OBJECTIVE: To prepare decellularized nerve grafts from alpha-1, 3-galactosyltransferase (GGTA1) gene-edited pigs and explore their biocompatibility for xenotransplantation. METHODS: The sciatic nerves from wild-type pigs and GGTA1 gene-edited pigs were obtained and underwent decellularization. The alpha-galactosidase (α-gal) content in the sciatic nerves of GGTA1 gene-edited pigs was detected by using IB4 fluorescence staining and ELISA method to verify the knockout status of the GGTA1 gene, and using human sciatic nerve as a control. HE staining and scanning electron microscopy observation were used to observe the structure of the nerve samples. Immunofluorescence staining and DNA content determination were used to evaluate the degree of decellularization of the nerve samples. Fourteen nude mice were taken, and subcutaneous capsules were prepared on both sides of the spine. Decellularized nerve samples of wild-type pigs ( n=7) and GGTA1 gene-edited pigs ( n=7) were randomly implanted in the subcutaneous capsules. Blood was drawn at 1, 3, 5, and 7 days after implantation to detect neutrophil counting. RESULTS: IB4 fluorescence staining and ELISA detection showed that GGTA1 gene was successfully knocked out in the nerves of GGTA1 gene-edited pigs. HE staining showed that the structure of the decellularized nerve from GGTA1 gene-edited pigs was well preserved; the nerve basement membrane tube structure was visible under scanning electron microscopy; no cell nuclei was observed, and the extracellular matrix components was retained in the nerve grafts by immunofluorescence staining; and the DNA content was significantly reduced when compared with the normal nerves ( P<0.05). In vivo experiments showed that the number of neutrophils in the two groups were similar at 1, 3, and 7 days after implantation, with no significant difference ( P>0.05); only at 5 days, the number of neutrophils was significantly lower in the GGTA1 gene-edited pigs than in the wild-type pigs ( P<0.05). CONCLUSION The decellularized nerve grafts from GGTA1 gene-edited pigs have well-preserved nerve structure, complete decellularization, retain the natural nerve basement membrane tube structure and components, and low immune response after xenotransplantation through in vitro experiments.
Animals ; Transplantation, Heterologous ; Galactosyltransferases/genetics* ; Sciatic Nerve/immunology* ; Swine ; Tissue Engineering/methods* ; Humans ; Graft Rejection/prevention & control* ; Gene Editing ; Mice ; Mice, Nude ; Heterografts/immunology* ; Animals, Genetically Modified ; Tissue Scaffolds ; Decellularized Extracellular Matrix

Objective:To investigate and summarize the imaging and pathological features of non-acute occlusion following flow diverter (FD) implantation in animal models.Methods:Four experimental pigs (experimental group) that experienced non-acute occlusion (occlusion time exceeding 24 hours) within the FD stent implanted in the common carotid artery, and 19 pigs (control group) that did not experience stent occlusion during the same period were involved. Using an interventional approach under digital subtraction angiography (DSA), the 4 occluded FD lumens were mechanically opened. Optical coherence tomography (OCT), intravascular ultrasound (IVUS) and histopathological examinations were performed to evaluate the intraluminal composition and characteristics of the occlusive tissues. These findings were compared with non-occluded FD stents to summarize the imaging and pathological changes within the occluded FD lumen.Results:The occlusion times of the FD stents in the 4 experimental pigs were 16 weeks, 20 weeks, 20 weeks, and 24 weeks postoperatively. All occluded stents were successfully recanalized under DSA, with a technical success rate of 4/4. Among the 19 non-occluded FD stents, OCT and IVUS revealed uniform (16 stents) or non-uniform (3 stents) neointimal coverage of the stent struts, presenting as homogeneous high/slightly high signal intensity or medium echogenicity. Histopathological examination indicated that the neointima was primarily composed of smooth muscle cells and a small amount of fibrous connective tissues. In contrast, the 4 occluded FD stents demonstrated excessive neointimal proliferation and plaque formation, leading to luminal loss, as shown by OCT and IVUS. The occlusion tissues predominantly presented as homogeneous high signal intensity with weak attenuation (fibrous plaques) on OCT, with some regions showing blurred low signal intensity and strong attenuation (lipid plaques). IVUS presented homogeneous echogenicity (fibrous plaques) and hypoechogenic zones (lipid plaques). Histopathological examination showed that the occlusion tissues mainly consisted of smooth muscle cells, fibrous connective tissues, and lipids, accompanied by numerous foam cells and a minor presence of inflammatory cells.Conclusions:Histopathological examinations confirm that non-acute occlusion of FD is mainly caused by excessive hyperplasia of intima along with the formation of fibrous plaques and lipid plaques. OCT and IVUS have typical finding in imaging that can assist in determining the cause of stent occlusion as well as the lesion's nature, thereby providing crucial guidance for subsequent clinical treatment and drug selection.

