Objective To explore the feasibility and reference value of allogeneic lung transplantation and postoperative monitoring in miniature pigs for lung transplantation research. Methods Two miniature pigs (R1 and R2) underwent left lung allogeneic transplantation. Complement-dependent cytotoxicity tests and blood cross-matching were performed before surgery. The main operative times and partial pressure of arterial oxygen (PaO₂) after opening the pulmonary artery were recorded during surgery. Postoperatively, routine blood tests, biochemical blood indicators and inflammatory factors were detected, and pathological examinations of multiple organs were conducted. Results The complement-dependent cytotoxicity test showed that the survival rate of lymphocytes between donors and recipients was 42.5%-47.3%, and no agglutination reaction occurred in the cross-matching. The first warm ischemia times of D1 and D2 were 17 min and 10 min, respectively, and the cold ischemia times were 246 min and 216 min, respectively. Ultimately, R1 and R2 survived for 1.5 h and 104 h, respectively. Postoperatively, in R1, albumin (ALB) and globulin (GLB) decreased, and alanine aminotransferase increased; in R2, ALB, GLB and aspartate aminotransferase all increased. Urea nitrogen and serum creatinine increased in both recipients. Pathological results showed that in R1, the transplanted lung had partial consolidation with inflammatory cell infiltration, and multiple organs were congested and damaged. In R2, the transplanted lung had severe necrosis with fibrosis, and multiple organs had mild to moderate damage. The expression levels of interleukin-1β and interleukin-6 increased in the transplanted lungs. Conclusions The allogeneic lung transplantation model in miniature pigs may systematically evaluate immunological compatibility, intraoperative function and postoperative organ damage. The data obtained may provide technical references for subsequent lung transplantation research.

Objective To establish a method for acquisition, perfusion, preservation and transportation of the genetically modified pig kidneys. Methods An eight genetically modified pig was utilized as experimental subject. Prior to kidneys procurement, the health status of the pig was assessed through hematology examination, and the vascular structure of the kidneys was examined using imaging techniques. Following kidneys acquisition, the pig kidneys were perfused and subsequently packaged into the cryogenic storage container labeled "For Organ Transportation Only" for interprovincial transport after communicating the transportation process with transportation department. To evaluate pathological damage to the pig kidneys, a serious of methods were employed such as hematoxylin-eosin (HE) staining, real-time fluorescent quantitative polymerase chain reaction (RT-qPCR), terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) fluorescence staining and enzyme-linked immune absorbent assay (ELISA). Results The preoperative examination of the eight genetically modified pig showed that the serum creatinine was 73.2 μmol/L, blood urea nitrogen was 2.8 mmol/L and hemoglobin was 116 g/L, all within the normal range, indicating normal renal function. CT angiography revealed no lesions in the pig kidneys, and no dilation, stenosis or premature branching of the blood vessels. The total time of obtaining the left and right kidneys from the eight genetically modified pig was (125 ± 10) min, with a blood loss of (20 ± 2) mL. The warm ischemia times were 3 min and 7 min, respectively. The perfusion and trimming times of the left and right kidneys were 36 min and 41 min, respectively. After perfusion, both kidneys were white and moist. The cold preservation and transportation time was 8 h. HE staining showed that some glomeruli were shrunk, and the lumens of the surrounding renal tubules were slightly depressed and swollen with partial inner membrane shedding and microvacuoles formed when the kidneys were preserved for 8 h. The level of cysteinyl aspartate-specific proteinase-3 messenger RNA in the kidneys tissue gradually increased with the extension of cold preservation time after 2 h (P<0.05). TUNEL fluorescence staining showed that only a small number of cells underwent apoptosis after 8 h of cold preservation, which was not significantly different from that at 0 h (P>0.05). ELISA results showed that the contents of lactate dehydrogenase (LDH) and creatinine in the preservation solution remained relatively stable, but the content of kidney injury molecule 1 (KIM-1) gradually increased with the extension of preservation time, suggesting that the pig kidneys had mild injury. Conclusions By establishing methods for acquisition, perfusion, preservation and transportation of the kidneys from genetically modified donor pig, it is possible to effectively and reliably use genetically modified pig kidneys for xenotransplantation.

