Inhibition of anlotinib-induced autophagy attenuates invasion and migration by regulating epithelial-mesenchymal transition and cytoskeletal rearrangement through ATG5 in human osteosarcoma cells

Abstract The cure rates for osteosarcoma have remained unchanged in the past three decades, especially for patients with pulmonary metastasis. Thus, a new and effective treatment for metastatic osteosarcoma is urgently needed. Anlotinib has been reported to have antitumor effects on advanced osteosarcoma. However, both the effect of anlotinib on autophagy in osteosarcoma and the mechanism of anlotinib-mediated autophagy in pulmonary metastasis are unclear. The effect of anlotinib treatment on the metastasis of osteosarcoma was investigated by transwell assays, wound healing assays, and animal experiments. Related proteins were detected by western blotting after anlotinib treatment, ATG5 silencing, or ATG5 overexpression. Immunofluorescence staining and transmission electron microscopy were used to detect alterations in autophagy and the cytoskeleton. Anlotinib inhibited the migration and invasion of osteosarcoma cells but promoted autophagy and increased ATG5 expression. Furthermore, the decreases in invasion and migration induced by anlotinib treatment were enhanced by ATG5 silencing. In addition, Y-27632 inhibited cytoskeletal rearrangement, which was rescued by ATG5 overexpression. ATG5 overexpression enhanced epithelial-mesenchymal transition (EMT). Mechanistically, anlotinib-induced autophagy promoted migration and invasion by activating EMT and cytoskeletal rearrangement through ATG5 both in vitro and in vivo. Our results demonstrated that anlotinib can induce protective autophagy in osteosarcoma cells and that inhibition of anlotinib-induced autophagy enhanced the inhibitory effects of anlotinib on osteosarcoma metastasis. Thus, the therapeutic effect of anlotinib treatment can be improved by combination treatment with autophagy inhibitors, which provides a new direction for the treatment of metastatic osteosarcoma.

Serum autophagy-related gene 5 level in stroke patients: correlation with CD4+ T cells and cognition impairment during a 3-year follow-up

Abstract Autophagy-related gene (ATG) 5 regulates blood lipids, chronic inflammation, CD4+ T-cell differentiation, and neuronal death and is involved in post-stroke cognitive impairment. This study aimed to explore the correlation of serum ATG5 with CD4+ T cells and cognition impairment in stroke patients. Peripheral blood was collected from 180 stroke patients for serum ATG5 and T helper (Th) 1, Th2, Th17, and regulatory T (Treg) cell detection via enzyme-linked immunosorbent assays and flow cytometry. The Mini-Mental State Examination (MMSE) scale was completed at enrollment, year (Y)1, Y2, and Y3 in stroke patients. Serum ATG5 was also measured in 50 healthy controls (HCs). Serum ATG5 was elevated in stroke patients compared to HCs (P<0.001) and was positively correlated to Th2 cells (P=0.022), Th17 cells (P<0.001), and Th17/Treg ratio (P<0.001) in stroke patients but not correlated with Th1 cells, Th1/Th2 ratio, or Treg cells (all P>0.050). Serum ATG5 (P=0.037), Th1 cells (P=0.022), Th17 cells (P=0.002), and Th17/Treg ratio (P=0.018) were elevated in stroke patients with MMSE score-identified cognition impairment vs those without cognition impairment, whereas Th2 cells, Th1/Th2 ratio, and Treg cells were not different between them (all P>0.050). Importantly, serum ATG5 was negatively linked with MMSE score at enrollment (P=0.004), Y1 (P=0.002), Y2 (P=0.014), and Y3 (P=0.001); moreover, it was positively related to 2-year (P=0.024) and 3-year (P=0.012) MMSE score decline in stroke patients. Serum ATG5 was positively correlated with Th2 and Th17 cells and estimated cognitive function decline in stroke patients.