Objective:To investigate the expression of indoleamine 2,3-dioxygenase 1(IDO1)in different types of breast cancer tissues and its relationship with patient prognosis and immune cell infiltration.Methods:RNA sequencing data of breast cancer and corresponding clinical information from the TCGA database were collected.The differential expression of IDO1 mRNA in various breast cancer tissues(different subtypes,stages,menopause statuses,and age groups)and adjacent normal tissues were analyzed.Breast cancer patients with significant differences in IDO1 mRNA expression were divided into high and low expression groups,and their disease-specific survival(DSS)was compared between the two groups.The relationship between IDO1 mRNA expression and immune cell infiltration in cancer tissues with significant DSS differences was analyzed.Immunohistochemistry was used to detect IDO1 protein expression in ER-negative,PR-negative,HER2-positive,and stage Ⅱ breast cancer tissues,to verify the data from the database.Results:IDO1 mRNA was highly expressed in breast cancer tissues but varied across different breast cancer types.IDO1 mRNA was highly expressed in breast cancer tissues of patients with ER-negative,PR-negative,HER2-positive,HER2-negative subtypes,stage Ⅱ,T2 stage,N0 stage,M0 stage,premenopausal,postmenopausal,and age≤60 years(P<0.05 or P<0.01,or P<0.001).In the ER-negative,PR-negative,HER2-positive,and stage Ⅱ subgroups,breast cancer tissues patients with high IDO1 mRNA expression had significantly higher DSS than those with low expression(P<0.05 or P<0.01).In ER-negative,PR-negative,HER2-positive,and stageⅡ breast cancer tissues,IDO1 mRNA expression was associated with immune cell infiltration,including activated dendritic cells(aDCs),Th1 cells,T cells,CD56dim NK cells,CTLs,and Treg cells(all P<0.001).IDO1 protein was highly expressed in ER-negative,PR-negative,HER2-positive,and stage Ⅱ breast cancer tissues(all P<0.001),consistent with the data from the database.Conclusion:IDO1 expression varies across different types of breast cancer tissues.The expression of IDO1 is associated with the prognosis and immune cell infiltration in ER-negative,PR-negative,HER2-positive,and at stage Ⅱ breast cancer patients.

Objective:To investigate and summarize the imaging and pathological features of non-acute occlusion following flow diverter (FD) implantation in animal models.Methods:Four experimental pigs (experimental group) that experienced non-acute occlusion (occlusion time exceeding 24 hours) within the FD stent implanted in the common carotid artery, and 19 pigs (control group) that did not experience stent occlusion during the same period were involved. Using an interventional approach under digital subtraction angiography (DSA), the 4 occluded FD lumens were mechanically opened. Optical coherence tomography (OCT), intravascular ultrasound (IVUS) and histopathological examinations were performed to evaluate the intraluminal composition and characteristics of the occlusive tissues. These findings were compared with non-occluded FD stents to summarize the imaging and pathological changes within the occluded FD lumen.Results:The occlusion times of the FD stents in the 4 experimental pigs were 16 weeks, 20 weeks, 20 weeks, and 24 weeks postoperatively. All occluded stents were successfully recanalized under DSA, with a technical success rate of 4/4. Among the 19 non-occluded FD stents, OCT and IVUS revealed uniform (16 stents) or non-uniform (3 stents) neointimal coverage of the stent struts, presenting as homogeneous high/slightly high signal intensity or medium echogenicity. Histopathological examination indicated that the neointima was primarily composed of smooth muscle cells and a small amount of fibrous connective tissues. In contrast, the 4 occluded FD stents demonstrated excessive neointimal proliferation and plaque formation, leading to luminal loss, as shown by OCT and IVUS. The occlusion tissues predominantly presented as homogeneous high signal intensity with weak attenuation (fibrous plaques) on OCT, with some regions showing blurred low signal intensity and strong attenuation (lipid plaques). IVUS presented homogeneous echogenicity (fibrous plaques) and hypoechogenic zones (lipid plaques). Histopathological examination showed that the occlusion tissues mainly consisted of smooth muscle cells, fibrous connective tissues, and lipids, accompanied by numerous foam cells and a minor presence of inflammatory cells.Conclusions:Histopathological examinations confirm that non-acute occlusion of FD is mainly caused by excessive hyperplasia of intima along with the formation of fibrous plaques and lipid plaques. OCT and IVUS have typical finding in imaging that can assist in determining the cause of stent occlusion as well as the lesion's nature, thereby providing crucial guidance for subsequent clinical treatment and drug selection.

