Design, synthesis and degradation activity of BRD4-targeting ATTECs / 药学实践杂志

Objective To design and synthesize autophagic degraders targeting BRD4 based on autophagosome tethering compound (ATTEC) strategy and test their BRD4 degradation activity. Methods BRD4-targeting ATTECs were constructed by conjugating ispinesib that used as a LC3 ligand and JQ1 through a variety of alkane linkers. The final compounds were confirmed by 1H NMR, 13C NMR and ESI-MS, and their degradation activity in different cell lines were tested by Western Blot. Results Five BRD4-ATTEC molecules were successfully synthesized for the first time. Compound 4 showed moderate BRD4 degradation activity in different cell lines. Conclusion The novel BRD4 autophagic degraders were discovered, which expanded the applicability of targeted autophagic degradation via ATTEC.

Research progress in role of autophagy in diabetic wound healing and traditional Chinese medicine intervention / 中国中药杂志

Diabetic ulcer(DU) is a chronic and refractory ulcer which often occurs in the foot or lower limbs. It is a diabetic complication with high morbidity and mortality. The pathogenesis of DU is complex, and the therapies(such as debridement, flap transplantation, and application of antibiotics) are also complex and have long cycles. DU patients suffer from great economic and psychological pressure while enduring pain. Therefore, it is particularly important to promote rapid wound healing, reduce disability and mortality, protect limb function, and improve the quality of life of DU patients. By reviewing the relevant literatures, we have found that autophagy can remove DU wound pathogens, reduce wound inflammation, and accelerate ulcer wound healing and tissue repair. The main autophagy-related factors microtubule-binding light chain protein 3(LC3), autophagy-specific gene Beclin-1, and ubiquitin-binding protein p62 mediate autophagy. The traditional Chinese medicine(TCM) treatment of DU mitigates clinical symptoms, accelerates ulcer wound healing, reduces ulcer recurrence, and delays further deterioration of DU. Furthermore, under the guidance of syndrome differentiation and treatment and the overall concept, TCM treatment harmonizes yin and yang, ameliorates TCM syndrome, and treats underlying diseases, thereby curing DU from the root. Therefore, this article reviews the role of autophagy and major related factors LC3, Beclin-1, and p62 in the healing of DU wounds and the intervention of TCM, aiming to provide reference for the clinical treatment of DU wounds and subsequent in-depth studies.

iPSCs ameliorate hypoxia-induced autophagy and atrophy in C2C12 myotubes via the AMPK/ULK1 pathway

Background Duchenne muscular dystrophy (DMD) is an X-linked lethal genetic disorder for which there is no effective treatment. Previous studies have shown that stem cell transplantation into mdx mice can promote muscle regeneration and improve muscle function, however, the specific molecular mechanisms remain unclear. DMD suffers varying degrees of hypoxic damage during disease progression. This study aimed to investigate whether induced pluripotent stem cells (iPSCs) have protective effects against hypoxia-induced skeletal muscle injury. Results In this study, we co-cultured iPSCs with C2C12 myoblasts using a Transwell nested system and placed them in a DG250 anaerobic workstation for oxygen deprivation for 24 h. We found that iPSCs reduced the levels of lactate dehydrogenase and reactive oxygen species and downregulated the mRNA and protein levels of BAX/BCL2 and LC3II/ LC3I in hypoxia-induced C2C12 myoblasts. Meanwhile, iPSCs decreased the mRNA and protein levels of atrogin-1 and MuRF-1 and increased myotube width. Furthermore, iPSCs downregulated the phosphorylation of AMPKA and ULK1 in C2C12 myotubes exposed to hypoxic damage. Conclusions Our study showed that iPSCs enhanced the resistance of C2C12 myoblasts to hypoxia and inhibited apoptosis and autophagy in the presence of oxidative stress. Further, iPSCs improved hypoxia-induced autophagy and atrophy of C2C12 myotubes through the AMPK/ULK1 pathway. This study may provide a new theoretical basis for the treatment of muscular dystrophy in stem cells.

Role of LC3-associated phagocytosis in regulating infection / 中华微生物学和免疫学杂志

LC3-associated phagocytosis (LAP) is a special phagocytosis occurring at the intersection of the two pathways of phagocytosis and autophagy. A hallmark event of the LAP process is the recruitment of microtubule-associated proteinⅠlight chain type 3-Ⅱ(LC3Ⅱ) to the phagosome surface of the monolayer membrane structure. The LAP pathway relies on the functions of the RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein (Rubicon) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The LC3-associated phagosome (LAPosome) binds to the lysosome to digest and degrade the contents. In recent years, increasing studies have found that LAP plays an important role in the infections caused by pathogenic microorganisms including fungi and bacteria. LAP is a crucial way in the host to resist and degrade the infection of pathogenic microorganisms. However, some pathogenic microorganisms can effectively escape from LAP in the host and even use LAPosome as a place for colonization and replication. This article summarized the recent progress in the role of LAP in host defense against pathogenic microorganism infection and the significance of it in the occurrence and development of diseases.