Objective To explore the construction of α-1,3-galactosyltransferase (GGTA1) gene-knockout (GTKO) Diannan miniature pigs and the kidney xenotransplantation from pigs to rhesus macaques, and to assess the effectiveness of GTKO pigs. Methods The GTKO Diannan miniature pigs were constructed using the CRISPR/Cas9 gene-editing system and somatic cell cloning technology. The phenotype of GTKO pigs was verified through polymerase chain reaction, Sanger sequencing and immunofluorescence staining. Flow cytometry was used to detect antigen-antibody (IgM) binding and complement-dependent cytotoxicity. Kidney xenotransplantation was performed from GTKO pigs to rhesus macaques. The humoral immunity, cellular immunity, coagulation and physiological indicators of the recipient monkeys were monitored. The function and pathological changes of the transplanted kidneys were analyzed using ultrasonography, hematoxylin-eosin staining, immunohistochemical staining and immunofluorescence staining. Results Single-guide RNA (sgRNA) targeting exon 4 of the GGTA1 gene in Diannan miniature pigs was designed. The pGL3-GGTA1-sgRNA1-GFP vector was transfected into fetal fibroblasts of Diannan miniature pigs. After puromycin selection, two cell clones, C59# and C89#, were identified as GGTA1 gene-knockout clones. These clones were expanded to form cell lines, which were used as donor cells for somatic cell nuclear transfer. The reconstructed embryos were transferred into the oviducts of trihybrid surrogate sows, resulting in 13 fetal pigs. Among them, fetuses F04 and F11 exhibited biallelic mutations in the GGTA1 gene, and F04 had a normal karyotype. Using this GTKO fetal pig for recloning and transferring the reconstructed embryos into the oviducts of trihybrid surrogate sows, seven surviving piglets were obtained, all of which did not express α-Gal epitope. The binding of IgM from the serum of rhesus monkey 20# to GTKO pig PBMC was reduced, and the survival rate of GTKO pig PBMC in the complement-dependent cytotoxicity assay was higher than that of wild-type pig. GTKO pig kidneys were harvested and perfused until completely white. After the left kidney of the recipient monkey was removed, the pig kidney was heterotopically transplanted. Following vascular anastomosis and blood flow restoration, the pig kidney rapidly turned pink without hyperacute rejection (HAR). Urine appeared in the ureter 6 minutes later, indicating successful kidney transplantation. The right kidney of the recipient was then removed. Seven days after transplantation, the transplanted kidney had good blood flow, the recipient monkey's serum creatinine level was stable, and serum potassium and cystatin C levels were effectively controlled, although they increased 10 days after transplantation. Seven days after transplantation, the levels of white blood cells, lymphocytes, monocytes and eosinophils in the recipient monkey increased, while platelet count and fibrinogen levels decreased. The activated partial thromboplastin time, thrombin time and prothrombin time remained relatively stable but later showed an upward trend. The recipient monkey survived for 10 days. At autopsy, the transplanted kidney was found to be congested, swollen and necrotic, with a small amount of IgG deposition in the renal tissue, and a large amount of IgM, complement C3c and C4d deposition, as well as CD68⁺ macrophage infiltration. Conclusions The kidneys of GTKO Diannan miniature pigs may maintain normal renal function for a certain period in rhesus macaques and effectively overcome HAR, confirming the effectiveness of GTKO pigs for xenotransplantation.

ObjectiveTo study the potential quality marker （Q-marker） of Tinosporae Radix associated with efficacy of "relieving sore throat" based on ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-Q-TOF-MS）， multivariate statistical analysis （MSA）， and network pharmacology. MethodUPLC-Q-TOF-MS was used to identify the main chemical components in 18 batches of Tinosporae Radix. On this basis， principal component analysis （PCA） and orthogonal partial least squares-discriminant analysis （OPLS-DA） were employed to screen out the main marker components that caused differences between groups. Moreover， network pharmacology technology was applied to predict the potential "sore throat-relieving" components， and the molecular docking between the common components resulting from MSA and network pharmacology and the core targets was carried out to verify the marker components. ResultA total of 17 compounds， including alkaloids， diterpenoid lactones， and sterols， were identified by UPLC-Q-TOF-MS. Five main differential components were found by MSA： Columbamine， jatrorrhizine， palmatine， menisperine， and columbin. Network pharmacology analysis yielded six compounds： tetrahydropalmatine， palmatine， menisperine， fibleucin， neoechinulin A， and columbin which were selected as potential "sore throat-relieving" components of Tinosporae Radix. They may relieve sore throat by acting on interleukin-6， epidermal growth factor receptor， prostaglandin G/H synthase 2， matrix metalloproteinase-9， proto-oncogene tyrosine-protein kinase Src and other targets， and regulating Hepatitis B， influenza A， human T-cell virus infection， human cytomegalovirus infection， coronavirus disease-2019， and other signaling pathways. The common active components in Tinosporae Radix resulting from MSA and network pharmacology analysis were palmatine， menisperine， and columbin， which had high binding affinity with six core targets and can be used as the Q-marker components of Tinosporae Radix in "relieving sore throat". ConclusionThis study predicts the "sore throat-relieving" Q-marker of Tinosporae Radix， which lays a basis for developing the quality standard of Tinosporae Radix based on the efficacy and improving the quality evaluation system of the medicinal.

