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1.
International Eye Science ; (12): 366-372, 2022.
Article in Chinese | WPRIM | ID: wpr-920400

ABSTRACT

@#AIM: To investigate the effect of modulated autophagy activity on subepithelial haze after photorefractive keratectomy(PRK)in rabbits.<p>METHODS: Totally 64 New Zealand white rabbits were randomly divided into 4 groups according to different postoperative medication after PRK operation, including simple PRK group, 14μmol/L DMSO group, 50μmol/L rapamycin group and 100μmol/L rapamycin group. According to the group situation, two hours after the operation, eye drops were given, 3 times a day for 7d. Another 16 rabbits were selected as normal control group. The postoperative inflammatory response and corneal epithelial healing were observed with slit-lamp microscope every day. Haze formation of each group at 1 and 4wk after PRK was collected by slit-lamp microscopy system. Eight rabbits in each group were killed by air embolization 1 and 4wk after PRK, and corneal tissue was extracted and frozen for later use. Immunohistochemistry was used to detect the expression levels of transforming growth factor-β1(TGF-β1), α-smooth muscle actin(α-SMA), and matrix metalloproteinase-2(MMP-2). Real-time PCR was used to detect the relative expression levels of autophage-5(ATG5), autophage-12(ATG12), B lymphocytoma gene-2(Bcl-2)and cysteine aspartic proteinase-3(Caspase3)genes.<p>RESULTS: Corneal epithelium of all operative rabbits healed completely at 3-4d and no significant difference in healing time between the groups after operation(<i>F</i>=0.745, <i>P</i>=0.530). During the observation period, haze was the most obvious at 4wk after operation in all groups. The haze symptoms were more serious in the simple operation group and the 14μmol/L DMSO group, followed by the 50μmol/L rapamycin group. The haze symptoms in the 100μmol/L rapamycin group were significantly relieved than those in other groups. There was no significant difference in the haze grading with different time points after operation among all groups(all <i>P</i><0.05). Immunohistochemistry showed that the expression of TGF-β1, MMP-2 and α-SMA was stronger in the operation group and 14μmol/L DMSO group, followed by 50μmol/L rapamycin group, and weakest in 100μmol/L rapamycin group than other groups at 1 and 4wk after operation(all <i>P</i><0.05). The results of PCR showed that the relative expression of ATG5, ATG12 and Bcl-2 mRNA in 50μmol/L rapamycin group and 100μmol/L rapamycin group were significantly higher than those in simple operation group and 14μmol/L DMSO group at 1 and 4wk after operation(all <i>P</i><0.05); The relative expression of Caspase3 mRNA in 50μmol/L rapamycin group and 100μmol/L rapamycin group was significantly lower than that in simple operation group and 14μmol/L DMSO group(all <i>P</i><0.05).<p>CONCLUSION: Rapamycin can enhance autophagy level and inhibit apoptosis level, thus reducing haze formation after PRK in rabbits.

2.
Journal of Clinical Hepatology ; (12): 210-214, 2022.
Article in Chinese | WPRIM | ID: wpr-913145

ABSTRACT

Hepatic ischemia-reperfusion injury (HIRI) is a common clinical problem after hepatectomy and liver transplantation and is the main cause of liver dysfunction and liver failure after transplantation. In recent years, autophagy-mediated pathways have become a research hotspot in alleviating HIRI. Autophagy refers to the process in which a large number of substrates such as cytoplasm and damaged organelles are transported into lysosomes for digestion and degradation, so as to constantly renew, reshape, and reuse cells. This article summarizes the research advances in the mechanism of targeting autophagy to alleviate HIRI from the aspects of gene, protein, signaling pathway, inflammatory response, oxidative stress, and mitochondrial and endoplasmic reticulum stress, as well as existing problems and prospects in research, in order to provide theoretical support for the future research on alleviating HIRI by targeting autophagy.

3.
Acta Pharmaceutica Sinica ; (12): 313-320, 2022.
Article in Chinese | WPRIM | ID: wpr-922930

ABSTRACT

In recent years, the targeted protein degradation technology has developed quickly, with proteolysis-targeting chimera (PROTAC) as the best-known strategy through exploring the ubiquitin-proteasome system. A number of new targeted protein degradation strategies have been emerging to expand the scope of protein degradation technology, including lysosome-targeting chimeras (LYTACs), autophagy-targeting chimeras (AUTACs), autophagosome-tethering compounds (ATTECs) and chimeras based on chaperone-mediated autophagy (CMA). The emerging methodologies have explored another important protein degradation system in eukaryotes-lysosomal systems, such as the endosome-lysosome pathway and the autophagy-lysosome pathway. This review summaries the mechanisms and features of different strategies for targeted protein degradation, with a special emphasis on the new targeted protein degradation technologies, such as their current status, advantages and limitations.

4.
Article in Chinese | WPRIM | ID: wpr-908318

ABSTRACT

BACKGROUND:The importance of autophagy for maintaining cellular homeostasis and stress response has long been recognized.As a way for cells to selectively clear their damaged organelles to achieve the recycling of cellular components,autophagy has a pivotal role in bone metabolism.OBJECTIVE:To review the role and possible mechanisms of autophagy in regulating bone-related cell activity and function among bone marrow mesenchymal stem cells,osteoblasts,osteocytes,and osteoclasts.METHODS:PubMed was searched for studies related to autophagy using the keywords of "autophagy;bone marrow mesenchymal stem cells;osteoblasts;osteocytes;osteoclasts."RESULTS AND CONCLUSION:We finally included 84 papers.Autophagy plays an important role in bone metabolism.Autophagy is involved in maintaining the balance between mineralization and absorption,and then maintaining bone homeostasis.An appropriate autophagy inducer may also benefit bone remodeling.Abnormal autophagy can lead to disorders of bone balance,leading to diseases such as osteoporosis.We may prevent or treat bone-related diseases by regulating the level of autophagy as its function in maintaining the balance of mineralization and resorption in bone homeostasis.

