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Cognitive impairment caused by chronic cerebral hypoperfusion (CCH) is associated with white matter injury (WMI), possibly through the alteration of autophagy. Here, the autophagy-lysosomal pathway (ALP) dysfunction in white matter (WM) and its relationship with cognitive impairment were investigated in rats subjected to two vessel occlusion (2VO). The results showed that cognitive impairment occurred by the 28th day after 2VO. Injury and autophagy activation of mature oligodendrocytes and neuronal axons sequentially occurred in WM by the 3rd day. By the 14th day, abnormal accumulation of autophagy substrate, lysosomal dysfunction, and the activation of mechanistic target of rapamycin (MTOR) pathway were observed in WM, paralleled with mature oligodendrocyte death. This indicates autophagy activation was followed by ALP dysfunction caused by autophagy inhibition or lysosomal dysfunction. To target the ALP dysfunction, enhanced autophagy by systemic rapamycin treatment or overexpression of Beclin1 (BECN1) in oligodendrocytes reduced mature oligodendrocyte death, and subsequently alleviated the WMI and cognitive impairment after CCH. These results reveal that early autophagy activation was followed by ALP dysfunction in WM after 2VO, which was associated with the aggravation of WMI and cognitive impairment. This study highlights that alleviating ALP dysfunction by enhancing oligodendrocyte autophagy has benefits for cognitive recovery after CCH.
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OBJE CTIVE To establish the finger prints for Yinhuang solution for inhalation and determine the contents of neochlorogenic acid ，chlorogenic acid and cryptochlorogenic acid simultaneously. METHODS Using baicalin as reference ，the fingerprints of Yinhuang solution for inhalation were established by high performance liquid chromatography （HPLC）. Relative correction factors of neochlorogenic acid and cryptochlorogenic acid were calculated by slope correction method ，using chlorogenic acid as reference ；the contents of them were calculated according to relative correction factor. The results of quantitative analysis of multi-components by single marker （QAMS）were compared with those of external standard method （ESM）. RESULTS There were 18 common peaks in the fingerprints of 10 batches of Yinhuang solution for inhalation ，and their similarities with reference fingerprint were higher than 0.90. A total of 7 common peaks were identified as baicalin ，neochlorogenic acid ，chlorogenic acid ， cryptochlorogenic acid ，isochlorogenic acid B ，3，5-di-O-caffeoylquinic acid and 4，5-di-O-caffeoylquinic acid. The linear range of neochlorogenic acid ，chlorogenic acid and cryptochlorogenic acid were 0.025 0-1.247 4 μg（r＝0.999 7），0.039 3-1.178 7 μg（r＝ 0.999 9），0.031 6-1.184 1 μg（r＝0.999 9），respectively. RSDs of precision ，reproducibility and stability tests （48 h）were all lower than 1.0％. The average recoveries were 93.92％（RSD＝1.32％ ，n＝6），94.46％（RSD＝1.45％，n＝6），93.93％（RSD＝ 1.57％，n＝6）. Relative correction factors of neochlorogenic acid and cryptochlorogenic acid were 1.068 and 1.233. The contents of neochlorogenic acid and cryptochlorogenic acid determined by QAMS method were 0.301 8-0.386 3 and 0.262 5-0.362 5 mg/mL， respectively. The contents of neochlorogenic acid ，chlorogenic acid and cryptochlorogenic acid by ESM were 0.302 6-0.387 2， 0.231 0- 0.334 0，0.261 6-0.361 3 mg/mL，respectively. The deviations of the content determination results of the two methods（except for chlorogenic acid ）were both not higher than 0.20％. CONCLUSIONS Established HPLC fingerprints are stable and feasible. Established QAMS method is accurate and rapid. HPLC fingerprint combined with QAMS can be used for the quality control for Yinhuang solution for inhalation ．