Down-regulation of autophagy proteins is associated with higher mTOR expression in the placenta of pregnant women with preeclampsia

Autophagy is a lysosomal degradation pathway that removes protein aggregates and damaged organelles maintaining cellular integrity. It seems to be essential for cell survival during stress, starvation, hypoxia, and consequently to the placenta implantation and development. Preeclampsia (PE) is a multisystemic disorder characterized by the onset of hypertension associated or not with proteinuria and other maternal complications. Considering that the placenta seems to play an important role in the pathogenesis of PE, the objective of the present study was to evaluate protein levels of light chain protein (LC3), beclin-1, and the mammalian target of rapamycin (mTOR) in the placenta of pregnant women with PE. Placental tissues collected from 20 women with PE and 20 normotensive (NT) pregnant women were evaluated for LC3, beclin-1, and mTOR expression by qPCR and immunohistochemistry. The mRNA for LC3 and beclin-1 were significantly lower, while mTOR gene expression was significantly higher in the placenta of pregnant women with PE than in the NT group. Placentas of PE women showed significantly decreased protein expression of LC3-II and beclin-1, whereas mTOR was significantly increased compared with the NT pregnant women. There was a negative correlation between protein expression of mTOR and LC3-II in the placental tissue of PE women. In conclusion, the results showed autophagy deficiency suggesting that failure in this degradation process may contribute to the pathogenesis of PE; however, new studies involving cross-talk between autophagy and inflammatory molecular mechanisms might help to better understand the autophagy process in this obstetric pathology.

Effect of Danggui Niantongtang on Expression of Autophagy-related Proteins LC3， Beclin1 and p62 in Rats with Adjuvant Arthritis Differentiated into Wind-damp-heat Impediment / 中国实验方剂学杂志

ObjectiveTo observe the preventive and control effects of Danggui Niantongtang against adjuvant arthritis differentiated into wind-damp-heat impediment in rats and its influences on the expression of autophagy-related proteins microtubule-associated protein 1 light chain 3 （LC3）， homolog of yeast Atg6 （Beclin1） and p62. MethodThe six-week-old male SD rats were randomly divided into the normal group， wind-damp-heat impediment model group， low-， medium-， and high-dose Danggui Niantongtang （5.67， 11.34， 22.68 g·kg-1） groups， and methotrexate （MTX， 1.35 mg·kg-1） group， with 10 rats in each group. A rat model of adjuvant arthritis was established by subcutaneous injection of inactivated Mycobacterium tuberculosis into the tail root， followed by exposure to the manual climatic box for 16 d for inducing the wind-damp-heat impediment. The drugs were administered intragastrically on the day of immunization for 28 d. The general conditions of rats were observed and the swelling degree of toes and arthritis index （AI） were detected. The pathological changes in the synovial tissues of the knee joints were observed by hematoxylin-eosin （HE） staining. The mRNA expression levels of LC3， Beclin1， and p62 in the synovial tissues were measured by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， followed by the assay of their protein expression by Western blot and immunohistochemistry. ResultCompared with the normal group， the wind-damp-heat impediment model group exhibited significantly increased swelling degree of toes （P<0.01）， increased AI （P<0.01）， proliferated synovial cells （P<0.01）， up-regulated LC3 and Beclin1 protein and mRNA expression （P<0.01）， and down-regulated p62 protein and mRNA expression （P<0.01） after 16， 20， 24， 28-d medication. Compared with the wind-damp-heat impediment model group， each medication group displayed alleviated toe swelling and synovial hyperplasia to different degrees， decreased mRNA and protein expression levels of LC3 and Beclin1 （P<0.01）， and increased p62 mRNA and protein expression （P<0.05，P<0.01）， with the best outcomes observed in the medium-dose Danggui Niantongtang group. ConclusionDanggui Niantongtang effectively relieves adjuvant arthritis due to wind-damp-heat impediment in rats， which may be related to its regulation of the expression of autophagy-related proteins LC3， Beclin1， and p62 and the inhibition of autophagy.