5.
Article in Chinese | WPRIM | ID: wpr-847212

ABSTRACT

BACKGROUND: The autophagy of macrophage cells plays a key role in regulating the immune system and inflammatory response, while knockdown Atg5 gene can specifically inhibit the autophagy. There are some shortcomings of traditional siRNA transfection methods such as transience and incompleteness. It is of great value and significance to construct a stable Atg5 gene knockdown cell line in RAW 264.7 cells for studying the macrophage autophagy and immune inflammation-related diseases and their pathogenesis. OBJECTIVE: To construct RAW 264.7 macrophage cell line stably knocking down the Atg5 gene by lentivirus infection, and to provide chassis cells for studying macrophage autophagy and immune inflammation-related diseases and their pathogenesis. METHODS: Recombinant expression vector (HBLV-m-Atg5-shRNA-GFP-Puro) with green fluorescence signal, Puro resistance gene, and knockdown Atg5 gene was constructed and transfected into 293T cells to obtain lentivirus plasmid system. The green fluorescence signal of the acquired lentivirus infected RAW 264.7 cells was observed under an inverted fluorescence microscope. Purinomycin resistance screening and flow cytometry were used to obtain high-purity infected cells. The RAW 264.7 cell line stably knocking down the Atg5 gene was identified by real-time quantitative polymerase chain reaction and western blot assay. RESULTS AND CONCLUSION: DNA sequencing results showed that Atg5 gene interfering sequences were correctly inserted into the expression vector, and the HBLV-m-Atg5-shRNA-GFP-Puro expression vector was successfully constructed. After infecting RAW 264.7 cell line with lentivirus plasmid, green fluorescence was observed under an inverted fluorescence microscope. Green fluorescent protein positive cell groups were observed by flow cytometry. The results of real-time quantitative polymerase chain reaction and western blot assay showed that the expression level of the Atg5 gene in RAW 264.7 cell line was significantly decreased (P < 0.05), indicating that the RAW 264.7 cell line with stable knockdown of Atg5 gene was successfully constructed.

6.
Article in Chinese | WPRIM | ID: wpr-847207

ABSTRACT

BACKGROUND: Yougui Decoction is an empirical prescription for the treatment of glucocorticoid-associated femoral head necrosis. Literature has shown that the pathogenesis of glucocorticoid-associated femoral head necrosis is associated with glucocorticoid-induced autophagy down-regulation and fate change in bone marrow mesenchymal stem cells. OBJECTIVE: To investigate the effect of Yougui Decoction on autophagy and fate of bone marrow mesenchymal stem cells in model rats of glucocorticoid-associated femoral head necrosis. METHODS: We used Escherichia coli endotoxin combined with high-dose dexamethasone to make the rat models of early femoral head necrosis. Forty SHR rats were randomly divided into five groups: blank control group, model group, high-dose Yougui Decoction group, medium-dose Yougui Decoction group and low-dose Yougui Decoction group. After 6 weeks of intervention, medullary cavity tissue of the rat proximal femur was taken for hematoxylin-eosin staining and immunohistochemical staining of autophagy proteins LC3 II, P53 and beclin-1. After culture and induction of bone marrow mesenchymal stem cells, alizarin red staining, bone alkaline phosphatase quantification, oil red staining and MTT determination were performed and western blot assay was used to quantitatively measure the expression of LC3 II, P53 and beclin-1 proteins. RESULTS AND CONCLUSION: (1) Immunohistochemistry and western blot assay results showed that Yougui Decoction significantly increased autophagy protein LC3 II, P53 and beclin 1 expression in a dose-dependent manner. (2) Alizarin red staining, oil red staining and bone alkaline phosphatase quantification suggested that Yougui Decoction could significantly interfere with the fate of bone marrow mesenchymal stem cells, up-regulate their osteogenic differentiation and down-regulate their adipogenic differentiation in a dose-dependent manner. (3) MTT results suggested that Yougui Decoction significantly improved the proliferation ability of bone marrow mesenchymal stem cells, but had no significant differences in different doses. (4) To conclude, Yougui Decoction can significantly improve the autophagy expression, change the cell fate, up-regulate osteogenic differentiation and down-regulate adipogenic differentiation of bone marrow mesenchymal stem cells in the rat models of glucocorticoid-associated femoral head necrosis, which provides certain basis for elucidating the mechanism of Yougui Decoction in treating glucocorticoid-associated femoral head necrosis.

7.
Article in Chinese | WPRIM | ID: wpr-847190

ABSTRACT

BACKGROUND: Abnormal autophagy in chondrocytes often leads to cartilage degeneration, thereby triggering osteoarthritis. Recent studies have found that microRNAs play an important role in chondrocyte autophagy; however, the molecular mechanism is yet unclear. OBJECTIVE: To investigate the role of microRNA-138-5p (miR-138-5p) in the regulation of chondrocyte proliferation and autophagy activities, and to reveal its mechanisms. METHODS: Chondrosarcoma cell line SW1353 were cultured in vitro and transfected with negative control miRNA or miR-138-5p mimic. Cell proliferation activity was measured by cell counting kit-8 assay, the expression of matrix metalloproteinases 1, 3, and 13 mRNA was measured by fluorogenic quantitative PCR. The endogenous LC3 subcellular location was detected by immunofluorescence staining. The miR-138-5p and SIRTI mRNA target sites were predicted using TargetScan 7.1 online tool. Autophagy-related proteins and AMPK signal proteins were detected by immunoblotting assay. RESULTS AND CONCLUSION: Cells transfected with miR-138-5p mimic, compared with those transfected with negative control miRNA, showed lower proliferation activity, less LC3 puncta, and reduced expression of SIRT1, LC3-II, p-AMPK, but increased protein expression of p62 and increased mRNA expression of matrix metalloproteinases 1, 3, 13. There was a conserved miR-138-5p binding site in the 3’UTR region of SIRT1 mRNA. To conclude, miR-138-5p regulates SW1353 cell autophagy and proliferation through the SIRT1/AMPK signaling pathway. The up-regulated expression of miR-138-5p promotes the secretion of matrix metalloproteinases from chondrocytes, indicating that miR-138-5p plays an important role in the progression of osteoarthritis.

