Objective To investigate the protective effect of high-altitude hypoxia acclimatization against hepatic ischemia-reperfusion injury(HIRI)in rats,as well as the mechanism of action of high-altitude hypoxia acclimatization in activating autophagy.Methods A total of 56 male Sprague-Dawley rats were randomly divided into plain sham-operation group(P-S group),plain model group(P-M group),acute high-altitude hypoxia sham-operation group(AHH-S group),acute high-altitude hypoxia model group(AHH-M group),high-altitude hypoxia acclimatization sham-operation group(HHA-S group),high-altitude hypoxia acclimatization model group(HHA-M group),and high-altitude hypoxia acclimatization model group with the adenosine monophosphate-activated protein kinase(AMPK)inhibitor compound C(HHA-M-CC group),with 8 rats in each group.The rats in the acute high-altitude hypoxia groups and the high-altitude hypoxia acclimatization groups were placed in a low-pressure oxygen chamber at an altitude of 5 000 meters for 1 week and 12 weeks,respectively;the rats in the sham-operation groups were given laparotomy to expose the portal vein without vascular clamping;the rats in the HHA-M-CC group were given abdominal injection of 20 mg/kg CC at 1 hour before surgery,while those in the other groups were given injection of an equal volume of normal saline.An automatic biochemical analyzer was used to measure the levels of liver function parameters including alanine aminotransferase(ALT),aspartate aminotransferase(AST),and total bilirubin(TBil);HE staining was used to observe liver histopathological changes;transmission electron microscopy was used to observe the formation of autophagosomes in liver tissue;RT-qPCR was used to measure the mRNA expression levels of AMPK and Unc-51 like autophagy activating kinase 1(ULK1)in liver tissue;Western Blot was used to measure the protein expression levels of phosphorylated AMPK(p-AMPK),phosphorylated ULK1(p-ULK1),Beclin-1,and microtubule-associated protein 1 light chain 3 Ⅱ(LC3Ⅱ).An analysis of variance was used for comparison of continuous data between multiple groups,and the least significant difference t-test was sued for comparison between two groups.Results Compared with the AHH-M and HHA-M-CC groups,the HHA-M group had significantly reductions in the levels of ALT,AST,and TBil(all P<0.05),alleviation of liver histopathological injury,a significant reduction in Suzuki score(all P<0.05),a reduction in the degree of abnormal morphological structure of hepatocytes under transmission electron microscopy,and significant increases in the number of autophagosomes,the mRNA expression levels of AMPK and ULK1(all P<0.05),and the protein expression levels of p-AMPK,p-ULK1,Beclin-1,and LC3Ⅱ(all P<0.05).Conclusion High-altitude hypoxia acclimatization can alleviate HIRI in SD rats by activating the AMPK/ULK1 signaling pathway and enhancing autophagy in hepatocytes.

Objective To investigate the protective effect of high-altitude hypoxia acclimatization against hepatic ischemia-reperfusion injury(HIRI)in rats,as well as the mechanism of action of high-altitude hypoxia acclimatization in activating autophagy.Methods A total of 56 male Sprague-Dawley rats were randomly divided into plain sham-operation group(P-S group),plain model group(P-M group),acute high-altitude hypoxia sham-operation group(AHH-S group),acute high-altitude hypoxia model group(AHH-M group),high-altitude hypoxia acclimatization sham-operation group(HHA-S group),high-altitude hypoxia acclimatization model group(HHA-M group),and high-altitude hypoxia acclimatization model group with the adenosine monophosphate-activated protein kinase(AMPK)inhibitor compound C(HHA-M-CC group),with 8 rats in each group.The rats in the acute high-altitude hypoxia groups and the high-altitude hypoxia acclimatization groups were placed in a low-pressure oxygen chamber at an altitude of 5 000 meters for 1 week and 12 weeks,respectively;the rats in the sham-operation groups were given laparotomy to expose the portal vein without vascular clamping;the rats in the HHA-M-CC group were given abdominal injection of 20 mg/kg CC at 1 hour before surgery,while those in the other groups were given injection of an equal volume of normal saline.An automatic biochemical analyzer was used to measure the levels of liver function parameters including alanine aminotransferase(ALT),aspartate aminotransferase(AST),and total bilirubin(TBil);HE staining was used to observe liver histopathological changes;transmission electron microscopy was used to observe the formation of autophagosomes in liver tissue;RT-qPCR was used to measure the mRNA expression levels of AMPK and Unc-51 like autophagy activating kinase 1(ULK1)in liver tissue;Western Blot was used to measure the protein expression levels of phosphorylated AMPK(p-AMPK),phosphorylated ULK1(p-ULK1),Beclin-1,and microtubule-associated protein 1 light chain 3 Ⅱ(LC3Ⅱ).An analysis of variance was used for comparison of continuous data between multiple groups,and the least significant difference t-test was sued for comparison between two groups.Results Compared with the AHH-M and HHA-M-CC groups,the HHA-M group had significantly reductions in the levels of ALT,AST,and TBil(all P<0.05),alleviation of liver histopathological injury,a significant reduction in Suzuki score(all P<0.05),a reduction in the degree of abnormal morphological structure of hepatocytes under transmission electron microscopy,and significant increases in the number of autophagosomes,the mRNA expression levels of AMPK and ULK1(all P<0.05),and the protein expression levels of p-AMPK,p-ULK1,Beclin-1,and LC3Ⅱ(all P<0.05).Conclusion High-altitude hypoxia acclimatization can alleviate HIRI in SD rats by activating the AMPK/ULK1 signaling pathway and enhancing autophagy in hepatocytes.