Effect of Aspergillus fumigatus on the autophagic flux in murine macrophages: a preliminary study / 中华皮肤科杂志

Objective:To explore the effect of Aspergillus fumigatus ( A. fumigatus) on the autophagic flux in murine bone marrow-derived macrophages (BMDM) . Methods:Murine BMDM were in vitro cultured with heat-killed A. fumigatus for 0, 0.5, 4, and 12 hours. Then, cellular proteins were extracted, and Western blot analysis was performed to detect the conversion of the key autophagy protein microtubule-associated protein 1 light chain 3 (LC3) -Ⅰ to LC3-Ⅱ, and to determine the protein expression of phosphorylated mammalian target of rapamycin (p-mTOR) Ser2481. Additionally, murine BMDM were in vitro cultured with A. fumigatus alone or in combination with different lysosomal inhibitors, including the cysteine cathepsin inhibitor E-64d + pepstatin, bafilomycin-A1 (BAF-A1) , ammonium chloride (NH 4Cl) , and chloroquine, for 4 or 12 hours. Then, Western blot analysis was performed to investigate the effect of A. fumigatus on newly formed LC3-Ⅱ and basal autophagic flux, and confocal laser scanning fluorescence microscopy to analyze the colocalization of A. fumigatus with LC3 and Rubicon (a RUN domain Beclin-1-interacting and cysteine-rich-domain-containing protein) . Experimental results at different treatment time points were analyzed by using unpaired t test, and results of experiments evaluating the effect of two factors ( A. fumigatus spores and autophagosome inhibitors) were analyzed by 2 × 2 factorial analysis. Results:After in vitro co-culture with A. fumigatus for 0.5, 4, 12 hours, Western blot analysis showed that the conversion of LC3-Ⅰ to LC3-Ⅱ increased over time in murine BMDM compared with the control (0 hour) group ( t = 6.58, 3.28, 3.02, respectively, all P < 0.05) , but the protein expression level of p-mTOR (Ser2481) did not significantly differ at different treatment time points ( t = 0.441, 0.477, 0.382, P = 0.682, 0.660, 0.722, respectively) . After 4- and 12-hour in vitro treatment, the accumulation levels of LC3-Ⅱ in BMDM significantly increased in the A. fumigatus + chloroquine group compared with the chloroquine-alone group ( t = 2.13, 2.78, respectively, both P < 0.05) , in the A. fumigatus + NH 4Cl group compared with the NH 4Cl-alone group ( t = 2.92, 2.92, respectively, both P < 0.05) , in the A. fumigatus + BAF-A1 group compared with the BAF-A1-alone group ( t = 2.13, 2.13, respectively, both P < 0.05) , and in the A. fumigatus + E-64d + pepstatin group compared with the E-64d + pepstatin group ( t = 2.13, 2.92, respectively, both P < 0.05) . After 8-hour treatment with calcofluor white-labeled A. fumigatus spores, confocal laser scanning fluorescence microscopy showed that LC3 and Rubicon mainly surrounded A. fumigatus, suggesting their colocalization with A. fumigatus. Conclusion:A. fumigatus can in vitro increase the basal autophagic flux in murine BMDM.

Inhibition of nitric oxide production of activated mice peritoneal macrophages is independent of the Toxoplasma gondii strain

BACKGROUND Toxoplasma gondii causes toxoplasmosis and is controlled by activated macrophages. However, infection of macrophages by tachyzoites induces TGF-β signaling (TGF-s) inhibiting nitric oxide (NO) production. NO inhibition may be a general escape mechanism of distinct T. gondii strains. OBJECTIVES To evaluate in activated macrophages the capacity of T. gondii strains of different virulence and genetics (RH, type I; ME-49, type II; VEG, type III; P-Br, recombinant) to evade the NO microbicidal defense system and determine LC3 loading to the parasitophorous vacuole. METHODS Activated peritoneal macrophages were infected with the different T. gondii strains, NO-production was evaluated by the Griess reagent, and inducible nitric oxide synthase expression, TGF-s, and LC3 localisation assayed by immunofluorescence. FINDINGS Only RH persisted in macrophages, while VEG was more resistant than P-Br and ME-49. All strains induced TGF-s, degradation of inducible nitric oxide synthase, and NO-production inhibition from 2 to 24 h of infection, but only RH sustained these alterations for 48 h. By 24 h of infection, TGF-s lowered in macrophages infected by ME-49, and P-Br, and NO-production recovered, while VEG sustained TGF-s and NO-production inhibition longer. LC3 loading to parasitophorous vacuole was strain-dependent: higher for ME-49, P-Br and VEG, lower for RH. All strains inhibited NO-production, but only RH sustained this effect probably because it persisted in macrophages due to additional evasive mechanisms as lower LC3 loading to parasitophorous vacuole. MAIN CONCLUSIONS These results support that T. gondii can escape the NO microbicidal defense system at the initial phase of the infection, but only the virulent strain sustain this evasion mechanism.