8.
Article in Chinese | WPRIM | ID: wpr-921778

ABSTRACT

This study investigated the effect of rhein(RH) on the apoptosis and autophagy of human umbilical vein endothelial cells(HUVECs) induced by hydrogen peroxide(H_2O_2) and its underlying mechanism. The oxidative damage model in HUVECs was established and the cells were divided into different treatment groups. Cell survival rate was detected by MTT assay, apoptosis by Annexin V-FITC/PI double staining and Hoechst 33258 fluorescence staining, autophagy by Ad-mCherry-GFP-LC3 B adenovirus transfection, and protein expression by Western blot. The results showed that RH could protect cells by increasing the cell survival rate in a dose-dependent manner, decreasing the expression of apoptosis-related proteins(Bax and cleaved caspase-3) and the ratio of Bax/Bcl-2, elevating the expression of Bcl-2, up-regulating the expression of microtubule-associated protein 1 light chain 3(LC3)-Ⅱ, and down-regulating the expression of p62. Adenovirus transfection results showed that RH could increase the green and red spots, as well as the yellow spots. However, after the addition of autophagy inhibitor 3-MA, autophagy was reduced and apoptosis was increased. RH could enhance the expression of silent information regulator 2 related enzyme 1(SIRT1). The addition of SIRT1 inhibitor EX-527 reduced the protective effect of RH and cell viability. The addition of 3-MA had no effect on the expression of SIRT1 protein, but the expression of SIRT1 and LC3-Ⅱ proteins decreased and the expression of p62 increased after the addition of EX-527. After RH treatment, the phosphorylation of adenosine monophosphate-activated protein kinase(AMPK) increased, while that of the mechanistic target of rapamycin(mTOR) decreased in a dose-dependent manner. Moreover, this effect could be weakened by the AMPK inhibitor compound C. RH may enhance autophagy through SIRT1/AMPK/mTOR pathway to reduce H_2O_2-induced apoptosis of HUVECs.


Subject(s)
Anthraquinones , Apoptosis , Autophagy , Human Umbilical Vein Endothelial Cells , Humans , Hydrogen Peroxide , Signal Transduction
9.
Article in Chinese | WPRIM | ID: wpr-921645

ABSTRACT

The present study investigated the effects of chikusetsu saponin Ⅳa(CHS Ⅳa) on isoproterenol(ISO)-induced myocardial hypertrophy in rats and explored the underlying molecular mechanism. ISO was applied to establish a rat model of myocardial hypertrophy, and CHS Ⅳa(5 and 15 mg·kg~(-1)·d~(-1)) was used for intervention. The tail artery blood pressure was measured. Cardiac ultrasound examination was performed. The ratio of heart weight to body weight(HW/BW) was calculated. Morphological changes in the myocardial tissue were observed by HE staining. Collagen deposition in the myocardial tissue was observed by Masson staining. The mRNA expression of myocardial hypertrophy indicators(ANP and BNP), autophagy-related genes(Atg5, P62 and beclin1), and miR199 a-5 p was detected by qRT-PCR. Atg5 protein expression was detected by Western blot. The results showed that the model group exhibited increased tail artery blood pressure and HW/BW ratio, thickened left ventricular myocardium, enlarged myocardial cells, disordered myocardial fibers with widened interstitium, and a large amount of collagen aggregating around the extracellular matrix and blood vessels. ANP and BNP were largely expressed. Moreover, P62 expression was up-regulated, while beclin1 expression was down-regulated. After intervention by CHS Ⅳa at different doses, myocardial hypertrophy was ameliorated and autophagy activity in the myocardial tissue was enhanced. Meanwhile, miR199 a-5 p expression declined and Atg5 expression increased. As predicted by bioinformatics, Atg5 was a target gene of miR199 a-5 p. CHS Ⅳa was capable of preventing myocardial hypertrophy by regulating autophagy of myocardial cells through the miR-199 a-5 p/Atg5 signaling pathway.


Subject(s)
Animals , Cardiomegaly/genetics , Isoproterenol , Myocardium , Myocytes, Cardiac , Oleanolic Acid/analogs & derivatives , Rats , Saponins/pharmacology
10.
Cancer Research and Clinic ; (6): 596-600, 2021.
Article in Chinese | WPRIM | ID: wpr-912930

ABSTRACT

Objective:To investigate the expressions and significances of autophagy-related genes Beclin-1, LC3 and p62 in esophageal squamous cell carcinoma (ESCC).Methods:The clinical data of 112 patients with primary ESCC who underwent surgery at the 81st Group Army Hospital of Chinese PLA from January 2015 to December 2016 were retrospectively analyzed. Immunohistochemistry was used to examine the expressions of Beclin-1, p62 and LC3 proteins in 112 ESCC tissues and 31 adjacent normal esophageal mucosa tissues. Furthermore, the expressions of the above three autophagy-related markers in ESCC and the relationship between their expressions and the clinicopathological characteristics of patients were analyzed.Results:The positive expression rates of Beclin-1, LC3 and p62 in ESCC tissues were 32.14% (36/112), 37.50% (42/112) and 63.39% (71/112), The positive expression rates of Beclin-1, LC3 and p62 in adjacent normal esophageal mucosa tissues were 61.29% (19/31), 64.52% (20/31) and 32.26% (10/31), and the differences were statistically significant ( χ2 values ??were 8.715, 7.216 and 9.584, all P < 0.01). The positive expression rates of Beclin-1 and LC3 in ESCC were lower than those in adjacent normal esophageal mucosa tissues, and the positive expression rate of p62 in ESCC was higher than that in adjacentnormal esophageal mucosa tissues. In ESCC patients, the expression of Beclin-1 was related to histological grade, infiltration depth, TNM staging and lymph node metastasis (all P < 0.05); the expression of LC3 was related to infiltration depth and TNM staging (both P < 0.01); the expression of p62 was related to lymph node metastasis ( P < 0.01). In ESCC, the expression of LC3 was positively correlated with the expression of Beclin-1 ( r = 0.731, P = 0.001), and negatively correlated with the expression of p62 ( r = -0.215, P = 0.023). Conclusions:Autophagy plays a certain role in the occurrence and development of ESCC. Combined detection of autophagy-related genes Beclin-1, p62 and LC3 can assist clinical diagnosis and guide follow-up comprehensive treatment.

