Objective To investigate the characteristics of macrophage depletion by clodronate liposomes(CL)in a carbon tetrachloride(CCl4)-induced liver fibrosis mouse model,and to analyze the transcriptomic features.Methods Thirty-two C57BL/6 mice were divided randomly into plain control liposomes for clophosome(PL)and clodronate liposome(CL)groups(n=16 mice per group),and administered intraperitoneal injections of PL and CL,respectively.On day 5,each group was further divided into normal(N)and model(M)subgroups(n=8 mice per subgroup).Mice in group M received 10％CCl4 intraperitoneally to induce liver fibrosis,while mice in group N received an equal volume of olive oil.After 4 weeks,serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels were measured,and hepatic inflammation and collagen deposition were evaluated by hematoxylin/eosin and Sirius red staining,respectively.Total RNA was extracted from liver tissues for transcriptomic sequencing and subsequent differential gene expression analysis.Results Serum ALT and AST levels were significantly elevated in the PL-M group(P＜0.01),with fibrosis staging primarily at S3,compared with S1 in the CL-M group.Totals of 1462 and 2119 differentially expressed genes(|log fold change|＞2 and P＜0.05)were identified in the PL and CL groups,respectively.Gene Ontology analysis revealed enrichment in multiple biological processes,cellular components,and molecular functions in both models,and Kyoto Encyclopedia of Genes and Genomes analysis identified 29 significantly enriched pathways(P＜0.05).The upregulation of genes including Lgals7 and Timp1 and the downregulation of Mup-ps16 and Mup15 were validated by reverse transcription-quantitative polymerase chain reaction,consistent with transcriptomic trends(P＜0.05).Conclusions This study highlights the characteristics and transcriptomic features of macrophage depletion in the CCl4-induced liver fibrosis model,providing a theoretical reference for research on the immune mechanisms of liver fibrosis.

Objective To investigate the characteristics of macrophage depletion by clodronate liposomes(CL)in a carbon tetrachloride(CCl4)-induced liver fibrosis mouse model,and to analyze the transcriptomic features.Methods Thirty-two C57BL/6 mice were divided randomly into plain control liposomes for clophosome(PL)and clodronate liposome(CL)groups(n=16 mice per group),and administered intraperitoneal injections of PL and CL,respectively.On day 5,each group was further divided into normal(N)and model(M)subgroups(n=8 mice per subgroup).Mice in group M received 10％CCl4 intraperitoneally to induce liver fibrosis,while mice in group N received an equal volume of olive oil.After 4 weeks,serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels were measured,and hepatic inflammation and collagen deposition were evaluated by hematoxylin/eosin and Sirius red staining,respectively.Total RNA was extracted from liver tissues for transcriptomic sequencing and subsequent differential gene expression analysis.Results Serum ALT and AST levels were significantly elevated in the PL-M group(P＜0.01),with fibrosis staging primarily at S3,compared with S1 in the CL-M group.Totals of 1462 and 2119 differentially expressed genes(|log fold change|＞2 and P＜0.05)were identified in the PL and CL groups,respectively.Gene Ontology analysis revealed enrichment in multiple biological processes,cellular components,and molecular functions in both models,and Kyoto Encyclopedia of Genes and Genomes analysis identified 29 significantly enriched pathways(P＜0.05).The upregulation of genes including Lgals7 and Timp1 and the downregulation of Mup-ps16 and Mup15 were validated by reverse transcription-quantitative polymerase chain reaction,consistent with transcriptomic trends(P＜0.05).Conclusions This study highlights the characteristics and transcriptomic features of macrophage depletion in the CCl4-induced liver fibrosis model,providing a theoretical reference for research on the immune mechanisms of liver fibrosis.