Role of autophagy-related molecules Beclin1 and LC3 in multidrug-resistant Staphylococcus aureus infection in diabetic foot / 中国医师杂志

Objective:To investigate the role of autophagy-related molecules Beclin1 and LC3 in diabetic foot infection with multidrug resistant (MDR) Staphylococcus aureus.Methods:From June 2016 to October 2017, 62 patients with diabetic foot infected by Staphylococcus aureus were selected as the diabetic foot infection group, and 38 patients with foot burns were selected as the control group. Staphylococcus aureus isolation, identification and drug sensitivity test were carried out. According to drug resistance, the patients with diabetic foot infection were further divided into multidrug resistance group and non-multidrug resistance group. The granulation tissue of foot wound was collected and the expression of Beclin1 and LC3 was detected by immunohistochemistry. The expression of LC3 in macrophages was detected by immunofluorescence double staining.Results:The levels of glycosylated hemoglobin, white blood cell count and erythrocyte sedimentation rate in diabetic foot infection group were significantly higher than those in control group ( P<0.05). Multidrug-resistant Staphylococcus aureus was detected in 30 cases (48.39%) of 62 patients with diabetic foot infection, 2 cases (5.26%) of 38 patients in the control group, and the detection rate of multidrug-resistant Staphylococcus aureus in the diabetic foot infection group was significantly higher than that in the control group ( P<0.05). The white blood cell count, neutrophil ratio and C-reactive protein levels in MDR group were significantly higher than those in non-MDR group ( P<0.05). Immunohistochemical examination showed that the expression levels of Beclin1 and LC3 in wound granulatin tissue of diabetic foot infection group were significantly lower than those in control group ( P<0.01); the expression levels of Beclin1 and LC3 in wound granulation tissue of multidrug resistance group were significantly lower than those in non-multidrug resistance group ( P<0.05). Immunofluorescence double staining showed that the co-expression intensity of LC3 and CD14 in wound granulation tissue of diabetic foot infection group was significantly lower than that of control group, suggesting that LC3 expression in macrophages was significantly reduced. Conclusions:The expression of Beclin1 and LC3 in the wound granulation tissue of diabetic foot patients infected with Staphylococcus aureus decreased significantly, especially in those infected with multidrug resistant Staphylococcus aureus. The occurrence and development of multidrug resistance of Staphylococcus aureus in diabetic foot patients may be related to the decrease of autophagy level.

Galangin enhances autophagy by inhibiting NF-κB pathway in gastric cancer MGC-803 cells / 中国中药杂志

This study aimed to explore the effects of galangin on energy metabolism and autophagy in gastric cancer MGC803 cells and the underlying mechanism. Cell counting kit-8(CCK-8) was used to detect the effects of galangin at different concentrations on via-bility of MGC803 cells after 48 h intervention. Western blot was carried out to measure the effects of galangin on expression of proteins related to autophagy, nuclear factor-κB(NF-κB) pathway and energy metabolism, followed by the determination of its effects on mRNA expression of energy metabolism-related proteins by Real-time quantitative PCR(qPCR). The impact of galangin on autophagy was explored using AutophagyGreen dye reagent, with autophagosomes and lysosomes observed under the transmission electron microscope(TEM). Nude mice transplanted with gastric cancer MGC803 cells via subcutaneous injection were randomly divided into the following three groups: control(0.5% sodium carboxymethyl cellulose, once a day), 5-fluorouracil(5-FU, 50 mg·kg~(-1), twice a week), and galangin(120 mg·kg~(-1), once a day) groups. The body weight and tumor volume were measured once every three days with a vernier caliper at the same time point by the same person. After 21-d treatment, the tumor tissue was isolated and weighed for the calculation of the tumor-suppressing rate. The comparison with the control group revealed that galangin inhibited the viability of MGC803 cells, up-regulated the protein expression of microtuble-associated protein 1 light chain 3 B(LC3 B) Ⅱ, inhibited the phosphorylation of NF-κB pathway-related proteins, and promoted the formation of autophagosomes in MGC803 cells. However, it did not obviously affect the expression of energy metabolism-related proteins. Furthermore, galangin at 120 mg·kg~(-1) significantly reduced the tumor weight and volume in mice, enhanced LC3 BⅡ protein expression, and inhibited the phosphorylation of NF-κB pathway-related proteins. All these have suggested that galangin inhibited the growth of gastric cancer MGC803 cells both in vivo and in vitro, possibly by inhibiting the NF-κB pathway and enhancing autophagy.