11.
Chinese Journal of Dermatology ; (12): 878-883, 2021.
Article in Chinese | WPRIM | ID: wpr-911545

ABSTRACT

Objective:To investigate the role of folliculin in apoptosis of and chemokine secretion by melanocytes mediated by interferon-γ (IFN-γ) .Methods:Normal primary melanocytes were isolated from circumcised foreskin tissues from a healthy male child, and primary vitiliginous melanocytes were isolated from normally pigmented suction-blistered epidermis from patients with vitiligo after suction blister epidermal grafting. Western blot analysis was performed to determine the folliculin protein expression in normal primary melanocytes, primary vitiliginous melanocytes and a human primary melanocyte line PIG1. PIG1 cells stimulated with 10 ng/ml IFN-γ for 48 hours served as induction group, and untreated PIG1 cells served as control group. Real-time quantitative RCR (qRT-PCR) was performed to determine the mRNA expression of folliculin, autophagy-related microtubule-associated protein 1 light chain 3 (LC3) -Ⅱ and Beclin genes, and Western blot analysis to determine the protein expression of folliculin, Beclin1 and LC3Ⅱ/Ⅰ, as well as phosphorylation levels of adenosine monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) in the above cells. Furthermore, the melanocytes stimulated with 10 ng/ml IFN-γ for 48 hours were divided into several groups: negative control group infected with an empty lentiviral vector, folliculin inhibition group infected with a folliculin-inhibiting lentivirus, autophagy enhancement group infected with a folliculin-inhibiting lentivirus followed by 2-hour treatment with a mTOR inhibitor, autophagy inhibition group infected with a folliculin-inhibiting lentivirus followed by 2-hour treatment with an AMPK inhibitor. Then, flow cytometry was conducted to detect apoptosis of PIG1 cells, and enzyme-linked immunosorbent assay to measure the concentration of chemokines CXCL10 and CCL20 in the culture supernatant of PIG1 cells in the above groups. Measurement data were compared among multiple groups by using one-way analysis of variance, and multiple comparisons were carried out by using least significant difference- t test. Results:The relative protein expression level of folliculin significantly differed among the normal primary melanocytes (0.850 ± 0.120) , primary vitiliginous melanocytes (1.507 ± 0.170) and PIG1 cells (0.697 ± 0.130; F = 50.09, P < 0.001) , and was significantly higher in the primary vitiliginous melanocytes than in the normal primary melanocytes and PIG1 cells ( t = 4.06, 5.89, respectively, both P < 0.01) . Compared with the control group, the induction group showed significantly increased relative mRNA and protein expression levels of folliculin (both P < 0.01) , but significantly decreased relative mRNA and protein expression levels of LC3Ⅱ and Beclin (all P < 0.01) ; moreover, the induction group showed significantly decreased LC3Ⅱ/Ⅰ levels (0.72 ± 0.02) and AMPK phosphorylation levels (0.714 ± 0.023) in the PIG1 cells compared with the control group (1.13 ± 0.02, 1.176 ± 0.002, t = 7.34, 6.67, respectively, both P < 0.01) , but significantly increased mTOR phosphorylation levels (1.051 ± 0.023) compared with the control Group (0.451 ± 0.016, t = 3.81, P = 0.009) . There were significant differences in the PIG1 cell apoptosis rate and concentrations of CXCL10 and CCL20 among the control group, induction group and other treatment groups (all P < 0.001) ; specifically, the PIG1 cell apoptosis rate and concentrations of CXCL10 and CCL20 were significantly higher in the induction group than in the control group, lower in the folliculin inhibition group than in the negative control group, lower in the autophagy enhancement group than in the folliculin inhibition group, and higher in the autophagy inhibition group than in the folliculin inhibition group (all P < 0.05) . Conclusions:Folliculin is highly expressed in vitiliginous melanocytes. Folliculin expression and downstream signaling pathways are regulated by IFN-γ, and folliculin may participate in IFN-γ-mediated melanocyte apoptosis and chemokine secretion via regulating autophagy.