Objective:To observe the effects of acupuncture and moxibustion on interleukin(IL)-9/IL-9 receptor(IL-9R)in the colon tissue of rats with ulcerative colitis(UC)and investigate the protective mechanism of acupuncture and moxibustion on the intestinal mucosal barrier in UC rats. Methods:Male Sprague-Dawley rats were randomly divided into a normal control(NC)group and a modeling group.UC models were prepared by giving 4%dextran sulfate sodium(DSS)water for 7 d.After the successful construction of the UC rat model,the modeling group was randomly divided into a UC group,a herb-insulated moxibustion(HM)group,and an electroacupuncture(EA)group.HM and EA interventions at bilateral Tianshu(ST25)were performed once a day for 7 d.Hematoxylin-eosin(HE)staining was used to observe the histopathological changes in the colon.The serum concentrations of IL-9,IL-6,IL-1β,and hemoglobin-H(HbH)were determined by enzyme-linked immunosorbent assay.The protein expression levels of IL-9,IL-9R,claudin-2,zonula occludens-1(ZO-1),and occludin in the colon tissue were measured by Western blotting or immuno-histochemistry.Immunofluorescence was used to detect the co-expression of PU.1 and CD4 with the IL-9 protein. Results:Compared with the NC group,the colon tissue of UC rats was severely damaged and ulcerated with congestion and edema,and the colonic histopathological score increased significantly(P<0.01).The serum HbH concentration decreased significantly(P<0.01),while the serum concentrations of IL-9,IL-6,and IL-1β increased(P<0.01).The protein expression of colonic ZO-1 and occludin decreased significantly(P<0.01),while the protein expression of colonic IL-9 and IL-9R increased(P<0.05).The positive co-expression levels of IL-9/PU.1 and IL-9/CD4 increased in the colon tissue(P<0.05).Compared with the UC group,the colonic mucosal structures were gradually repaired in both HM group and EA group,and healed ulcers could be observed,the colonic histopathological score decreased significantly(P<0.05).The serum concentration of HbH increased(P<0.01),while the serum concentrations of IL-9,IL-6,and IL-1β decreased(P<0.05).The protein expression levels of ZO-1 and occludin increased(P<0.05),while the protein expression levels of IL-9 and IL-9R decreased(P<0.01).The positive co-expression levels of IL-9/PU.1 and IL-9/CD4 decreased in the colon tissue(P<0.05). Conclusion:Both HM and EA can inhibit the protein expression levels of IL-9 and IL-9R in the UC colon by regulating the transcription factor PU.1,promote the repair of intestinal mucosal barrier,and down-regulate protein contents of proinflammatory factors IL-9,IL-6,and IL-1β in the serum,which may be one of the key mechanisms of acupuncture and moxibustion in reducing the inflammation of UC colonic mucosa and protecting the intestinal mucosal barrier.

Objective:In order to obtain safer HBV DNA vaccine,recombinant kanamycin resistance eukaryotic expression plasmid containing the gene of HBV surface antigen was constructed and used to study humoral immune response in BALB/c mice.Methods:HBV antigen middle protein(preS2+S) encoding gene fragment was isolated from the recombinant eukaryotic expression plasmid pcDNA-S 2S;subcloned into eukaryotic expression vector pVAX1.After confirmed the presence of the gene by restriction endonuclease analysis,it was used to immunize the healthy BALB/c mice at different doses,and the levels of anti-HBs in serum was determined by ELISA.Results:Restriction endonuclease analysis confirmed recombinant plasmid pVAX-S 2S was what was expected.Serum anti-HBs was detected at the 2nd week after DNA vaccination at all three doses(100 ?g,50 ?g,10 ?g per mice)and their titers were increased with time.Quantitative comparison of the serum anti-HBs levels revealed significant(P