Effect of advanced glycation end products on autophagic ability in osteoblasts / 中南大学学报(医学版)

OBJECTIVES@#Excessive production of AGEs in diabetic patients will affect the normal function of osteoblasts, and this process may be related to autophagy of osteoblasts. This study aims to explore the effect of advanced glycation end products (AGEs) on autophagic activity during osteogenic differentiation in rat bone marrow mesenchymal stem cells (BMSCs).@*METHODS@#BMSCs were isolated and cultured in vitro, treated with different concentrations (0, 50, 100, 200, and 400 mg/L) of AGEs for different time (3, 6, 12, 24, 48, and 72 h). The proliferation activity was detected by CCK-8 method. The mRNA and protein expression levels of Beclin1 and LC3 in cells were detected by real-time PCR and Western blotting, respectively.The autophagic vacuoles were observed under the transmission electron microscope. The cells were treated with autophagy promoter rapamycin or autophagy inhibitor 3MA. After 7 days of osteogenic induction, we performed alkaline phosphatase (ALP) staining and real-time PCR to detect the mRNA expression levels of osteogenesis-related genes.@*RESULTS@#In the low-concentration groups, the proliferation activity in BMSCs was increased (@*CONCLUSIONS@#Low concentration of AGEs can enhance the proliferative activity of BMSCs and promote osteogenic differentiation by accelerating autophagy. High concentration of AGEs can suppress the proliferation of BMSCs and inhibit osteogenic differentiation by reducing autophagy.

Effect of Qiyu Sanlong Decoction in Inducing Autophagy of A549 Cells by Regulating Molecular Expression Related to Signal Axis of mTOR-Beclin1-LC3 / 中国实验方剂学杂志

Objective:To observe the effect of Qiyu Sanlong decoction （QYSL） on the expressions of key molecules in signal axis of mammalian rapamycin target protein （mTOR）/yeast Atg6 homologous （Beclin1）/ microtubule-associated protein1 light chain3 （LC3） in A549 cells. Method:With A549 cells as the research object， the effect of QYSL medicated serum on cell viability of A549 cells were detected by cell counting kit-8 （CCK-8） method. The effect of QYSL decoction on A549 cell apoptosis， autophagosome formation and the expression of autophagy markers were detected by Terminal-deoxynucleoitidyl transferase mediated nick end labeling （TUNEL） method， transmission electron microscope （TEM）， Real-time polymerase chain reaction （Real-time PCR） and Western blot. Result:QYSL medicated serum could inhibit the viability of A549 cells in a concentration-dependent manner. Compared with the blank serum group， the number of apoptotic A549 cells in the QYSL medicated serum group was significantly increased （P<0.01）， and the formation of autophagosome was significantly increased. Compared with the blank serum group， the mRNA and protein expressions of mTOR in A549 cells in the QYSL serum group were significantly decreased （P<0.01）， while mRNA and protein expressions of Beclin-1， autophagy related genes 5 （ATG5）， autophagy related genes 13 （ATG13） were significantly increased （P<0.01）. Conclusion:QYSL decoction can induce autophagy in A549 cells， and its specific mechanism may be related to the down-regulation of mTOR expression， the up-regulation of Beclin1， ATG5， ATG13 and LC3 expression， and the promotion of LC3Ⅰ conversion to LC3Ⅱ.

Regulation of Buyang Huanwu Decoction extracts on apoptosis and autophagy of PC12 cells model with oxidative stress injury / 中草药

Objective: To explore the underlying mechanism of Buyang Huanwu Decoction extracts on apoptosis and autophagy in PC12 cells model with oxidative stress injury. Methods: Different level oxidative stress injury models with H2O2 at various concentrations were established. The effective concentrations of Buyang Huanwu Decoction extracts were determined by MTT method in the initial stage and the intensifying period after oxidative stress injury. The apoptosis of PC12 cells were evaluated by FCM and TUNNEL, the autophagy situations were observed by TEM and mRFP-GFP-LC3. Furthermore, the proteins of Bax, Bcl-2, Beclin1, LC3A, and LC3B related to apoptosis were determined by Western blotting. Results: The initial stage and the intensifying period of oxidative stress injury cell models were established by H2O2 at the concentration of 1.5 and 2.0 mmol/L, respectively. Compared with the control group, model group appeared increasing apoptosis and autophagy levels, and model group had higher expressions of Bax/Bcl-2, Beclin1, LC3B and lower expression of LC3A (P < 0.05). Compared with the initial stage of oxidative stress injury cell models, Buyang Huanwu Decoction extracts could reduce the Bax/Bcl-2 and restrain apoptosis rates, while the autophagy was activated by up-regulation Beclin1 and LC3B/LC3A (P < 0.05). When the serious apoptosis and excessive autophagy were observed in the intensifying period of oxidative stress injury cells, the extracts could play the protective effect by apoptosis restraining and autophagy alleviating. Conclusion: Buyang Huanwu Decoction extracts can play the protective effects on oxidative stress injury cell models in different period by regulating apoptosis and autophagy.