12.
Chinese Journal of Dermatology ; (12): 861-868, 2021.
Article in Chinese | WPRIM | ID: wpr-911543

ABSTRACT

Objective:To investigate the effect of pterostilbene on the growth, apoptosis and autophagy of a human papillomavirus type 16 (HPV-16) -immortalized cervical epithelial cell line H8.Methods:H8 cells were treated with pterostilbene at different concentrations of 0 (control group) , 25, 50, 75, 100 μmol/L for 24 and 48 hours. Cell counting kit-8 (CCK8) assay was performed to evaluate the cellular proliferative activity, flow cytometry was conducted to detect apoptosis and cell cycle, monodansylcadaverine (MDC) staining and fluorescence microscopy were performed to detect autophagy, and Western blot analysis was conducted to determine the expression of the cell cycle-related protein cyclinD1, apoptosis-related proteins caspase-3 and caspase-9, autophagy-related proteins Beclin1, microtubule-associated protein 1 light chain 3 (LC3) -Ⅱ/Ⅰ, ATG5 and P62, as well as HPV oncoproteins E6 and E7. Statistical analysis was carried out by using one-way analysis of variance, repeated measures analysis of variance and least significant difference- t test. Results:After 48-hour treatment with pterostilbene at different concentrations of 0, 25, 50, 75, 100 μmol/L, the relative cellular proliferation rate significantly differed among the groups (100.00% ± 1.56%, 99.02% ± 4.97%, 93.59% ± 2.01%, 81.28% ± 4.90%, 69.17% ± 7.56%, respectively; F = 77.22, P < 0.05) , and gradually decreased along with the increase in the concentration of pterostilbene; compared with the control group, the pterostilbene groups all showed significantly decreased cellular proliferation rate (all P < 0.05) . After 24-hour treatment with pterostilbene, the proportions of H8 cells at G1, G2 and S phases significantly differed among the above groups ( F = 7 845.00, 51.14, 266.50, respectively, all P < 0.05) ; compared with the control group, the pterostilbene groups showed significantly increased proportions of H8 cells at G1 and G2 phases (all P < 0.05) , but significantly decreased proportions of H8 cells at S phase ( P < 0.05) . After 48-hour treatment with pterostilbene, the apoptosis rate was significantly higher in the 25-, 50-, 75- and 100-μmol/L pterostilbene groups (14.66% ± 0.22%, 13.50% ± 0.49%, 14.56% ± 0.19%, 15.30% ± 0.76%, respectively) than in the control group (11.58% ± 0.50%, all P < 0.05) . After 24-hour treatment with pterostilbene, MDC staining showed only a small number of H8 cells with bright dot-like fluorescence in the control group, but increased number of autophagosome-positive H8 cells with bright dot-like fluorescence in the pterostilbene groups. Western blot analysis revealed that there were significant differences in the protein expression of cyclin D1, caspase-3, caspase-9, Beclin1, LC3-Ⅱ/Ⅰ, ATG5, P62, E6 and E7 among the control and pterostilbene groups after 24- and 48-hour treatment with pterostilbene (all P < 0.05) . The treatment with pterostilbene could down-regulate the expression of cyclin D1, E6 and E7, and up-regulate the expression of caspase-3, caspase-9, Beclin1, LC3-Ⅱ/Ⅰ, ATG5 and P62, with significant differences between the control group and most pterostilbene groups in expression of the above proteins (all P < 0.05) . Conclusion:Pterostilbene can inhibit the proliferation of H8 cells, promote their apoptosis and autophagy, and down-regulate the expression of oncogenes E6 and E7.

13.
Chinese Journal of Dermatology ; (12): 683-687, 2021.
Article in Chinese | WPRIM | ID: wpr-911506

ABSTRACT

Objective:To detect the mRNA expression profile of CD4 + T cells in the peripheral blood of leprosy patients, and to screen and identify genes that may be closely related to the pathogenesis of leprosy. Methods:From July 2018 to May 2020, 45 leprosy patients were collected from Hunan Province, and 45 healthy volunteers from Health Examination Center of Changsha Central Hospital. CD4 + T cells were isolated from peripheral blood samples by using magnetic beads, and then RNA was extracted. Solexa sequencing was performed to screen differentially expressed genes between 6 patients and 6 healthy controls, who were randomly selected from the above subjects. Differentially expressed genes were defined as those with a fold change greater than 2 and a P value below 0.05, and then Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis was performed. Real-time fluorescence-based quantitative PCR was conducted to verify the gene expression. Results:Genetic screening revealed 4 831 newly-discovered transcripts with protein-coding potential. Eight differentially expressed genes were screened out between the two groups. Among them, the mRNA expression of CXCL8, PPBP, RPS18 and IL-1β genes was up-regulated, while the mRNA expression of RNH1, RPL39, RPL15 and AMBRA1 genes was down-regulated. Verification results of real-time fluorescence-based quantitative PCR were consistent with the above-mentioned genetic screening results. KEGG analysis showed that the differentially expressed genes between leprosy patients and healthy controls were mainly enriched in mitochondrial autophagy, autophagy-related pathways and human papillomavirus infection pathways.Conclusion:Down-regulated mRNA expression of AMBRA1 and RNH1 genes and up-regulated mRNA expression of CXCL8, PPBP and IL-1β genes were identified in patients with leprosy, which may be involved in the pathogenesis of leprosy through the mitochondrial autophagy pathway and chemokine-mediated signaling pathway, respectively.

14.
Chinese Journal of Anesthesiology ; (12): 1252-1255, 2021.
Article in Chinese | WPRIM | ID: wpr-911353

ABSTRACT

Objective:To evaluate the role of histone deacetylase 3 (HDAC3) in high glucose hypoxia/reoxygenation (H/R) injury to primary rat cardiomyocytes and the relationship with autophagy.Methods:The primary cardiomyocytes extracted from newborn Sprague-Dawley rats, aged about 1-3 days, were divided into 5 groups ( n=24 each) according to the random number table method: control group (C group, glucose concentration 5.5 mmol/L), H/R group, high glucose group (H group, glucose concentration 30 mmol/L), high glucose H/R group (HH/R group), and high glucose H/R + HDAC3 inhibitor RGFP966 group (HH/R+ RG group). Fifty percent glucose injection was used to prepare high-glucose medium (final concentration 30 mmol/L). Cells were cultured in a hypoxic environment (5% CO 2-0.9% O 2-94.1% N 2) for 6 h, followed by reoxygenation in a normoxic environment for 2 h to establish the cardiomyocyte H/R model in H/R group.RGFP966 at a final concentration of 10 μmol/L was added at 24 h before H/R in HH/R+ RG group.At 2 h of reoxygenation, the cell viability was measured using CCK-8 kit, the activity of lactic dehydrogenase (LDH) in the cell supernatant was determined using enzyme-linked immunosorbent assay, the level of autophagy was detected with a confocal microscope after cells were transfected with autophagy double-labeled adenovirus (mRFP-GFP-LC3), and the expression of HDAC3, p62, LC3 Ⅱ and LC3 Ⅰ was detected using Western blot.LC3Ⅱ/LC3Ⅰ ratio was calculated. Results:Compared with group C, the cell viability was significantly decreased, and the activity of LDH in supernatant was increased in H/R and H groups, the number of autophagosomes was significantly increased, the expression of HDAC3 in cardiomyocytes was up-regulated, the expression of p62 was down-regulated, and the LC3 Ⅱ/I ratio was increased in group H/R, and the number of autophagosomes was significantly decreased, the expression of HDAC3 and p62 in cardiomyocytes was up-regulated, and the LC3 Ⅱ/I ratio was decreased in group H ( P<0.05). Compared with group H/R, the cell viability was significantly decreased, the activity of LDH in supernatant was increased, the number of autophagosomes was decreased, the expression of HDAC3 and p62 in cardiomyocytes was up-regulated, and the LC3 Ⅱ/I ratio was decreased in group HH/R ( P<0.05). Compared with group H, the cell viability was significantly decreased, the activity of LDH in supernatant was increased, the number of autophagosomes was increased, the expression of HDAC3 and p62 in cardiomyocytes was up-regulated, and the LC3 Ⅱ/I ratio was increased in group HH/R ( P<0.05). Compared with group HH/R, the cell viability was significantly increased, the activity of LDH in supernatant was decreased, the number of autophagosomes was increased, the expression of HDAC3 and p62 in cardiomyocytes was down-regulated, and the LC3 Ⅱ/I ratio was increased in group HH/R+ RG ( P<0.05). Conclusion:Up-regulation of HDAC3 expression is involved in high glucose H/R injury to primary rat cardiomyocytes, which is related to decreasing the level of autophagy.