Objective To investigate the effects of cultured mycelium Cordyceps sinensis(CMCS)on the AMPK/SirT1 signaling pathway in carbon tetrachloride(CCl4)-induced liver fibrosis in mice.Methods Forty male SPF-grade C57BL/6 mice were divided randomly into a normal control group,CMCS control group(3.0 g/kg),model control group,CMCS1.5 g/kg group,and CMCS 3.0 g/kg group.Mice were injected intraperitoneally with 10%CCl4(2 mL/kg)to induce liver fibrosis.Two weeks later,serum levels of alanine transaminase(ALT),aspartate transaminase(AST),and total bilirubin(TBil)were measured.Inflammation and collagen deposition in liver tissue were observed by hematoxylin and eosin(HE)and Sirius red staining,respectively.The content of hydroxyproline in liver tissue was detected by Jamall's hydrochloric acid hydrolysis method.Levels of interleukin(IL)-6,monocyte chemoattractant protein-1(MCP-1),interferon,tumor necrosis factor(TNF),IL-10,and IL-12p70 in liver tissue were detected using a cytometric bead array analysis system.Collagen Ⅰ and SirT1 expression in liver tissue were detected by immunohisotochemistry,and Prkaa1,Prkaa2,Lkb1,and p53 gene expression were detected by real-time fluorescent quantitative reverse transcriptase-polymerase chain reaction.Results Serum levels of ALT,AST,and TBil were significantly increased in the model control group compared with those in the normal control group(P＜0.05).HE and Sirius red staining showed extensive inflammatory cell infiltration and collagen deposition in the liver,respectively.Hydroxyproline content and expression levels of IL-6,MCP-1,and TNF in the liver were significantly increased(P＜0.05),while IL-10 and IL-12p70 levels were significantly decreased(P＜0.01).Immunohistochemical staining revealed an increase in Collagen Ⅰ expression and SirT1 staining was decreased in the hepatic sinusoidal space,while collagen deposition was increased.Prkaa1,Prkaa2,and Lkb1 gene expression levels were decreased and p53 was increased in liver tissue(P＜0.05).CMCS significantly reduced serum ALT and AST levels,decreased IL-6,MCP-1,and TNF expression in liver tissue(P＜0.05),up-regulated IL-10 and IL-12p70(P＜0.05),alleviated liver inflammation,collagen deposition,and hydroxyproline content,up-regulated the expression of SirT1 in the hepatic sinusoidal space,enhanced Prkaa1,Prkaa2,and Lkb1 expression(P＜0.05),and down-regulated Collagen Ⅰ and p53(P＜0.05)in the liver.Compared with CMCS 1.5 g/kg,CMCS 3.0 g/kg significantly inhibited liver inflammation and collagen deposition and up-regulated AMPK/SirT1 expression(P＜0.05).Conclusions CMCS could improve CCl4-induced liver fibrosis via up-regulation of the AMPK/SirT1 signaling pathway.

Objective To assess transcriptomic differences between carbon tetrachloride(CCl4)-induced and diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate(DDC)diet-induced mouse models of liver fibrosis to provide a framework for future research using mouse liver fibrosis models.Methods Mouse models of liver fibrosis were induced by a 10％ CCl4(2 mL/kg)injection or a 0.1％DDC diet.After 4 weeks of induction,serum levels of ALT,AST,and TBil were measured.HE and Sirius red staining were used to observe hepatic inflammation and collagen deposition.Jamall's method was used to evaluate hydroxyproline(Hyp)content in liver tissues.Hepatic tumor necrosis factor-α(TNF-α),interleukin(IL)-6,and IL-1β were measured by ELISA.Total RNA was extracted from murine liver tissues for RNA-sequencing(RNA-seq).Differentially expressed genes of the two models were analyzed by R software and then GO and KEGG enrichment was performed.Then,genes with significant differences were verified.Results Compared with normal mice,serum levels of ALT,AST,and TBil and hepatic expression of TNF-α,IL-6,and IL-1β were significantly increased in mice that received CCl4 and DDC,while the Alb serum level was decreased.Pathological staining showed that the structures of liver tissues were destroyed and a large number of hepatocytes around the central vein were hyalinized and necrotic in CCl4-treated mice.In DDC diet-treated mice,a large amount of porphyrins had been deposited in the liver and a large number of inflammatory cells had infiltrated into the portal area and bile duct.Different degrees of collagen deposition were observed in the liver tissues of the two model mice.Different genes(DEGs)of CCl4-and DDC diet-treated mice were screened using a filter(|logFC|＞2-fold and P＜0.05).As a result,1820 and 2373 DEGs in CCl4-and DDC diet-treated mice were analyzed,including 1302 and 1978 upregulated genes,and 518 and 395 downregulated genes,respectively.GO annotation