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Objective:To explore the diagnostic value of contrast-enhanced ultrasound combined with fine-needle aspiration biopsy and BRAF gene detection for TI-RADS category 4 nodules.Methods:The clinical datas of 80 patients who underwent surgery in the First Affiliated Hospital, Zhejiang University School of Medicine and Lishui People′s Hospital and diagnosed with TI-RADS 4 thyroid nodules from January 2019 to January 2020 were retrospectively analyzed. All patients received contrast-enhanced ultrasound combined fine-needle aspiration biopsy and BRAF gene detection, the ROC curves were plotted, the area under the ROC curve(AUC) and the best diagnostic cut-off values were calculated, and the application value of ultrasound-enhanced contrast, fine-needle aspiration biopsy and BRAF gene detection were compared.Results:Based on the results of pathological diagnosis, in diagnosing TI-RADS 4 thyroid nodules, the sensitivity, specificity and accuracy were 77.61%, 70.97% and 75.51% for contrast-enhanced ultrasound, respectively; 80.60%, 74.19%, and 78.57% for ultrasound-guided fine-needle aspiration biopsy, respectively; 79.10%, 96.77%, and 84.69% for the BRAF gene test, respectively; and 98.51%, 70.97% and 89.80% for the combined diagnosis, respectively. The AUC was 0.790 for contrast-enhanced ultrasound, and 0.774 for ultrasound-guided fine-needle aspiration biopsy, 0.799 for BRAF genetic testing, and 0.847 for combined testing. The diagnostic value of combined diagnosis was significantly higher than other diagnostic methods ( P<0.05). Conclusions:Contrast-enhanced ultrasound combined with fine-needle aspiration biopsy and BRAF gene detection is valuable for the diagnosis of TI-RADS 4 class thyroid nodules and improves the preperative diagnosis.
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Objective:To explore the value of constrast-enhanced ultrasound-micro flow imaging (CEUS-MFI) in the differential diagnosis of benign and malignant renal tumors.Methods:Totally 96 patients with renal space occupying found in two-dimensional gray-scale ultrasound examination in the First Affiliated Hospital, College of Medicine, Zhejiang University from November 2020 to August 2021 were collected, and 97 lesions were examined by CEUS-MFI and constrast-enhanced ultrasound(CEUS), respectively. The microvascular morphology and contrast-enhanced characteristics of renal tumors were recorded, ROC curves were constructed, and the diagnostic efficacies of the two methods were compared.Results:In the CEUS examination, the enhancement modes of malignant tumors were mainly fast wash-in(52/66, 78.8%) and fast wash-out (49/66, 74.2%) and high perfusion (56/66, 84.8%), and ring enhancement can be seen in 48.5%(32/66) of the lesions. The enhancement patterns of benign tumors were mainly slow wash-in (17/31, 54.8%) and slow wash-out (20/31, 64.5%) and low perfusion (18/31, 58.1%), and no circular blood flow was found in 31 lesions.In the CEUS-MFI examination, the vascular morphology of malignant tumors was mainly irregular (46/66, 69.7%), 93.9%(62/66) of malignant tumors had circular blood flow. Most of benign tumors were of linear vascular structure(12/31, 38.7%) and dendritic vascular structures (14/31, 45.2%), 93.5% (29/31) of benign tumors showed no circular blood flow. The detection rate of the annular blood flow in malignant tumors by CEUS-MFI was higher than that by CEUS, and the difference was statistically significant (93.9% vs 48.5%, P<0.001). The accuracy, sensitivity, and specificity of diagnosing renal tumors for using CEUS-MFI were 90.7%, 93.9% and 83.8%, respectively; and 84.5%, 92.4% and 67.7%, respectively, for using CEUS. The areas under the ROC curve were 0.898 and 0.814 for using CEUS-MFI and CEUS, respectively, the difference between the two techniques was not significant ( P=0.151). Conclusions:CEUS-MFI can sensitively and clearly display the microvascular morphology inside the tumor, and greatly improve the detection rate of annular blood flow in renal malignant tumors, which provides a new method for clinicians to identify benign and malignant renal tumors.