15.
Chinese Journal of Anesthesiology ; (12): 1000-1004, 2021.
Article in Chinese | WPRIM | ID: wpr-911317

ABSTRACT

Objective:To evaluate the effect of esketamine on acute kidney injury (AKI) in the rats with sepsis and the role of autophagy.Methods:Forty SPF healthy adult male Sprague-Dawley rats, weighing 200-240 g, were divided into 5 groups ( n=8 each) by a random number table method: control group (Con group), esketamine group (Con+ Ket group), sepsis group (lipopolysaccharide [LPS] group), sepsis plus esketamine group (LPS+ Ket group), and sepsis plus esketamine plus 3-methyladenine (3MA) group (LPS+ Ket+ 3MA group). The model of AKI was established by intraperitoneal injection of LPS in anesthetized rats.Normal saline 10 ml/kg was intraperitoneally injected in Con group.In Con+ Ket group, normal saline 10 ml/kg was intraperitoneally injected, and 30 min later esketamine 10 mg/kg was injected via the tail vein.LPS 10 mg/kg was intraperitoneally injected in LPS group.In LPS+ Ket group, LPS 10 mg/kg was intraperitoneally injected, and 30 min later esketamine 10 mg/kg was injected via the tail vein.In LPS+ Ket+ 3MA group, LPS 10 mg/kg was intraperitoneally injected, and 30 min later esketamine 10 mg/kg and 3-MA 15 mg/kg were injected via the tail vein.The rats were anesthetized at 24 h after intraperitoneal injection of LPS and then sacrificed, and renal tissues were removed for microscopic examination of the pathological changes which were scored and for determination of contents of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, interleukin-1beta (IL-1β) and IL-18 (by enzyme-linked immunosorbent assay) and expression of LC3, P62 and Beclin-1 (by Western blot). Results:Compared with Con group, the score for pathological damage to renal tissues and contents of NLRP3, ASC, caspase-1, IL-1β and IL-18 were significantly increased, LC3-Ⅱ/LC3-Ⅰ ratio was decreased, the expression of Beclin-1 was down-regulated, and the expression of P62 was up-regulated in LPS group ( P<0.05). Compared with LPS group, the score for pathological damage to renal tissues and contents of NLRP3, ASC, caspase-1, IL-1β and IL-18 were significantly decreased, LC3-Ⅱ/LC3-Ⅰ ratio was increased, the expression of Beclin-1 was up-regulated, and the expression of P62 was down-regulated in LPS+ Ket group ( P<0.05). Compared with LPS+ Ket group, the score for pathological damage to renal tissues and contents of NLRP3, ASC, caspase-1, IL-1β and IL-18 were significantly increased, LC3-Ⅱ/LC3-Ⅰ ratio was decreased, the expression of Beclin-1 was down-regulated, and the expression of P62 was up-regulated in LPS+ Ket+ 3MA group ( P<0.05). Conclusion:Esketamine can reduce AKI and autophagy is involved in the process, which is related to inhibiting the activation of NLRP3 inflammasomes and decreasing inflammatory responses in rats with sepsis.

16.
Article in Chinese | WPRIM | ID: wpr-911316

ABSTRACT

Objective:To evaluate the effect of TBK1 overexpression on hypoxia-reoxygenation (H/R) injury in isolated mouse cardiomyocytes subjected to high glucose and the relationship with mitochondrial autophagy.Methods:Normally cultured log-phase HL-1 mouse cardiomyocytes were inoculated in a 6-well plate at a density of 1×10 6 cells/ml and were divided into 4 groups ( n=10 each) using a random number table method: control group (group C), high glucose group (group HG), high glucose and H/R group (group HG+ H/R), and TBK1 overexpression group (group TBK1). The cells were incubated in culture medium with 1% fetal bovine serum and 1% double antibody for 24 h when the cell density reached 50%.When the cell density reached 80%, pcDNA3.1 (+ ) was used as a vector to achieve TBK1 overexpression.The cells were cultured with high glucose medium (33 mmol/L) for 24 h, exposed to 94% N 2+ 5% CO 2+ 1% O 2 for 24 h in an incubator at 37℃ followed by 12 h reoxygenation in an incubator containing 5% CO 2 at 37°C to establish the model of H/R injury to cardiomyocytes subjected to high glucose.After reoxygenation, CCK-8 assay was used to detect cell viability, the activity of lactic dehydrogenase (LDH) in supernatant was detected using LDH kit, mitochondrial contents were determined using Mito-Tracter green fluorescent probe, and the expression of TBK1 and mitophagy-related proteins PINK1, Parkin, LC3B and P62 was detected by Western blot. Results:Compared with group C, the cell viability was significantly decreased, the activity of LDH in supernatant was increased, mitochondrial contents were decreased, the expression of TBK1, PINK1, Parkin and LC3B was down-regulated, and the expression of P62 was up-regulated in HG group and HG+ H/R group ( P<0.05). Compared with group HG, the cell viability was significantly decreased, the activity of LDH in supernatant was increased, mitochondrial contents were decreased, the expression of TBK1, PINK1, Parkin and LC3B was down-regulated, and the expression of P62 was up-regulated in group HG+ H/R ( P<0.05). Compared with group HG+ H/R, the the cell viability was significantly increased, the activity of LDH in supernatant was decreased, mitochondrial contents were increased, the expression of TBK1, PINK1, Parkin and LC3B was up-regulated, and the expression of P62 was down-regulated in group TBK1 ( P<0.05). Conclusion:The mechanism by which TBK1 overexpression reduces the H/R injury is related to restoring mitophagy in isolated mouse cardiomyocytes subjected to high glucose.