showed that the two models had important functions in molecular function,biological process,and cell component.KEGG analysis showed that 22 and 29 signaling pathways were activated in CCl4-and DDC diet-induced models,respectively.Among them,16 signaling pathways,such as extracellular matrix receptor interaction,cell cycle,protein digestion and absorption,focal adhesion,and PI3K-Akt,were significantly enriched in the two models(P＜0.05).Cluster analysis showed that Mup11,Mup15,Mup17,and Mup1 were significantly down-regulated in both models,which were identified by RT-qPCR(P＜0.05).Conclusions This study conducted a comparative analysis of the RNA-Seq transcriptomic features of liver fibrosis models induced by exposure to CCl4 and a DDC diet.It examined the gene expression patterns and the pathways influenced by gene expression.The findings serve as a valuable resource for selecting appropriate animal models for future research on the pathogenesis and treatment of liver fibrosis.

Objective To investigate the expression and clinical pathological significance of PIWI-interacting RNA-47851 (piR-47851) in gastric adenocarcinoma and its influence on proliferation. Methods The expression of piR-47851 was detected in 79 gastric adenocarcinoma tissues by real time fluorescence quantitative polymerase chain reaction (qRT-PCR), and the correlation of piR-47851 expression level and clinical features with survival and prognosis were analyzed. The effect of piR-47851 on proliferation activity of gastric cancer cells was observed by cell proliferation experiments. Informatics websites were used to predict the downstream target genes of piR-47851. The wild-type and mutant plasmids for the 3'untranslated region (UTR) of MAPK1 gene were established, and a dual luciferase reporting system was used to verify that piR-47851 binded to the 3'UTR of MAPK1 gene, thereby inhibiting MAPK1 protein synthesis. The effect of overexpression/silencing of piR-47851 on MAPK1 expression level was examined through qRT-PCR experiment. Rescue experiment was used to confirm the direct regulatory effect of piR-47851 on MAPK1 and explore the effect of piR-47851/MAPK1 on the proliferation of gastric cancer cells. The effect of reduced piR-47851 expression on the volume and weight of transplanted tumors was observed in nude mice by xenograft tumor experiments; the effect of reduced piR-47851 expression on the cell proliferation activity of xenograft tumors was observed by Ki-67 staining. Results The qRT-PCR experiment result showed that the expression level of piR-47851 in 79 gastric adenocarcinoma tissues was significantly higher than that in normal gastric tissues adjacent to the cancer (P < 0.05). The expression level of piR-47851 was significantly correlated with tumor size, degree of differentiation, and survival prognosis (P < 0.05). Bioinformatics suggested that there was a complementary binding site between piR-47851 and MAPK1 gene 3'UTR. The dual luciferase reporter gene experiment showed that piR-47851 was able to significantly inhibit fluorescence enzyme activity in the MAPK1 wild-type transfection group, while this inhibitory effect was not observed in the MAPK1 mutant group. The CCK-8 proliferation activity experiment showed that overexpression of piR-47851 was able to significantly increase the proliferation activity of gastric cancer cells; after reducing piR-47851, the proliferation activity of cells decreased significantly (P < 0.05). Rescue experiment indicated that after changing the expression of piR-47851 and (or) MAPK1 and then detecting the expression level of MAPK1 by qRT-PCR, the MAPK1 could be regulated by piR-47851. Overexpression of MAPK1 could functionally reduce the proliferation promoting effect of piR-47851 on gastric cancer cells, while reducing MAPK1 expression could further enhance the proliferation activity of gastric cancer cells. In xenograft tumor experiments, reducing the expression of piR-47851 significantly resulted in smaller tumor growth volume and weight (P < 0.05). The proliferation index Ki-67 staining showed that the number of proliferative active cells in the reduced piR-47851 expression group was significantly lower than that in the control group (P < 0.05), which further validated the promotional effect of piR-47851 on cell proliferation. Conclusion The expression level of piR-47851 is increased in gastric adenocarcinoma tissues, which is closely related with tumor size and survival prognosis. The piR-47851 downregulates MAPK1 protein expression by binding to the 3'UTR of MAPK1, thereby promoting the proliferation of gastric cancer cells.