Detection of autophagy in peripheral CD4+ T lymphocytes and skin lesions of patients with herpes zoster / 中华皮肤科杂志

Objective@#To observe changes in expression of autophagy proteins in peripheral CD4+ T lymphocytes and the epidermis of skin lesions, as well as generation of autophagy vesicles in epidermal cells in skin lesions of patients with herpes zoster, and to explore the relationship between varicella-herpes zoster virus (VZV) infection and autophagy.@*Methods@#Totally, 35 patients with herpes zoster were enrolled from Department of Dermatology, General Hospital of Southern Theater Command of PLA between December 2017 and December 2018, including 20 males and 15 females. Their age ranged from 18 to 79 (59.23 ± 9.27) years, pain duration was 5.14 ± 2.28 days, and lesion duration (from the onset of the lesion to the clinic visit) was 3.45 ± 1.77 days. Flow cytometry was performed to determine the expression of autophagy proteins including microtubule-associated protein 1 light chain 3B (LC3B) , Beclin-1 and p62 in peripheral blood CD4+ T lymphocytes of these patients. Thirty healthy adults served as control group. Lesional skin tissues were obtained from 12 patients with herpes zoster, and perilesional normal skin tissues of the same patient served as the control. Immunohistochemical study was conducted to determine the expression of autophagy proteins LC3B, Beclin-1 and p62 in epidermal tissues, and transmission electron microscopy to observe the generation of autophagy vesicles in epidermal cells. Two independent-sample t-test was carried out for intergroup comparison.@*Results@#The expression rates of autophagy proteins LC3B and Beclin-1 in peripheral CD4+ T lymphocytes were significantly higher in the herpes zoster group (61.23% ± 7.61%, 35.84% ± 4.22%, respectively) than in the control group (36.56% ± 4.27%, 15.34% ± 1.89%, respectively; t = 15.75, 24.56 respectively, both P < 0.01) , while the expression rate of p62 (5.75% ± 0.67%) was significantly lower in the herpes zoster group than in the control group (10.03% ± 1.15%, t = 18.65, P < 0.01) . Among the 12 patients with herpes zoster, the expression levels of LC3B and Beclin-1 in the epidermis were significantly higher in the skin lesions than in the perilesional normal skin tissues (t = 2.86, 4.58, P < 0.05) , but the expression level of p62 was significantly lower in the skin lesions than in the perilesional normal skin tissues (t = 2.43, P < 0.05) . Transmission electron microscopy showed formation of autophagy vesicles containing virus particles in epidermal cells in the skin lesions of 12 patients with herpes zoster, and vesicle counts were significantly higher in the skin lesions than in perilesional normal skin tissues (t = 9.67, P < 0.01) .@*Conclusion@#The autophagy level was elevated in peripheral CD4+ T lymphocytes and epidermis of skin lesions of patients with herpes zoster.

Effect of Qiyu Sanlong Decoction in Inhibiting Growth of Lung Cancer by Regulating Expressions of Beclin1, Atg5 and LC3B / 中国实验方剂学杂志