ObjectiveTo investigate the effect of phytoestrogen biochanin A （BCA） on liver fibrosis induced by CCl₄ in female mice with bilateral oophorectomy （ovariectomized） and its mechanism. MethodsA total of 50 ovariectomized Kunming mice were selected and given intraperitoneal injection of CCl₄ to establish a model of liver fibrosis， and then according to body weight， they were randomly divided into model group， positive control group， and low-， middle-， and high-dose BCA groups， with 10 mice in each group. In addition， 10 female mice in the same litter were given resection of a small amount of adipose tissue near both ovaries to establish the sham-operation group. The mice in the positive control group were given estradiol 2 mg/kg by gavage， and those in the low-， middle-， and high-dose BCA groups were given BCA by gavage at a dose of 25， 50， and 100 mg/kg， respectively， once a day for 7 consecutive weeks； the mice in the sham-operation group and the model group were given an equal volume of 0.5% sodium carboxymethyl cellulose solution by gavage. The mice were anesthetized and sacrificed after administration to collect samples. Liver index and uterus index were measured； HE staining and Masson staining were used to observe liver histopathological changes； the biochemical analysis was used to measure the activity of aspartate aminotransferase （AST） and alanine aminotransferase （ALT）； ELISA was used to measure the levels of interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） in liver tissue， and Western blot was used to measure the relative protein expression levels of collagen Ⅰ， transforming growth factor-beta 1 （TGF-β₁）， alpha-smooth muscle actin （α-SMA）， estrogen receptor beta （ERβ）， and p-NF-κBp65/NF-κBp65 in liver tissue. The t-test was used for comparison of continuous data between two groups； a one-way analysis of various was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. The Kruskal-Wallis H test was used for comparison of non-normally distributed continuous data between multiple groups and further comparison between two groups. ResultsCompared with the sham-operated group， the model group had a significant increase in liver index and a significant reduction in uterus index， as well as significant increases in the activities of serum AST and ALT， the levels of IL-6 and TNF-α in liver tissue， and the protein expression levels of collagen Ⅰ， TGF-β₁， α-SMA， and p-NF-κBp65/NF-κBp65 in liver tissue （all P<0.05）， with no significant change in the expression of ERβ in liver tissue （P>0.05）， and the model group showed significant fibrosis lesions in the liver， such as hepatocyte edema， steatosis， and necrosis with inflammatory cell infiltration and hyperplasia， deposition， and staggered distribution of collagen fibers. Compared with the model group， the low-， middle-， and high-dose BCA groups had significant reductions in liver index， the activities of serum AST and ALT， the levels of IL-6 and TNF-α， and the protein expression levels of collagen Ⅰ， TGF-β₁， α-SMA， and p-NF-κBp65/NF-κBp65 in liver tissue （all P<0.05）， with no significant change in uterine index （P>0.05）， as well as a significant increase in the protein expression level of ERβ in liver tissue （P<0.05） and varying degrees of improvement in liver fibrosis lesions. ConclusionBCA can effectively improve CCL₄-induced liver fibrosis in ovariectomized female mice， possibly by upregulating ERβ to inhibit the NF-κB signaling pathway and then alleviating inflammatory response.