Objective To investigate the role of hsa_circ_0013058 in esophageal squamous cell carcinoma (ESCC) and the mechanism in promoting the invasion and migration of ESCC cells. Methods ESCC tissues and adjacent normal tissues of 75 patients were collected. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) and RNA in situ hybridization (RISH) were used to detect the expression of hsa_circ_0013058, and the relationship between hsa_circ_0013058 and clinicopathological data of ESCC patients was analyzed. The invasion and migration ability of ESCC cells were detected by scratch test and Transwell test. The target gene of hsa_circ_0013058 was predicted by bioinformatics. The regulation of hsa_circ_0013058 on downstream genes and its effect on invasion and migration of ESCC cells were detected by Rescue experiment. Results The results of qRT-PCR showed that the expression level of hsa_circ_0013058 in the ESCC tissues was significantly higher than that in the adjacent normal tissues (t=5.078, P < 0.05). The expression level of hsa_circ_0013058 in KYSE70 was significantly higher than that in human normal esophageal epithelial cells HTT-1A (P < 0.05). At the RNA level, hsa_circ_0013058 was negatively correlated with miR-548p (r=-0.254 2, P < 0.05). The positive expression rate of hsa_circ_0013058 RNA in the ESCC tissues was 72.00%, which was significantly higher than 17.33% in the adjacent normal tissues (χ²=6.862, P < 0.05). The positive expression of hsa_circ_0013058 was closely related to the depth of tumor invasion, differentiation degree and lymph node metastasis (P < 0.05). Scratch test results showed that overexpression of hsa_circ_0013058 enhanced the migration ability of KYSE70 cells (P < 0.05); knockdown of hsa_circ_0013058 expression decreased the migration ability of KYSE70 cells (P < 0.05). After overexpression of hsa_circ_0013058, the number of invasion and migration cells of ESCC increased, and after knockdown of hsa_circ_0013058 expression, the number of invasion and migration cells decreased (P < 0.05). The qRT-PCR confirmed that hsa_circ_0013058 could regulate the expression of microRNA-548p (miR-548p). Dual luciferase reporter gene assay showed that miR-548p was specifically bound to LPAR3, which was the direct target gene of miR-548p. Rescue experiment results indicated that hsa_circ_0013058 regulated the invasion and migration of ESCC cells through the miR-548p/LPAR3 signaling axis. Conclusion In ESCC, hsa_circ_0013058 inhibits the expression of miR-548p, thereby activating the LPAR3 signaling pathway and promoting the invasion and migration of ESCC cells.