Silencing of matrix metalloprotease-12 delays the progression of castration-resistant prostate cancer by regulating autophagy and lipolysis

Abstract The complex pathogenesis of castration-resistant prostate cancer (CRPC) makes it challenging to identify effective treatment methods. Matrix metalloproteinase (MMP)-12 can degrade elastin as well as various extracellular matrix (ECM) components, which is associated with cancer progression. However, the relationship between MMP-12 and CRPC progression is poorly understood. In this study, we observed the effect of MMP-12 on the progression of CRPC and further explored its potential mechanism of action. High levels of MMP-12 were observed in patients with CRPC. We therefore developed cell co-culture and mouse models to study the function of MMP-12. Silencing MMP-12 in CRPC cells disrupted lipid utilization and autophagy marker expression via the CD36/CPT1 and P62/LC3 pathways, respectively, leading to reduced CRPC cell migration and invasion. Moreover, animal experiments confirmed that MMP-12-knockdown CRPC xenograft tumors exhibited reduced tumor growth, and the mechanisms involved the promotion of cancer cell autophagy and the inhibition of lipid catabolism. According to our results, MMP-12 played important roles in the progression of CRPC by disrupting adipocyte maturation and regulating cancer migration and invasion via the modulation of autophagy and lipid catabolism pathways.

Electroacupuncture Promotes Functional Recovery after Facial Nerve Injury in Rats by Regulating Autophagy via GDNF and PI3K/mTOR Signaling Pathway / 中国结合医学杂志

OBJECTIVE@#To explore the mechanism of electroacupuncture (EA) in promoting recovery of the facial function with the involvement of autophagy, glial cell line-derived neurotrophic factor (GDNF), and phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway.@*METHODS@#Seventy-two male Sprague-Dawley rats were randomly allocated into the control, sham-operated, facial nerve injury (FNI), EA, EA+3-methyladenine (3-MA), and EA+GDNF antagonist groups using a random number table, with 12 rats in each group. An FNI rat model was established with facial nerve crushing method. EA intervention was conducted at Dicang (ST 4), Jiache (ST 6), Yifeng (SJ 17), and Hegu (LI 4) acupoints for 2 weeks. The Simone's 10-Point Scale was utilized to monitor the recovery of facial function. The histopathological evaluation of facial nerves was performed using hematoxylin-eosin (HE) staining. The levels of Beclin-1, light chain 3 (LC3), and P62 were detected by immunohistochemistry (IHC), immunofluorescence, and reverse transcription-polymerase chain reaction, respectively. Additionally, IHC was also used to detect the levels of GDNF, Rai, PI3K, and mTOR.@*RESULTS@#The facial functional scores were significantly increased in the EA group than the FNI group (P<0.05 or P<0.01). HE staining showed nerve axons and myelin sheaths, which were destroyed immediately after the injury, were recovered with EA treatment. The expressions of Beclin-1 and LC3 were significantly elevated and the expression of P62 was markedly reduced in FNI rats (P<0.01); however, EA treatment reversed these abnormal changes (P<0.01). Meanwhile, EA stimulation significantly increased the levels of GDNF, Rai, PI3K, and mTOR (P<0.01). After exogenous administration with autophagy inhibitor 3-MA or GDNF antagonist, the repair effect of EA on facial function was attenuated (P<0.05 or P<0.01).@*CONCLUSIONS@#EA could promote the recovery of facial function and repair the facial nerve damages in a rat model of FNI. EA may exert this neuroreparative effect through mediating the release of GDNF, activating the PI3K/mTOR signaling pathway, and further regulating the autophagy of facial nerves.