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Objective To observe the expression of mRNA of podocin,nephrin,CD2AP and α - actin - 4 in Doxorubicin - induced nephrotic(ADN)rats,and explore the possible mechanisms of podocyte molecule during the de-velopment of proteinuria. Methods Forty - eight Sprague - Dawley(SD)rats were divided into ADN model group(in-jected with 6. 5 mg/ kg Doxorubicin in tail vein,n = 24)and control group(injected with saline solution in tail vein,n =24). After the nephropathy model was established,6 rats were killed at the end of 1st ,2nd ,4th ,6th week in each group. The changes of the following indicators were observed:(1)24 - hour urinary protein,serum albumin and cholesterol were detected;(2)mRNA expression of nephrin,podocin,CD2AP and α - actin - 4 in cortex of kidney were examined by real time fluorescence quantification PCR. Results The model group came out massive proteinuria(15. 66 ± 1. 50) mg/ 24 h,(45. 98 ± 1. 45)mg/ 24 h,(65. 58 ± 4. 68)mg/ 24 h,(82. 83 ± 8. 43)mg/ 24 h in 1,2,4,6 weeks respec-tively,hypoalbuminemia(27. 4 ±2. 5)g/ L,(23. 6 ±2. 9)g/ L,(20. 6 ±1. 5)g/ L,(6. 9 ± 2. 3)g/ L in 1,2,4,6 weeks respectively and hypercholesterolaemia(2. 00 ± 0. 25)mmol/ L,(2. 16 ± 0. 44)mmol/ L,(4. 02 ± 0. 81)mmol/ L, (7. 54 ± 1. 12)mmol/ L in 1,2,4,6 weeks respectively,and the differences of proteinuria,plasma albumin and total cholesterol compared with control group at each time point had statistical significance(all P ﹤ 0. 01). Compared with the control group,podocin mRNA expression in the model group decreased at the end of 1st week(10. 56 ± 3. 62),de-creased significantly at the end of 2nd week(20. 44 ± 9. 03),and decreased at the end of 4th week(2. 19 ± 0. 18)com-pared with the control group;nephrin mRNA expression decreased at the end of 1st week(2. 41 ± 1. 10)and reached to the peak value,decreased at the end of 4th week(0. 52 ± 0. 18);CD2AP mRNA expression did not change significantly in the 1st week(4. 17 ± 0. 79),increased at the end of 2nd week(6. 74 ± 1. 53),reached to the peak value at the end of 4th week(6. 91 ± 1. 13),but did not change significantly at the end of 6th week(4. 04 ± 0. 82);α - actin - 4 mRNA ex-pression did not change significantly at the end of 1st week(1. 75 ± 0. 48),decreased at the end of 2nd week(2. 01 ± 0. 55),reached to the peak value at the end of 4th week(2. 24 ± 0. 81),but did not change significantly at the end of 6th week(1. 39 ± 0. 18). Compared with the control group,the difference had statistical significance( all P ﹤ 0. 05). Conclusion The abnormal expression of podocyte molecules mRNA in ADN rats may be an important molecular mechanism in the development of proteinuria.