Objective:To analyze the diagnostic and prognostic values of the red blood cell distribution width-to-platelet count ratio (RPR) for hepatitis B and liver cirrhosis.Methods:The clinical data of 80 patients with hepatitis B and liver cirrhosis who were diagnosed and treated in Yiwu Central Hospital from June 2020 to August 2021 were retrospectively analyzed. These patients were included in the hepatitis B and liver cirrhosis group. They were subdivided into survival ( n = 69) and death ( n = 11) groups according to their prognosis outcomes. Eighty patients with chronic hepatitis B were included in the chronic hepatitis B group. Eighty healthy controls who concurrently underwent physical examination were included in the control group. The diagnostic and prognostic values of RPR, aspartate aminotransferase-to-platelet ratio index (APRI), and fibrosis index based on four factors (FIB-4) for hepatitis B and liver cirrhosis were analyzed. Results:Red blood cell distribution width, alanine transaminase, and aspartate transaminase in the hepatitis B and liver cirrhosis group and chronic hepatitis B group were significantly higher compared with the control group (all P < 0.05). Platelet count in the hepatitis B and liver cirrhosis group and chronic hepatitis B group was significantly lower than that in the control group (both P < 0.05). Red blood cell distribution width in the hepatitis B and liver cirrhosis group was significantly higher than that in the chronic hepatitis B group [(18.25 ± 3.28)% vs. (14.67 ± 2.15)%, t = 8.16, P < 0.05]. Platelet count, alanine transaminase, and aspartate transaminase levels in the hepatitis B and liver cirrhosis group were (78.47 ± 11.43) × 10 9/L, (49.48 ± 6.85) U/L, (45.86 ± 6.28) U/L, respectively, which were significantly lower than (133.36 ± 18.42) × 10 9/L, (128.36 ± 15.40) U/L, (98.67 ± 14.41) U/L in the chronic hepatitis B group ( t = -22.65, -41.86, -30.05, all P < 0.05). PRP, APRI, and FIB-4 in the hepatitis B and liver cirrhosis group were (0.23 ± 0.05), (1.85 ± 0.44), (4.25 ± 0.81) respectively, which were significantly higher than (0.11 ± 0.02), (1.46 ± 0.33), (3.38 ± 0.63) in the chronic hepatitis B group ( t = 19.93, 6.34, 7.58, all P < 0.001). The RPR, APRI, and FIB-4 in the death group were (0.25 ± 0.08), (1.97 ± 0.48), (4.52 ± 1.31), respectively, which were significantly higher than (0.18 ± 0.05), (1.68 ± 0.40), (3.69 ± 1.21) in the survival group ( t = 3.94, 2.17, 2.09, all P < 0.05). The receiver operating characteristic curve revealed that PRP has an extremely high value in diagnosing hepatitis B and liver cirrhosis and predicting the death of patients with hepatitis B and liver cirrhosis. Conclusion:RPR has an extremely high value in diagnosing hepatitis B and liver cirrhosis and predicting the prognosis of this disease.