Quercetin Alleviates Lipopolysaccharide-Induced Cardiac Inflammation via Inhibiting Autophagy and Programmed Cell Death / 生物医学与环境科学（英文）

OBJECTIVE@#The aim of this study is to explore the potential modulatory role of quercetin against Endotoxin or lipopolysaccharide (LPS) induced septic cardiac dysfunction.@*METHODS@#Specific pathogen-free chicken embryos ( n = 120) were allocated untreated control, phosphate buffer solution (PBS) vehicle, PBS with ethanol vehicle, LPS (500 ng/egg), LPS with quercetin treatment (10, 20, or 40 nmol/egg, respectively), Quercetin groups (10, 20, or 40 nmol/egg). Fifteen-day-old embryonated eggs were inoculated with abovementioned solutions via the allantoic cavity. At embryonic day 19, the hearts of the embryos were collected for histopathological examination, RNA extraction, real-time polymerase chain reaction, immunohistochemical investigations, and Western blotting.@*RESULTS@#They demonstrated that the heart presented inflammatory responses after LPS induction. The LPS-induced higher mRNA expressions of inflammation-related factors (TLR4, TNFα, MYD88, NF-κB1, IFNγ, IL-1β, IL-8, IL-6, IL-10, p38, MMP3, and MMP9) were blocked by quercetin with three dosages. Quercetin significantly decreased immunopositivity to TLR4 and MMP9 in the treatment group when compared with the LPS group. Quercetin significantly decreased protein expressions of TLR4, IFNγ, MMP3, and MMP9 when compared with the LPS group. Quercetin treatment prevented LPS-induced increase in the mRNA expression of Claudin 1 and ZO-1, and significantly decreased protein expression of claudin 1 when compared with the LPS group. Quercetin significantly downregulated autophagy-related gene expressions (PPARα, SGLT1, APOA4, AMPKα1, AMPKα2, ATG5, ATG7, Beclin-1, and LC3B) and programmed cell death (Fas, Bcl-2, CASP1, CASP12, CASP3, and RIPK1) after LPS induction. Quercetin significantly decreased immunopositivity to APOA4, AMPKα2, and LC3-II/LC3-I in the treatment group when compared with the LPS group. Quercetin significantly decreased protein expressions of AMPKα1, LC3-I, and LC3-II. Quercetin significantly decreased the protein expression to CASP1 and CASP3 by immunohistochemical investigation or Western blotting in treatment group when compared with LPS group.@*CONCLUSION@#Quercetin alleviates cardiac inflammation induced by LPS through modulating autophagy, programmed cell death, and myocardiocytes permeability.

MiRNA Regulating Autophagy Signaling Pathway Induced by Cerebral Ischemia/Reperfusion / 中山大学学报(医学科学版)

Ischemia and hypoxia cause functional damage to brain tissues during stroke， and when blood supply is restored to brain tissues after ischemia， a large number of free radicals and calcium overload cause cerebral ischemia-reperfusion injury， which further aggravates the condition. Autophagy is a self-protection mechanism that maintains the homeostasis of the intracellular environment， but excessive autophagy causes brain tissue damage. MiRNA is a small endogenous non-coding RNA molecule that regulate various physiological activities at the gene level by binding to complementary sequences in the 3 '- UTR of its target gene mRNA， leading to translation inhibition or mRNA degradation. MiRNA not only directly acts on autophagy related proteins， but also participates in autophagy regulation induced by ischemia/reperfusion through various signaling pathways. However， there is still a lack of systematic induction and analysis of miRNA regulation of autophagy signaling pathways induced by cerebral ischemia/reperfusion. This article reviews the regulation of cellular autophagy during cerebral ischemia/ reperfusion by miRNA-124， miRNA-298， miRNA-202-5p， miRNA-142， miRNA-26b and so on through different signaling pathways， providing a systematic and theoretical approach for the study of autophagy in stroke.