Objective To investigate the expression level of piR-35296 in clear cell renal cell carcinoma (ccRCC) and its pathological significance, as well as its effect on ccRCC cell proliferation. Methods Surgical resection specimens and clinicopathological data from 67 ccRCC patients were collected. Differential expression profiles of piRNAs in ccRCC were detected using piRNA microarray analysis, and validated by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The effect of piR-35296 on the proliferative activity of ccRCC cells was examined by CCK-8 assay. Bioinformatic analysis predicted TAB2 as a potential downstream target gene of piR-35296; qRT-PCR was performed to detect theinfluence of piR-35296 overexpression or knockdown on TAB2 expression levels. Rescue experiments were conducted to verify the regulation of TAB2 by piR-35296 and its impact on ccRCC cell proliferation. The effect of piR-35296 knockdown on the growth of subcutaneous tumors in experimental nude mice was analyzed, and Ki-67 immunohistochemical staining was used to validate the influence of piR-35296 on ccRCC cell proliferation. Results Both piRNA expression profiling and qRT-PCR revealed an elevated expression level of piR-35296 in ccRCC (P < 0.05). The expression of piR-35296 was closely associated with tumor size and differentiation degree in ccRCC patients (χ²=3.984, 3.974, P < 0.05). CCK-8 proliferation assays demonstrated that piR-35296 overexpression enhanced the proliferative activity of ccRCC cells, whereas its downregulation inhibited cell proliferation (P < 0.05). qRT-PCR experiments showed that TAB2 expression was upregulated after piR-35296 overexpression and downregulated upon piR-35296 knockdown (P < 0.05). Rescue experiments indicated that piR-35296 promoted TAB2 mRNA and its protein expression in ccRCC cells, thereby stimulating cell proliferation; TAB2 downregulation inhibited ccRCC cell proliferation. In experimental nude mice, piR-35296 knockdown reduced the mass andvolume of subcutaneous tumors (P < 0.05). Immunohistochemical staining for Ki-67 revealed a decreased percentage of Ki-67-positive cells after piR-35296 downregulation, suggesting reduced cell proliferation capacity (P < 0.05). Conclusion The piR-35296 serves as a novel oncogenic growth promoter in ccRCC. By elevating TAB2 expression, piR-35296 promotes the proliferative growth of ccRCC cells.

Objective:To determine the molecular role of DNA methyltransferase (DNMT) in kidney tumorigenesis. Methods:Tissue samples consisted of 15 cancer tissues and 15 matched adjacent tissues from clear cell renal cell carcinoma (ccRCC) patients who had undergone nephrectomy in 2012 at the First Affiliated Hospital of Baotou Medical College, Science and Technology University of Inner Mongolia were collected. Real-time PCR, Western blot, combined bisulfite restriction analysis (COBRA) and methylation specific PCR were used in this study. Real-time PCR was used to examine the mRNA expression levels of DNMT. The global methylation level, DNA methylation level, and the expression of the antioncogene RASSF1A in ccRCC tissues were concurrently detected. Results:Both mRNA and protein levels of DNMT3B4, a splice variant of DNMT3B, were elevated in renal cell carcinoma tissue compared with those in con-troll tissue. Additionally, Alu was hypomethylated in ccRCC tissue (0.106±0.04) compared with control tissue (0.115±0.03) (P<0.05). Fur-thermore, the methylation of the promoter for RASSF1A, a tumor-suppressor gene, moderately increased in renal cell carcinoma tis-sue. By contrast, RASF1A expression decreased. Conclusion:DNMT3B4 overexpression may play an important role in human kidney tu-morigenesis via chromosomal instability and the decreased expression of RASSF1A.

Objective To explore effect of increasing cerebrospinal fluid (CSF) leptin level by administration from the foramen magnum to the cerebellum medulla oblongata pool in osteoporosis in rabbit model established by ovariectomy (OVX) and methylprednisolones.Methods According to certain inclusion and exclusion criteria,30 adult rabbits which were healthy 5-month-old female New Zealand white rabbits with the same batch and weight were divided into 5 groups (n=6) with random number table,including control group,normal saline group,Low dose group,middle dose group and high dose group.The animal model of osteoporosis (OP) was established 8 weeks after ovariectomy (bilateral ovarian resection) and intramuscular injection of methylprednisolone daily for 8 consecutive weeks in all groups.The control group were only simulated puncture and didn't inject any drugs or normal saline,the normal saline group were intracerebroventricular (ICV) injection with NS and Low,Middle and High dose group injected with low,middle and high dose of leptin respectively at 0,3,7 days after OVX.Before (0 week) and 4,8 weeks after the operation,the measure of dual energy X-ray absorptiometry was done on all the rabbits femur and bone mineral density (BMD) was delivered.Before (0 week) and 4,8 