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Objective To observe the changes of foot processes,expression and distribution of transient receptor potential cation channel 6 (TRPC6) in podocytes by puromycin aminonucleoside (PAN) and dexamethasone (DEX) intervention,then to investigate the function of TRPC6 in podocytes and its relation to proteinuria in kidney diseases.Methods Podocytes cultured in vitro were divided into three group:control group,PAN stimulation group and DEX intervention group.Mouse podocyte cell line (MPC5) were cultured in 0.02％ dimethyl sulfoxide (DMSO) in control group,subjected to PAN (50 μg/ml) treatment alone or with DEX (1 μmol/L) in other two groups for 8 h,24 h,48 h.The podocyte morphology was observed and took pictures by phase-contrast microscope,then the differences of morphology and areas were analyzed.The distribution,mRNA expression and protein expression of TRPC6 were detected by indirect immunocyto-fluorescence,real-time quantitative PCR and Western blotting,respectively.Results The well-developed podocyte arborization and interconnection was formed in control group,but PAN led to significant shrinkage of podocytes (P ＜ 0.05),together with podocyte foot process retraction,effacement and loss of cell contact.DEX significantly prevented the shrinkage and apoptosis of podocytes.The apoptosis rate was significantly increased after PAN stimulated 48 h (P ＜ 0.05).Real-time quantitative PCR and Western blotting found TRPC6 mRNA and protein expression were prone to increase in PAN group compared with control group (P ＜ 0.05).The distribution of TRPC6 becamed abnormal in PAN group.DEX decreased TRPC6 mRNA and protein expression at 48 h compared with PAN group (P ＜ 0.05).The abnormal distribution of TRPC6 was also alleviated by the protection of DEX.Conclusion DEX exerts a direct action to podocyte which retains the integrity of slit diaphragm against podocyte injury,and alleviates proteinuria via stabilizing mRNA,protein expression and distribution of TRPC6.
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Objective To observe the expression and distribution of α-actin-4 mRNA through puromycin aminonueleoside(PAN) injury podocyte,and discuss the relation between α-actin-4 and podocyte damage.Methods Podocytes were cultured in vitro,and 2 groups were set up:control group and PAN stimulation group.The control group was cultured with concentration of 100 mL/L FBS RPMI 1640 nutrient solution culture,while the PAN group was cultivated to PAN(50 mg/L) treatment,and cell morphology,extraction of total RNA of α-actin-4 were observed in 8 h,24 h and 48 h.The podocyte morphology was observed and pictures were taken through phase-contrast microscope,then the differences of morphology and areas between the 2 groups were analyzed.The distribution and mRNA expression of α-actin-4 were detected by indirect immunocyto-fluorescence and real-time quantitative PCR,respectively.Results The well-developed podocyte arborization was formed after the in vitro induction,and the PAN treatment led to the podocyte foot process retraction and effacement together with the mouse podocyte cell line shrinkage and the loss of cell contact.The above time point α-actin-4 mRNA expressions between the 2 groups were compared,and there was no significant difference in 8 h (P ＞ 0.05),but significant difference was found in 24 h,48 h,α-actin-4 higher mRNA expression,with statistical significance(all P ＜0.01).α-actin-4 in the control group had thin filaments evenly distributed in the cytoplasm,but a radioactive distribution in foot process.In the experimental group,α-actin-4 pressure silk fiber was shorter,with disordered arrangement,and PAN stimulus after 24 h,α-actin-4 distribution in cytoplasm was decreased significantly,while cytoplasmic distribution was missing after 48 h.Conclusions The abnormal of distribution and mRNA expression of α-actin-4 is time-related to the PAN injury podocyte,and α-actin-4 is an important part of podocyte damage mechanism.
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Autophagy is the cytoplasm and organelles of eukaryotic cells through degradation of itself,aself digest,a series of biochemical process is a kind of programmed cell death,its main function is to remove and degrade its damaged organelles and redundant biological macromolecules,and use the degradation products provide energy,reconstruction and cellular structure,in the steady state of cell and cell life activities play an important role.In recent years,autophagy in glomerular cells play a role of regulation and control have become the focus in the confrontation podocyte damage,including Beclin-1 autophagy related genes in the role of autophagy regulation has caused wide public concern.This article chooses of Beclin-1 in sertoli cell autophagy related research in the role of regulation are reviewed.
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With the development and progress of genetic testing,genetic testing of kidney disease has become an important method for diagnosis and prognosis evaluation.Some abnormal protein coding genes related to pediatric kidney diseases have been discovered in recent years.Fully and deeply understanding of these genes will provide a basis for applications of gene diagnosis,genetic counseling,prenatal diagnosis,disease prognosis valuation and clinical treatment guidance.In this article,the relationship between kidney diseases and relative genes had been reviewed.