17.
Article in Chinese | WPRIM | ID: wpr-911246

ABSTRACT

Objective:To evaluate the effect of activating adenosine A2B receptors on autophagy during myocardial ischemia-reperfusion (I/R) and the role of phosphatidylinositol 3-kinase/serine/threonine protein kinase (PI3K/Akt) signaling pathway in rats.Methods:Forty-eight clean-grade healthy male Sprague-Dawley rats, weighing 220-280 g, were divided into 4 groups ( n=12 each) using a random number table method: sham operation group (group Sham), myocardial I/R group (group I/R), adenosine A2B receptor agonist BAY 60-6583 group (group BAY) and BAY 60-6583+ PI3K inhibitor LY 294002 group (group BAY+ LY). Myocardial I/R was induced by occlusion of the anterior descending branch of the left coronary artery for 30 min followed by 120-min reperfusion.BAY 60-6583 1 mg/kg was intraperitoneally injected at 5 min before reperfusion in group BAY.BAY 60-6583 1 mg/kg was intraperitoneally injected at 5 min before reperfusion and LY 294002 10 mg/kg was intraperitoneally injected at 10 min before reperfusion in group BAY+ LY.Blood samples were obtained at the end of reperfusion for determination of concentrations of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) in serum (by enzyme-linked immunosorbent assay). The animals were sacrificed, and myocardial tissues were obtained for measurement of the percentage of myocardial infarct size (by Evan Blue and TTC double-staining) and for determination of the expression of microtubule-associated protein 1 light chain 3 (LC3Ⅰ), LC3Ⅱ, Beclin-1 and phosphorylated Akt (p-Akt) (by Western blot). The ratio of LC3Ⅱ/LC3Ⅰ was calculated. Results:Compared with group Sham, the serum LDH and CK-MB concentrations and percentage of myocardial infarct size were significantly increased, the expression of p-Akt was down-regulated, the expression of Beclin-1 and LC3Ⅱ was up-regulated, and the ratio of LC3Ⅱ/LC3Ⅰ was increased in group I/R ( P<0.05). Compared with group I/R, the concentrations of serum LDH, CK-MB and percentage of myocardial infarct size were significantly decreased, the expression of p-Akt was up-regulated, the expression of Beclin-1 and LC3Ⅱ was down-regulated, and the ratio of LC3Ⅱ/LC3Ⅰ was decreased in the group BAY ( P<0.05), and no significant change was found in the parameters mentioned above in group BAY+ LY ( P>0.05). Compared with group BAY, the concentrations of serum LDH, CK-MB and percentage of myocardial infarct size were significantly increased, the expression of p-Akt was down-regulated, the expression of Beclin-1 and LC3Ⅱ was up-regulated and the ratio of LC3Ⅱ/LC3Ⅰ was increased in group BAY+ LY ( P<0.05). Conclusion:Activating adenosine A2B receptors can decrease autophagy of myocardial cells during myocardial I/R injury, and the mechanism may be related to activating PI3K/Akt signaling pathway in rats.

18.
Article in Chinese | WPRIM | ID: wpr-911215

ABSTRACT

Objective:To evaluate the relationship between the mechanism of protective effect of hydromorphone postconditioning on myocardium and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway-mediated autophagy in rats.Methods:Forty healthy male Sprague-Dawley rats, weighing 220-250 g, were divided into 5 groups ( n=8 each) using a random number table method: sham operation group (Sham group), ischemia-reperfusion (I/R) group (group IR), hydromorphone postconditioning group (group HP), PI3K inhibitor group (group W) and hydromorphone postconditioning+ PI3K inhibitor group (group HP+ W). Myocardial ischemia was induced by 30 min occlusion of left anterior descending branch of coronary artery followed by 120 min reperfusion.In group HP, hydromorphone 0.1 mg/kg was injected via femoral vein at 5 min before reperfusion in group HP.In group HP+ W, hydromorphone 0.1 mg/kg and wortmannin (PI3K inhibitor) 15 μg/kg were injected via femoral vein at 5 min before reperfusion.In group W, wortmannin 15 μg/kg was injected via femoral vein at 5 min before reperfusion.At the end of reperfusion, the myocardial infarct size (IS) was determined by TTC staining, the activities of serum lactate dehydrogenase (LDH) was detected by colorimetry, myocardial specimens were collected for microscopic examination of the ultrastructure (with a electron microscope), the expression of phosphorylated Akt (p-Akt) and microtubule-associated protein 1 light chain 3 (LC3) was determined by Western blot and the ratio of LC3-Ⅱ/Ⅰwas calculated. Results:Compared with Sham group, IS and the activities of serum of LDH were significantly increased, p-Akt expression in myocardial tissues was up-regulated, the ratio of LC3-Ⅱ/Ⅰwas increased ( P<0.05), autophagic vacuoles were increased and the damage of ultrastructure of cardiomyocytes was obvious in group IR.Compared with group IR, IS and the activities of serum of LDH were significantly decreased, p-Akt expression in myocardial tissues was up-regulated, the ratio of LC3-Ⅱ/Ⅰwas decreased ( P<0.05), autophagic vacuoles were decreased and the damage of ultrastructure of cardiomyocytes was attenuated in group HR.Compared with group HR, IS and the activities of serum of LDH were significantly increased, p-Akt expression in myocardial tissues was down-regulated, the ratio of LC3-Ⅱ/Ⅰwas increased ( P<0.05), autophagic vacuoles were increased and the damage of ultrastructure of cardiomyocytes was aggravated in group HR+ W. Conclusion:The mechanism of protective effect of hydromorphone postconditioning on myocardium is related to activation of PI3K/Akt signaling pathway and inhibition of autophagy in rats.