BACKGROUND:At present,many drugs used in the treatment of polycystic ovary syndrome are super-designated drugs,and the treatment of patients with polycystic ovary syndrome still faces great challenges.Studies have shown that human umbilical cord mesenchymal stem cells can repair ovarian function,but few studies have reported their therapeutic effect on polycystic ovary syndrome. OBJECTIVE:To investigate the therapeutic effect of human umbilical cord mesenchymal stem cells on polycystic ovary syndrome,and to preliminarily explore the correlation between mitochondrial autophagy and the improvement of polycystic ovary syndrome by human umbilical cord mesenchymal stem cells. METHODS:Polycystic ovary syndrome mouse model was established by subcutaneous injection of dehydroepiandrosterone for 20 days into C57BL/6J mice.Human umbilical cord mesenchymal stem cells(2×106)were injected through the caudal vein.After treatment,vaginal secretions were collected for 10 consecutive days to detect the estrus cycle of mice.At 2 weeks after treatment,the levels of sex hormones in the peripheral blood of mice,including luteinizing hormone and follicle-stimulating hormone,were detected by ELISA.Hematoxylin-eosin staining was used to evaluate ovarian histopathology.Finally,mitochondrial autophagy in ovaries was observed by transmission electron microscopy. RESULTS AND CONCLUSION:(1)After human umbilical cord mesenchymal stem cell therapy,follicles at different stages(primitive follicles,primary follicles,and secondary follicles)appeared in the ovary of polycystic ovary syndrome mice,and luteal tissue could be seen,indicating that ovulation function of mice was effectively improved.(2)Polycystic ovary syndrome mice treated with human umbilical cord mesenchymal stem cells had sex hormone levels.(3)Untreated polycystic ovary syndrome mice were found to be in the estrous stage for a long time,lacking estrous interphase and estrous phase,but after human umbilical cord mesenchymal stem cell therapy,the estrous cycle returned to a normal level.(4)After treatment with human umbilical cord mesenchymal stem cells,the mitochondrial autophagy of polycystic ovary syndrome mice was significantly reduced.(5)The results show that human umbilical cord mesenchymal stem cells can effectively improve the symptoms of endocrine disorders and promote ovulation in polycystic ovary syndrome mice,which may be related to the inhibition of mitochondrial autophagy.

Non-alcoholic fatty liver disease is a common chronic liver disease in clinical practice. In recent years, with increasing social attention to the health of women and the elderly, the prevention and treatment of non-alcoholic fatty liver disease after menopause has increasingly become a research hotspot in metabolic diseases. This study explores the pathogenesis and treatment method of non-alcoholic fatty liver disease in postmenopausal women based on the theory of "deficient qi and stagnation" and combined with the physiological and pathological characteristics of postmenopausal women and the Western medicine understanding of non-alcoholic fatty liver disease. We believe that the pathogenesis of non-alcoholic fatty liver disease in postmenopausal women is rooted in the "deficient qi" caused by depletion of liver and kidney essence and blood. The imbalance between the physical and functional aspects of the liver due to this "deficient qi" is the primary factor, while the "stagnation" of phlegm and blood stasis is the manifestation. Furthermore, the "deficient qi" and "stagnation" reinforce each other, with the deficiency leading to stagnation and stagnation exacerbating the deficiency, thus accelerating the progression of the disease. The treatment approach should be one that combines nourishing deficiency and resolving stagnation, addressing both root cause and maifestations. Given the female characteristic of "the liver as the innate organ" and the post-menopausal physiological state of "gradual decline of kidney essence", it is important to focus on nourishing the liver and kidneys, nurturing the liver′s physical body while maintaining its function, and also promoting the circulation of qi, resolving phlegm, and invigorating blood circulation to remove blood stasis. This approach aims to reduce the accumulation of lipids in the liver, offering a new perspective and approach for the treatment of non-alcoholic fatty liver disease in post-menopausal women with traditional Chinese medicine.

