an impact on downstream analysis of cell clustering, differential genes, cell annotation, and pseudotime, hindering the discovery of meaningful biological signals. The main idea to solve this problem is to make use of the potential correlation between cells and genes, and to impute the technical zeros through the observed data. Based on this, this paper reviewed the basic methods of imputing technical zeros in the scRNA-seq data and discussed the advantages and disadvantages of the existing methods. Finally, recommendations and perspectives on the use and development of the method were provided.
Cluster Analysis ; Transcriptome

Objective:To investigate the efficacy of a SARS-CoV-2 recombinant protein vaccine as a booster dose.Methods:A new immunogen, namely RBD-sc-trimer, was designed by tandem repeating of single receptor binding domain (RBD) of SARS-CoV-2 spike (S) protein to mimic the trimeric form of RBD presented by the virus. The RBD-sc-trimer protein was expressed as a His-tagged fusion protein using a baculovirus expression system and purified by nickel affinity column. The purified protein was identified by Western blot. Its in vitro binding activity to human angiotensin converting enzyme 2 (hACE2) was analyzed by ELISA. The immunogenicity of RBD-sc-trimer as well as RBD proteins of other forms including RBD dimer (RBD-Fc), RBD monomer (RBD) and S protein trimer (S trimer) as a booster dose was evaluated in BALB/c mice. Results:In terms of both binding and neutralizing antibodies against SARS-CoV-2, RBD-sc-trimer showed an immunogenicity that was superior to that of RBD-Fc and RBD and close to the level of S trimer. The antibody response induced by RBD-sc-trimer was characterized as Th1-biased. Moreover, it displayed a stronger cross-neutralization activity against SARS-CoV-2 Beta, Delta and Omicron variants. The titer of neutralizing antibody against Omicron induced by RBD-sc-trimer only decreased by 9.1 folds relative to the prototype strain, while the antibody response induced by RBD-Fc and S trimer decreased by 68.4 and 70.8 folds, respectively.Conclusions:The recombinant protein, RBD-sc-trimer, which was capable of eliciting stronger humoral response in mice as a booster dose and showed the superiority in raising cross-reactive antibodies against SARS-CoV-2 variants over non-trimeric RBD forms, should be considered as an optimal immunogen for the development of more effective SARS-CoV-2 vaccines.

Objective:To investigate the role of growth arrest specific protein 6 (Gas6) in regulating macrophage polarization in wound healing.Methods:Clean male B6 mice were randomly(random number) divided into the normal group, skin defect group, skin defect group + normal saline group (PBS group), skin defect + Gas6 (1 μg) group, skin defect + Gas6 (5 μg) group, and skin defect + Gas6 (10 μg) group. Ten mice in each group were used to observe the healing of skin wounds. Macrophages were isolated from the wound tissues of the remaining 6 mice on the fifth day after modeling. The levels of IL-6 and IL-10 were detected by enzyme-linked immunosorbent assay (ELISA), the mRNA expression levels of arginase-1 (Arg-1) and inducible nitric oxide synthase (iNOS) were detected by RT-PCR, and flow cytometry was used to detect the expression of M1 marker CD197, M2 marker CD163 and F4/80. HE staining was used to detect the pathological changes of skin wounds. Masson staining was used to analyze the granulation tissue and collagen deposition.Results:Scab began to form on the surface of the wound on the third day after the skin defect model was established. The wound area of the Gas6 treatment group was smaller than that of the PBS group, and the wound healing was better than that of the PBS group. Compared with the normal group, the proportion of CD197 in macrophages of the skin defect group was significantly increased ( P=0.00 49), the proportion of CD163 and F4/80 double positive was significantly decreased ( P=0.00 86), the level of IL-6 was significantly increased ( P=0.00 13), the level of IL-10 was significantly increased ( P=0.00 14), the level of iNOS mRNA was significantly increased ( P=0.00 8), and Arg-1 was significantly increased in the skin defect group The mRNA level was significantly decreased ( P=0.01 21), and the inflammatory infiltration was aggravated. Compared with the PBS group, the proportion of CD197 in the Gas6 treatment group was significantly decreased ( P=0.00 0), the double positive rates of CD163 and F4/80 were significantly increased ( P = 0.00 0), the level of IL-6 was significantly decreased (P = 0.00 0), the level of IL-10 was significantly increased ( P=0.00 03), the level of iNOS mRNA was significantly decreased ( P=0.00 18), the level of Arg-1 mRNA was significantly increased ( P=0.00 1), and the number of inflammatory cells and the number of collagen fibers were increased. Conclusions:Gas6 can promote the transformation of macrophages from M1 to M2 in mice with skin defect, which is beneficial to the wound healing of skin defect.