Metabolic-associated fatty liver disease (MAFLD), which is previously known as non-alcoholic fatty liver disease (NAFLD), represents a major health concern worldwide with limited therapy. Here, we provide evidence that ferroptosis, a novel form of regulated cell death characterized by iron-driven lipid peroxidation, was comprehensively activated in liver tissues from MAFLD patients. The canonical-GPX4 (cGPX4), which is the most important negative controller of ferroptosis, is downregulated at protein but not mRNA level. Interestingly, a non-canonical GPX4 transcript-variant is induced (inducible-GPX4, iGPX4) in MAFLD condition. The high fat-fructose/sucrose diet (HFFD) and methionine/choline-deficient diet (MCD)-induced MAFLD pathologies, including hepatocellular ballooning, steatohepatitis and fibrosis, were attenuated and aggravated, respectively, in cGPX4-and iGPX4-knockin mice. cGPX4 and iGPX4 isoforms also displayed opposing effects on oxidative stress and ferroptosis in hepatocytes. Knockdown of iGPX4 by siRNA alleviated lipid stress, ferroptosis and cell injury. Mechanistically, the triggered iGPX4 interacts with cGPX4 to facilitate the transformation of cGPX4 from enzymatic-active monomer to enzymatic-inactive oligomers upon lipid stress, and thus promotes ferroptosis. Co-immunoprecipitation and nano LC-MS/MS analyses confirmed the interaction between iGPX4 and cGPX4. Our results reveal a detrimental role of non-canonical GPX4 isoform in ferroptosis, and indicate selectively targeting iGPX4 may be a promising therapeutic strategy for MAFLD.

Objective:To evaluate the effect of self-made lithotomy heating mask on intraoperative and postoperative body temperature and short-term postoperative outcome indicators in patients undergoing radical resection of rectal cancer.Methods:Using the method of quasi experimental research design, 100 patients with open rectal cancer in Ningbo Huamei Hospital of the University of Chinese Academy of Sciences from February to July 2021 were selected as the research objects. The patients were divided into experimental group and control group with 50 cases in each group. The control group was kept warm by routine methods, and the experimental group was kept warm by self-made lithotomy heating hood. The changes of core temperature at different time points before, during and after operation were compared between the two groups. The incidence of accidental hypothermia and shivering, the recovery time of anesthesia, and the incidence of various complications within 48 hours after operation were compared between the two groups after operation from the beginning of the operation to 6 hours after returning to the ward.Results:From 30 minutes after anesthesia to 3 hours after entering the ward, the core temperatures of the experimental group at 10 time points were higher than that of the control group, and the differences were statistically significant ( t values were 3.48-37.30, all P<0.01). From the beginning of surgery to 6 h after returning to the ward, the incidence of perioperative accidental hypothermia in the experimental group was 2% (1/50), lower than 24% (12/50) in the control group, and the difference was statistically significant ( χ2=11.06, P<0.05) . The number of cases of shivering in the experimental group was 10, lower than that in the control group of 22, the difference was statistically significant ( χ2=6.62, P<0.05) . The recovery time, extubation time and stay time in anesthesia recovery room of the experimental group were (8.44 ± 2.83), (13.05 ± 4.72), (74.51 ± 11.82) min, which were shorter than those of the control group (15.35 ± 2.09), (17.62 ± 3.28), (89.14 ± 9.19) min, and the difference was statistically significant ( t=-13.89, -5.62, -6.91, all P<0.01). The number of cases of agitation, delirium and nausea and vomiting in the experimental group was 3, 1 and 2 respectively, which was lower than 13, 7 and 8 in the control group, and the difference were statistically significant ( χ2=7.44, 4.89, 4.00, all P<0.05). There was no significant difference in the incidence of adverse cardiac events between the two groups ( P>0.05). Conclusions:The application of self-made lithotomy heating mask in open rectal cancer surgery can effectively improve the risk of hypothermia at different time points during and after surgery, reduce the incidence of shivering, restlessness, postoperative nausea, vomiting and delirium, shorten the time of awakening and extubation, and prevent postoperative complications. It has practical value in clinic.

Objective: To investigate the application of low molecular weight heparin modified injection in the nursing of patients with acute myocardial infarction. Methods: A total of 90 patients with myocardial infarction who underwent subcutaneous injection of low molecular weight heparin from May 2017 to May 2018 were enrolled in the study. The patients were divided into the control group and the observation group according to the different injection and treatment methods. The control group used the traditional injection method of low molecular weight heparin, and the observation group used the low molecular weight heparin modified injection method. Forty-five patients were observed and compared for the degree of subcutaneous hemorrhage, incidence of induration and pain. Results: The incidence of subcutaneous induration and subcutaneous hemorrhage in the observation group were 10.16% (64/630) and 19.84% (125/630), respectively. The control group was 16.19% (102/630) and 31.11% (196/630), respectively. The difference between the groups was statistically significant (χ²=10.019, 21.073, both P<0.05). The pain score of the observation group was 1.29±0.21, and the control group was 1.86±0.28. The difference between the two groups was statistically significant (t=40.877, P<0.05); the subcutaneous mild, moderate, and severe bleeding in the observation group were 17.30% (109/630), 2.54% (16/630), and 0, respectively, and the control group was 21.90% (138/). 630), 8.41% (53/630), 0.79% (5/630), the difference between the two groups was statistically significant (χ²=4.235, 20.990, 4.019, all P < 0.05). Conclusions Low molecular weight heparin modified injection method for myocardial infarction patients can effectively reduce the incidence of subcutaneous hemorrhage and induration, reduce the degree of subcutaneous hemorrhage and pain, suitable for clinical promotion.