19.
Article in Chinese | WPRIM | ID: wpr-911201

ABSTRACT

Objective:To investigate the relationship between the mechanism of exogenous hydrogen sulfide (H 2S)-induced reduction of apoptosis in neurons during focal cerebral ischemia-reperfusion (I/R) and PINK1/Parkin pathway-mediated mitochondrial autophagy in rats. Methods:Two hundred and sixteen healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 250-270 g, were divided into 4 groups ( n=54 each) using a random number table method: control group (group C), I/R group, H 2S group and H 2S plus 3-methyladenine (3-MA) group (H 2S+ 3-MA group). Focal cerebral ischemia was induced by middle cerebral artery occlusion in anesthetized rats.In group H 2S+ 3-MA, 3-MA 10 mg/kg was intraperitoneally injected at 15 min before the onset of reperfusion, while the equal volume of normal saline was given instead in the other groups.In H 2S and H 2S+ 3-MA groups, 0.25% NaSH (a donor of exogenous H 2S) 10 mg/kg was intraperitoneally injected at the onset of reperfusion, while the equal volume of normal saline was given instead in the other groups.At 1, 3 and 7 days of reperfusion, neural function was scored, and corner test (the percentage of left turn was calculated) was performed.Brains were removed and brain tissues were obtained for determination of the cerebral infarct size, Bax, Bcl-2 and caspase-3 positive cells, cell apoptosis, and expression of mitophagy-related protein microtubule-associated protein 1 light chain 3 (LC3), PINK1 and Parkin (by Western blot). The percentage of cerebral infarct size, rate of Bax, Bcl-2 and caspase-3 positive cells and apoptosis rate were calculated.The ratio of LC3-Ⅱexpression to LC3-Ⅰexpression (LC3-Ⅱ/LC3-Ⅰ) was also calculated. Results:Compared with group C, the neural function score was significantly decreased, the percentage of left turn, percentage of cerebral infarct size, rate of Bax, Bcl-2 and caspase-3 positive cells, apoptosis rate of neurons, and LC3-Ⅱ/LC3-Ⅰ were increased, and the expression of PINK1 and Parkin was up-regulated at each time point of reperfusion in group I/R ( P<0.05). Compared with group I/R, the neural function score and rate of Bcl-2 positive cells were significantly increased, the percentage of left turn, percentage of cerebral infarct size, rate of Bax and caspase-3 positive cells, and apoptosis rate of neurons were decreased, the expression of PINK1 and Parkin was up-regulated, and LC3-Ⅱ/LC3-Ⅰ were increased at each time point of reperfusion in group H 2S ( P<0.05), and no significant change was found in the parameters mentioned above in group H 2S+ 3-MA ( P>0.05). Compared with group H 2S, the neural function score and rate of Bcl-2 positive cells were significantly decreased, the percentage of left turn, percentage of cerebral infarct size, rate of Bax and caspase-3 positive cells, and apoptosis rate of neurons were increased, the expression of PINK1 and Parkin was down-regulated, and LC3-Ⅱ/LC3-Ⅰ was decreased at each time point of reperfusion in H 2S+ 3-MA group ( P<0.05). Conclusion:The mechanism by which exogenous H 2S inhibits apoptosis in neurons during focal cerebral I/R is related to enhancing mitochondrial autophagy mediated by the PINK1/Parkin pathway in rats.

20.
Article in Chinese | WPRIM | ID: wpr-911193

ABSTRACT

Objective:To evaluate the effect of methane on acetaminophen-induced acute liver injury (ALI) in mice and the role of autophagy.Methods:Forty clean-grade SPF healthy adult male C57BL/6 mice, aged 8-10 weeks, weighing 20-25 g, were divided into 4 groups ( n=10 each) using a random number table method: control group (group C), group ALI, methane-rich saline group (group MS) and methane-rich saline plus 3-methyladenine (3-MA) group (group MS+ 3-MA). Acetaminophen 300 mg/kg was injected intraperitoneally to establish ALI model.Group MS was injected intraperitoneally with methane-rich saline 10 ml/kg immediately after establishing the model and at 12 h after establishing the model.Group MS+ 3-MA was injected intraperitoneally with methane-rich saline 10 ml/kg and autophagy inhibitor 3-MA 30 mg/kg immediately after establishing the model and was injected intraperitoneally with methane-rich saline 10 ml/kg at 12 h after establishing the model.The equal volume of sterile saline was given intraperitoneally at the same time points in C and ALI groups.At 24 h after establishment of the model, blood samples from the eyeball were taken for measuring concentrations of alanine transaminase (ALT) and aspartate transaminase (AST) in serum (using biochemistry analyzer) and the concentrations of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in serum (using enzyme-linked immunosorbent assay). The animals were then sacrificed and liver tissues were removed for determination of microtubule-associated protein 1 light chain 3 (LC3) and p62 (by Western blot) and for the examination of the number of autophagosomes (under a transmission electron microscope). Results:Compared with group C, the concentrations of ALT, AST, TNF-α and IL-6 in serum were significantly increased, LC3Ⅱ/LC3Ⅰ ratio was increased, expression of p62 was up-regulated, and the number of autophagosomes was increased in liver tissues in ALI, MS and MS+ 3-MA groups ( P<0.05). Compared with group ALI, the concentrations of ALT, AST, TNF-α and IL-6 in serum were significantly decreased, LC3Ⅱ/LC3Ⅰ ratio was increased, expression of p62 was down-regulated, and the number of autophagosomes in liver tissues was increased in group MS, and AST concentration in serum was decreased and LC3Ⅱ/LC3Ⅰ ratio was increased in group MS+ 3-MA ( P<0.05). Compared with group MS, the concentrations of ALT, AST, TNF-α and IL-6 were significantly increased, the LC3 II/LC3 I ratio and the number of autophagosomes were decreased in lung tissues in group MS+ 3-MA ( P<0.05). Conclusion:The mechanism by which methane can reduce acetaminophen-induced ALI is related to enhancement of the level of autophagy in liver cells in mice.

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