Objective ::To investigate the role of trehalose in hepatic ischemia-reperfusion injury and its underlying mechanisms.Methods:C57BL/6J mice were randomly divided into no-ischemia group, ischemia-reperfusion group, trehalose-treated group and normal saline control group. After ischemia for 90 minutes, reperfusion immediately or 6h, blood and liver tissues were collected, and serum was separated. The liver function parameters of ALT, AST, the inflammatory factors of TNF-α, IL-1β and IL-2, and the pathological changes of liver were detected to study the role of trehalose during hepatic ischemia-reperfusion injury. Hypoxia-reoxygenation cell model was established by AML12 mouse hepatocyte line, and divided into experimental group and control group. The experimental group was divided into low dose group and high dose group according to the concentration of trehalose administrated. And the control group had no use of trehalose. The level of apoptosis was measured to study the effect of trehalose on apoptosis induced by hepatic ischemia-reperfusion injury with flow cytometry. Western blot was utilized for detecting the levels of Caspase-3, Cleaved Caspase-3 and Bcl-2 protein to understand the molecular mechanisms of trehalose in apoptosis during hepatic ischemia-reperfusion injury.Results:In vivo animal experiments showed that liver function and such inflammatory factors as ALT, AST, TNF-α, IL-1β and IL-2 increased in ischemia-reperfusion group after hepatic ischemia-reperfusion ( P<0.05), and liver tissue became necrotic. After a treatment of trehalose, the levels of ALT, AST, TNF-α, IL-1β and IL-2 were lower than those of normalsaline control group and the area of liver tissue necrosis also decreased ( P<0.05). In vitro cell experiments showed that the apoptosis level of hepatocytes in the experimental group decreased compared with the control group.And the level of activated pro-apoptotic protein Cleaved Caspase-3 decreased, the level of anti-apoptotic protein Bcl-2 increased. Conclusions:Trehalose has protective effects on hepatic ischemia-reperfusion injury in vivo and in vitro. The mechanism may be involved in inhibiting inflammation induced by hepatic ischemia-reperfusion injury, suppressing the activation of Caspase-3 and promoting the expression of Bcl-2, thus played a protective role by extenuation of hepatocyteapoptosis.

Cordycepin,which has great immunomodulatory activities such as anticancer,antifungal,antivirus,antileukemia and lipid-lowering ones,is the secondary metabolite of Cordyceps militaris (C.militaris).Liquid submerged fermentation is the common cultivation process to produce cordycepin.To optimize the fermentation process and improve production,monitoring the cordycepin secretion in the fermen-tation is essential.The measurement based on chromatography-mass spectrometry methods is generally involved in the complex sample pretreatments and time-consuming separation,so more rapid and convenient methods are required.Matrix-assisted laser desorption ionization mass spectrometry(MALDI-MS) is more attractive for faster and direct detection.Therefore,MALDI-MS detection combined with isotope-labeled internal standard was applied to the measurement of cordycepin content in the fermentation broth and mycelium.This method made accurate quantification of cordycepin in the range of 5-400 μg/mL with a relative standard deviation of 5.6％.The recovery rates of fermentation samples after the 1,13,and 25 days were 90.15％,94.27％,and 95.06％,respectively.The contents of cordycepin in the mycelium and fermentation broth were 136 mg/g and 148.39 mg/mL on the 20th culture day,respectively.The cordycepin secretion curve of the liquid fermentation of C.militaris was real-time traced over 25 days.

ObjectiveTo investigate the serological markers associated with posthepatectomy recurrence in patients with hepatocellular carcinoma, and to establish a prognostic model to evaluate whether palliative hepatectomy is suitable for such patients. MethodsA total of 111 patients with hepatocellular carcinoma who underwent hepatectomy in the Affiliated Cancer Hospital of Zhengzhou University from February 2009 to July 2013 and received follow-up were enrolled. Basic clinical data were collected and the patients were divided into recurrence group and non-recurrence group according to whether recurrence was observed during follow-up. The t-test was used for comparison of normally distributed continuous data between two groups and the Wilcoxon rank sum test was used for comparison of non-normally distributed continuous data between two groups; the chi-square test was used for comparison of categorical data between two groups. Survival curves were plotted using the Kaplan-Meier method, and survival differences were analyzed using the log-rank test. A Cox regression analysis was used to perform univariate and multivariate analyses, and the area under the ROC curve (AUC) was used to evaluate prediction efficiency. ResultsThe Kaplan-Meier survival curves showed that the patients with low alpha-fetoprotein (AFP), alkaline phosphatase, gamma-glutamyl transpeptidase (GGT), and fibrinogen and high CXCL13 had a longer median time to recurrence (P＜0.05). AFP (hazard ratio ［HR］［95%CI］=1.69(1.03~2.79), P=0.039), GGT (HR［95%CI］=1.89(1.14~3.14), P=0.014), and CXCL13 (HR［95%CI］=0.54(0.33~0.89), P=0.015) were independent factors associated with posthepatectomy recurrence. The prognostic index PI=0.526×AFP+0.637×GGT-0.616×CXCL13 established based on these factors had an AUC of 0.87, a sensitivity of 93.75%, and a specificity of 63.64% in predicting recurrence within 0-3 months after palliative hepatectomy, with a significant reduction in prediction efficiency for recurrence within 0-6 months (AUC=0.68) or a longer period of time. The recurrence prediction efficiency of this model for palliative hepatectomy was significantly higher than that for radical resection. ConclusionThe prognostic model established based on CXCL13, AFP, and GGT can be used to evaluate the risk of early recurrence after palliative hepatectomy and thus helps clinicians to make diagnosis and treatment decisions based on patients’ benefits.