The clinical data, laboratory test, and gene mutations were collected from a family with pseudohypoaldosteronism type II(PHA2). The proband, aged 1 year and 7 months, presented with hyperkalemia(6.69 mmol/L; reference range 3.5-5.3 mmol/L), blood pressure of 110/68 mmHg(normal<106/61 mmHg, 1 mmHg=0.133 kPa), blood chloride of 111.5 mmol/L(reference 99-110 mmol/L), blood HCO 3- of 17.1 mmol/L(reference 22-29 mmol/L), estimated glomerular filtration rate of 128.5 mL·min -1·(1.73 m 2) -1[>90 mL·min -1·(1.73 m 2) -1], and blood renin concentration of 0.30 μIU/mL(reference 4.2-45.6 μIU/mL). The mother and maternal grandfather also exhibited normal renal function with hyperkalemia, hypertension, hyperchloremia, metabolic acidosis, and low renin. Genetic testing revealed a heterozygous missense mutation(c.1685A>G, p. E562G) in exon 7 of the no-lysine kinase 4(WNK4) gene. Treatment with hydrochlorothiazide was effective. Literature review comparing this E562G pedigree with other WNK4 variants suggested clinical heterogeneity of WNK4 mutations. For unexplained hyperkalemia, especially with concurrent hypertension, PHA2 should be considered early for genetic screening to prevent misdiagnosis or delayed diagnosis.

OBJECTIVE To study the protective effects of ligustrazine-scutellarein twin drug ST-11 on rat adrenal medullary pheochromocytoma PC12 cell injury induced by oxygen-glucose deprivation/reperfusion （OGD/R） and its mechanism. METHODS PC12 cells were divided into blank group， model group， nimodipine group （positive control， 5 μmol/L） and different concentration groups of ST-11 （5， 10， 20 μmol/L）. After 24 hours of pre-administration intervention， all the other groups except the blank group were cultured in glucose-free DMEM culture medium containing 10 mmol/L Na2S2O4 for 4 hours with glucose deficiency and hypoxia. After 4 hours of glucose and oxygen re-introduction， the survival rate of cells in each group， the contents of lactate dehydrogenase （LDH）， catalase （CAT）， glutathione （GSH）， malondialdehyde （MDA） and superoxide dismutase （SOD） in cell supernatant， apoptosis rate， the levels of reactive oxygen species （ROS） and mitochondrial membrane potential （MMP）， the protein expressions of B-cell lymphoma 2 related X protein （Bax）， B-cell lymphoma 2 （Bcl-2） and caspase-3 were all detected in each group. RESULTS Compared with blank group， the cell survival rate， the contents of CAT， GSH and SOD in cell supernatant， MMP level， relative expression of Bcl-2 and Bcl-2/Bax ratio in model group decreased significantly （P＜0.05）， while the contents of LDH and MDA， ROS level， apoptosis rate， relative expressions of Bax and caspase-3 were significantly increased （P＜0.05）. Compared with model group， above indexes of ST-11 groups （except for the protein expression of caspase-3 in 5 μmol/L ST-11 group） were reversed signifi-cantly （P＜0.05）. CONCLUSIONS ST-11 has a certain protec-tive effect on OGD/R-injured PC12 cells， and its effects may be related to reduction of oxidative stress and inhibition of cell apoptosis.

Objective:To explore any effect of following an individualized exercise program on the cardio-pulmonary health of elderly persons with stable chronic obstructive pulmonary disease (COPD).Methods:A total of 120 elderly COPD patients were randomly divided into an experimental group and a control group, each of 60. Both groups were given medication and routine rehabilitation treatment for 3 months, but the experimental group also followed an individualized exercise program based on the cardiopulmonary exercise test. Before and after the treatment, Borg scoring was used to assess the subjects′ degree of dyspnea. Forced expiratory volume (FEV 1), forced vital capacity (FVC), maximum oxygen uptake (VO 2max), anaerobic threshold (AT), heart rate (HR) and a heart index (CI) were also recorded. The 6-minute walk test (6 MWD) was administered to evaluate the rehabilitation effect. Results:There were no significant differences in the average Borg scores, cardiopulmonary function indexes or 6MWD distances between the two groups before treatment. After the treatment significant improvement was observed in all of the measurements in both groups, but at that point all of the experimental group′s averages were better than those of the control group.Conclusion:Individualized exercise based on the cardiopulmonary exercise test can alleviate the symptoms of dyspnea in elderly COPD patients, improve their cardiopulmonary functioning and exercise endurance and improve the rehabilitation effect.