weeks after the first administration,leptin in cerebrospinal fluid (CSF) and serum growth hormone (GH),insulin growth factor 1 (IGF-1) and osteocalcin (OT/BGP) were tested with Enzyme linked immunosorbent assay and serum calcium,phosphorus and alkaline phosphatase (ALP) with automatic biochemical analyzer (ACA).The rabbits were sacrificed and the thalamus and femoral neck were harvested to test the expression of leptin receptor (LEPR) in thalamus and bone morphogenetic protein-2 (BMP-2) and type Ⅰ collagen (Col Ⅰ) at 8 weeks after the first administration.Results Compared with the control group and NS group,the cerebrospinal fluid (CSF) leptin concentration of leptin administration groups kept in a higher level significantly at 4 and 8 weeks after leptin administration,it was positively correlated with the concentration of administration.After administration 4 and 8 weeks,the bone mineral density (BMD) of leptin administration groups were not significantly lower than that of non-administration groups,and the high dose group was unchanged.The serum osteocalcin/bone glad protein (OT/BGP) concentration of leptin administration groups decreased more significantly compared with the non-administration groups at 4 weeks after leptin administration;and it rebounded more significantly than the non-administration groups and low dose group rebounded more significantly than other non-administration groups at 8 weeks.The concentration of serum growth hormone (GH) of 5 groups showed a downward trend at 4 and 8 weeks,the serum GH concentration of leptin administration groups decreased more significantly than the non-administration groups.The concentration of serum insulin-like growth IGF-1 of 5 groups were decreased significantly at 4 weeks,and the serum IGF-1 concentration of control and low dose groups were significantly higher than other 3 groups at 8weeks.Compared with the non-administration groups,the concentration of serum Calcium of leptin administration groups were increased more obvious,and it was positively correlated with the concentration of administration.Serum phosphorus of 5 groups were decreased at 4 weeks and elevated at 8 weeks;there was no difference among the groups.Serum ALP showed a continuous downward trend,and the middle dose group decreased significantly compared with other groups at 4 and 8 weeks.RTPCR results showed that the BMP-2 and Col Ⅰ mRNA expression increased and positively correlated with leptin administration concentration at 8weeks.Conclusion The increase of cerebrospinal fluid leptin level can promote the bone metabolism,reduce bone loss and precaution the occurrence of osteoporosis in rabbit model caused by OVX and methylprednisolone,and it was positively correlated with the concentration of administration.That provides a new method for the prevention and treatment of osteoporosis in rabbit model.

ObjectiveTo investigate the clinical promoting effect of crossing nape electroacupuncture on the recovery of swallowing function and recovery from pulmonary infection in post-cerebral infarction patients with tracheotomy.MethodSixty post-cerebral infarction patients with cough reflex disorder and swallowing dysfunction associated with pulmonary infection receiving tracheotomy and tracheal intubation were subjects. They were allocated, using a random number table, to three groups, 20 cases each. In each group, the patients were enrolled in order of visits. The three groups were given the same basic treatment for fighting inflammation, resolving phlegm and improving blood supply. The crossing nape electroacupuncture group received bilateral points Fengchi (GB20), Yifeng (TE17), Dicang (ST4)-to-Jiache (ST6) and Lianquan (CV23) acupuncture with electrodes connected by left-right crossing. The acupuncture group received bilateral points Fengchi, Yifeng, Dicang-to-Jiache and Lianquan acupuncture without electrodes connected. The control group received basic treatment with Western drugs without acupuncture therapy. Observations were carried out using the Kubota’s water drinking test, the Toshima Ichiro Swallowing Assessment and the Clinical Pulmonary Infection Score. The clinical therapeutic effects were evaluated in the three groups.ResultThe therapeutic effects evaluated using the Kubota’s water drinking test and the Toshima Ichiro Swallowing Assessment were better in the crossing nape electroacupuncture group than in the acupuncture group and better in the acupuncture group than in the control group (P<0.05). The score of the Clinical Pulmonary Infection Score decreased in all the three groups. The promoting effect on recovery from pulmonary infection was marked in the crossing nape electroacupuncture group (P<0.01).ConclusionCrossing nape electroacupuncture has a marked improving effect on dysphagia in post-cerebral infarction patients with tracheotomy and tracheal intubation. It can promote recovery from pulmonary infection in post-cerebral infarction patients with cough reflex disorder receiving tracheotomy and tracheal intubation.