Objective::To study the effect of Qiyu Sanlong decoction on the growth of subcutaneous tumor in lung cancer mice and the expressions of key autophagy molecule, yeast Atg6 homologous (Beclin1), autophagy related genes5 (Atg5), and microtubule-associated protein1 light chain3 (LC3B). Method::Lewis lung carcinoma cells (LLC) were used to reproduce the lung cancer mice transplanted model. After the modeling, the mice were randomly divided into model group, Qiyu Sanlong decoction group, chemotherapy group and combination group, with 18 transplanted mice in each group. In model group, mice were fed with 0.9% saline 20 mL·kg-1 daily. In Qiyu Sanlong decoction group, mice were fed with Qiyu Sanlong decoction 80.48 g·kg-1 daily. The chemotherapy group was intraperitoneally injected with 0.4 mL cisplatin solution (DDP) at the 1st, 3rd and 5th day. The combination group was orally given the drugs at the concentration of 80.48 g·kg-1, and 0.4 mL DDP solution was intraperitoneally injected at the 1st, 3rd and 5th day. After 21 days of continuous treatment, tumor tissue was exfoliated and weighed, and the tumor inhibition rate was calculated. Hematoxylin-eosin (HE) staining was used to observe the histological changes of tumor. The expressions and localizations of Beclin1 and LC3B in tumor tissues were detected by immunohistochemical staining. Protein expressions of Beclin1, Atg5, LC3B-Ⅰand LC3B-Ⅱ were determined by Western blot, and the ratio of LC3B-Ⅱ/LC3B-Ⅰ was calculated. The transcription levels of Beclin1, Atg5 mRNA in tumor tissues were detected by Real-time PCR. Result::Qiyu Sanlong decoction had a mild inhibitory effect on transplanted tumor, with an inhibitory rate of 31.2%. Under microscope, patchy necrotic tumor cells were observed in the tumor tissues of Qiyu Sanlong decoction group. Immunohistochemical staining and Western blot analysis showed that Qiyu Sanlong decoction could up-regulate the expressions of Beclin1, Atg5 and LC3B protein (P<0.01), and promote the conversion from LC3B-Ⅰ into LC3-Ⅱ compared with the model group. Real-time PCR results showed that Qiyu Sanlong decoction could promote the transcription of Beclin1 mRNA and Atg5 mRNA compared with the model group (P<0.01). Conclusion::Qiyu Sanlong decoction has a mild inhibitory effect on lung tumors, and its mechanism may be related to up-regulating the expressions of autophagy key proteins Beclin1, Atg5 and LC3B, and promoting the conversion from LC3B-Ⅰ to LC3B-Ⅱ.

Effect of Danggui Yinzi on Relieving Allergic Reaction in CU Model Mice by Autophagy Pathway / 中国实验方剂学杂志

Objective:To investigate the effect of Danggui Yinzi on allergic reaction in chronic urticaria (CU) mice model and the mechanism of autophagy intervention. Method:The SPF BALB/c mice were used to replicate the CU mice model by intraperitoneal injection of ovalbumin and aluminum hydroxide suspension. The animals were randomly allocated into six groups: a normal group (normal saline 20 mL·kg-1·d-1), a model group (normal saline 20 mL·kg-1·d-1), a loratadine group(0.001 3 g·kg-1·d-1), a Danggui Yinzi high，medium and low-dose group（39.3，19.6，9.8 g·kg-1·d-1）. The pathological changes of skin tissues were observed by hematoxylin-eosin (HE) staining. Morphological changes of autophagy in skin tissues epithelial cells were observed by transmission electron microscope. The mRNA levels of microtubule-associated protein 1 light chain 3B(LC3B) and ubiquitin-binding protein p62 mRNA in skin tissues were detected by real-time quantitative polymerase chain reaction (Real-time PCR). The expressions of LC3B and p62 in skin tissues were detected by immunohistochemistry (IHC). Result:Danggui Yinzi can significantly improve the pathological manifestations of dermal edema, collagen bundles separation, telangiectasia in CU mice， it can also improve autophagosomes formation and abnormal cell ultrastructure such as nuclear chromatin condensation, mitochondrial swelling, endoplasmic reticulum expansion, etc. Compared with the normal group, the protein expressions of LC3B in skin tissues of the model group was significantly increased (P<0.01), LC3B mRNA level was increased too, while p62 mRNA levels and its protein expressions were decreased-regulated (P<0.01). Compared with the model group, levels of LC3B mRNA and protein expressions of the Danggui Yinzi groups were significantly increased (P<0.05,P<0.01), while p62 mRNA levels and its protein expressions were significantly decreased-regulated (P<0.05,P<0.01). Conclusion:Danggui Yinzi can regulate the expression of LC3B, p62 mRNA and protein expressions, enhance the level of autophagy, and improve the pathological state of CU mice.

Escin alleviates chemotherapy-induced peripheral neuropathic pain by inducing autophagy in the spinal cord of rats / 南方医科大学学报

OBJECTIVE@#To investigate the effect of escin in relieving chemotherapy-induced peripheral neuropathic pain in rats and explore and the underlying mechanism.@*METHODS@#Eighteen SD rats were randomly divided into 3 groups (@*RESULTS@#The rats in both the escin preconditioning group and escin postconditioning group showed obviously increased thresholds of mechanical allodynia and thermal hyperalgesia as compared with those in the control group (@*CONCLUSIONS@#Escin can alleviate chemotherapy-induced peripheral neuropathic pain in rats possibly by upregulating the expressions of autophagy-related proteins in the spinal cord.