Objective To study the resting-state functional connectivity( FC) changes of anterior cingulate cortex(ACC) and amygdala in adolescent patients with medication-naive first-episode major de-pressive disorder(MDD).Methods Medication-naive first-episode MDD adolescents( n＝30) and pair-wise matched healthy controls(HCs,n＝29) underwent resting-state functional magnetic resonance images(fM-RI).To study whether there were different in FC changes between amygdala and ACC and whole brain re-gions,and whether abnormal FC was related to clinical characteristics.Results Compared with HCs,the ado-lescent MDD patients showed decreased FC between the left amygdala and right thalamus( MNI x,y,z:15,－21,18)(t＝－2.85)and increased FC between the left amygdala and the bilateral temporal middle gyrus,left occipital gyrus,right medial prefrontal cortex(MPFC)(MNI x,y,z:9,66,15) ( t＝2.80).The right amygdale showed decreased FC with bilateral middle frontal gyrus,left caudate(MNI x,y,z:－15,－9,21)(t＝－2.53), right thalamus(MNI x,y,z:12,－36,12)(t＝－3.01),right cerebellum(MNI x,y,z:3,－84,－33)(t＝－2.45) and increased FC with right temporal middle gyrus,right MPFC( MNI x,y,z:6,69,12) ( t＝2.41).The left ACC showed decreased FC with left cerebellum( MNI x,y,z:12,－87,－24) ( t＝－2.36),left frontal inferior triangle gyrus,right frontal inferior orbital gyrus and increased FC with right frontal middle orbital gyrus.The right ACC showed decreased FC with left caudate(MNI x,y,z:－15,21,6)(t＝－2.29),right temporal middle gyrus,right MPFC(MNI x,y,z:9,54,36)(t＝－2.29),right frontal inferior orbital gyrus,right angular(MNI x,y,z:45,－51,21)(t＝－3.01),left dorsolateral prefrontal cortex(MNI x,y,z:－21,33,36) ( t＝－2.61).The increased FC between the left amygdale and bilateral temporal middle gyrus showed positive correlation with the score of HAMD(r＝0.50,P＜0.05;r＝0.45,P＜0.05).And the FC between the right amygdale and right middle frontal gyrus was positive correlation with duration of disease( r＝0.63,P＜0.05).Conclusion There is widespread FC disorder in amygdala and ACC in adolescents patients with first-episode depression.Abnor-mal FC changes may be the neuropathological mechanism of depression.

Objective To investigate the diagnostic value of the partial least square discriminant analysis (PLS-DA) model based on seven serum cytokines for children patients with mycoplasma pneumonia,the 7 cytokines includ interleukin-2 (IL-2),IL-4,IL-6,IL-8,IL-10,tumor necrosis factor alpha (TNF-α) and interferonγ(IFN-γ).Methods Serum levels of cytokines were measured by the double antibody sandwich method of ELI-SA in 140 patients with pneumoniae infection and 135 normal healthy controls,and data was analyzed with the receiver operation characteristic (ROC) curve and the PLS-DA.Results The area under the ROC curve (AUC) of serum IL-10 and IFN-γfor the diagnosis of MPP was 0.84 (95％ CL:0.79 ～ 0.89).The sensitivity of IL-10 and IFN-γ for the diagnosis of MPP was 91.4％ and 82.1％,with the specificity was 77.0％ and 82.2％,respectively.The diagnostic accuracy of the PLS-DA model based on seven serum cytokines for children MPP and the controls was 90.0％ and 88.15％,and the prediction accuracy was 86.4％ and 87.4％,respectively.Conclusion Seven serum cytokines based on PLS-DA model was helpful for the diagnosis of children patients with MPP.