High expression of LC3A, LC3B, and p62/SQSTM1 autophagic proteins in human colonic ganglion cells

@#Introduction: Autophagy is a mechanism that degrades large damaged organelles and misfolded proteins to maintain the homeostasis in all cells. It plays double-faceted roles in tumourigenesis and prevention of various cancers. In our side observation of investigating the prognostic value of autophagy in colorectal cancer (CRC), we found high expression of autophagy proteins (LC3A, LC3B, and p62/SQSTM1) in the colonic ganglion cells. To our best understanding, this is the first paper reporting such finding. Materials and Methods: Formalin-fixed paraffin-embedded (FFPE) CRC tissues blocks were retrieved and confirmed by haematoxylin & eosin (H&E) staining. Immunohistochemistry (IHC) targeting autophagy proteins (LC3A, LC3B, and p62/SQSTM1) was then performed followed by pathological examination. Results: All three autophagy proteins were present in both normal and tumour tissues of CRC patients. Interestingly, high expression of autophagy proteins in colonic ganglion cells was consistently seen regardless of tissue type (normal or cancer) or tumour site (caecum, ascending, transverse, descending, sigmoid colon and rectum). Conclusions: This work highlights the high autophagic activities in human colonic ganglion cells.

Detection of autophagy in peripheral CD4+ T lymphocytes and skin lesions of patients with herpes zoster / 中华皮肤科杂志

Objective To observe changes in expression of autophagy proteins in peripheral CD4+ T lymphocytes and the epidermis of skin lesions,as well as generation of autophagy vesicles in epidermal cells in skin lesions of patients with herpes zoster,and to explore the relationship between varicella-herpes zoster virus (VZV) infection and autophagy.Methods Totally,35 patients with herpes zoster were enrolled from Department of Dermatology,General Hospital of Southern Theater Command of PLA between December 2017 and December 2018,including 20 males and 15 females.Their age ranged from 18 to 79 (59.23 ± 9.27) years,pain duration was 5.14 ± 2.28 days,and lesion duration (from the onset of the lesion to the clinic visit) was 3.45 ± 1.77 days.Flow cytometry was performed to determine the expression of autophagy proteins including microtubule-associated protein 1 light chain 3B (LC3B),Beclin-1 and p62 in peripheral blood CD4 + T lymphocytes of these patients.Thirty healthy adults served as control group.Lesional skin tissues were obtained from 12 patients with herpes zoster,and perilesional normal skin tissues of the same patient served as the control.Immunohistochemical study was conducted to determine the expression of autophagy proteins LC3B,Beclin-1 and p62 in epidermal tissues,and transmission electron microscopy to observe the generation of autophagy vesicles in epidermal cells.Two independent-sample t-test was carried out for intergroup comparison.Results The expression rates of autophagy proteins LC3B and Beclin-1 in peripheral CD4 + T lymphocytes were significantly higher in the herpes zoster group (61.23％ ± 7.61％,35.84％ ± 4.22％,respectively) than in the control group (36.56％ ± 4.27％,15.34％ ± 1.89％,respectively;t =15.75,24.56 respectively,both P ＜ 0.01),while the expression rate of p62 (5.75％ ± 0.67％) was significantly lower in the herpes zoster group than in the control group (10.03％ ± 1.15％,t =18.65,P ＜ 0.01).Among the 12 patients with herpes zoster,the expression levels of LC3B and Beclin-1 in the epidermis were significantly higher in the skin lesions than in the perilesional normal skin tissues (t =2.86,4.58,P ＜ 0.05),but the expression level of p62 was significantly lower in the skin lesions than in the perilesional normal skin tissues (t =2.43,P ＜ 0.05).Transmission electron microscopy showed formation of autophagy vesicles containing virus particles in epidermal cells in the skin lesions of 12 patients with herpes zoster,and vesicle counts were significantly higher in the skin lesions than in perilesional normal skin tissues (t =9.67,P ＜ 0.01).Conclusion The autophagy level was elevated in peripheral CD4+ T lymphocytes and epidermis of skin lesions of patients with herpes zoster.

Effects of fine Particulate Matter(PM2.5) on the autophagy in human skin melanocytes / 公共卫生与预防医学

Objective  To study the effects of fine particulate matter(PM2.5) on the autophagy in human melanocytes. Methods PM2.5 was collected in Huanggang city. Then the water-soluble extracts and non-water-soluble extracts were isolated and the melanocytes were exposed to a W-PM2.5 or NW-PM2.5 with various concentrations. Autophagy of melanocytes was observed by MDC staining, and the expression of LC3-II /I were detected by immunohistochemistry and western blot. Results With the increase of concentration of W-PM2.5 and NW-PM2.5, the expression of melanocyte autophagosome and LC3-II /I increased, and the autophagy response was significantly enhanced（P2.5 components had a stronger effect on promoting LC3II/I expression than W-PM2.5 components（P2.5 can induce oxidative stress injury and excessive autophagy of melanocytes.