Dihydroartemisinin Suppresses Proliferation of Oral Squamous Cell Carcinoma Cells by Promoting Autophagy / 肿瘤防治研究

Objective To investigate the effect of dihydroartemisinin (DHC) on the proliferation capacity of human oral squamous carcinoma cells and its mechanism of action. Methods The viability and colony formation ability of CAL27 cells treated with different concentrations of dihydroartemisinin was measured by CCK-8 and colony formation assay. The expression of proteins related to proliferation and autophagy was determined by Western blot. Potential targets for DHA inhibition of the biological behavior of oral cancer were screened based on network pharmacology and bioinformatics. Measurement was conducted after the cells were cotreated with autophagy blocker 3-methyladenine and autophagy inducers rapamycin and dihydroartemisinin. Results Dihydroartemisinin significantly reduced the proliferation viability and clone formation ability of CAL27 cells in a concentration-dependent manner. The PCNA expression level also decreased substantially. DHA suppressed oral cancer targets involving autophagy-related pathways. DHA intervention increased the expression of intracellular autophagy-related proteins Beclin-1 and LC3. After co-treatment of DHA combined with autophagy blocker, the proliferation viability and clone formation ability of CAL27 cells decreased. The expression of PCNA increased, and the expression of Beclin-1 and LC3 decreased. Conclusion Dihydroartemisinin could inhibit the proliferative capacity of oral squamous carcinoma cells in vitro, and its effect may be correlated with the induction of autophagy.

Attenuation and synergism of Hugan buzure recipe combined with oxaliplatin on hepatocellular carcinoma tumor bearing nude mice and its mechanism / 中国药房

OBJECTIVE To investigate the attenuation and synergism of Hugan buzure recipe （HBR） combined with oxaliplatin on hepatocellular carcinoma tumor bearing nude mice and its mechanism. METHODS Eight nude mice were selected from 40 nude mice as the blank group （normal saline）， and the remaining nude mice were inoculated with hepatoma cells Huh7 to establish the tumor-bearing model. The 32 modeled nude mice were randomly allocated to four groups： model group （normal saline， ig）， HBR group （0.69 g/kg， ig）， oxaliplatin group （10 mg/kg， ip）， and combination group （intraperitoneal injection of 0.69 g/kg HBR+intragastric administration of 10 mg/kg oxaliplatin）， with 8 mice in each group. Administer drug/normal saline once a day for 32 consecutive days； administer subcutaneous injection once every 7 days for a total of 5 times. During the experiment， the general condition of nude mice in each group was observed， and the tumor volume was measured every 4 days. On the 30th day of administration， the thermal stimulation paw withdrawal latency of nude mice in each group were detected. The tumor inhibition rate， spleen coefficient， the number of red blood cells， white blood cells and platelets in the whole blood of nude mice in each group， and the content of aspartate aminotransferase （AST） and creatinine in serum were detected after the end of administration. HE staining was used to observe the pathological changes in tumor tissues in nude mice in each group. The expression of microtubule-associated protein 1 light chain 3 （LC3），selective autophagy adaptor protein p62， B-cell lymphoma-2 （Bcl-2）， Bcl-2 associated X protein （Bax）， and Caspase-3 protein in tumor tissues. RESULT Compared with the model group， the tumor volume， tumor weight， white blood cells，red blood cells in the whole blood and spleen coefficients of nude mice in the oxaliplatin group were significantly decreased （P＜0.01）； the thermal stimulation paw withdrawal latency， AST and creatinine in serum were significantly increased （P＜0.05 or P＜0.01）. Compared with the oxaliplatin group， the tumor volume and tumor weight of nude mice in the combination group were significantly decreased （P＜0.01）； the white blood cells， red blood cells and platelets in the whole blood and spleen coefficients of nude mice were significantly increased （P＜0.05 or P＜0.01）； the thermal stimulation paw withdrawal latency， AST and creatinine in serum were significantly decreased （P＜0.01）； the expression levels of LC3， Bax and Caspase-3 proteins in tumor tissues of nude mice were significantly increased （P＜0.01）， and the expression levels of p62 and Bcl-2 proteins were significantly decreased （P＜0.01）. CONCLUSIONS HBR enhances the tumor inhibition rate of oxaliplatin by inducing apoptosis and autophagy， and can alleviate the peripheral neurotoxicity， hematological toxicity， hepatorenal toxicity， and immune organ toxicity caused by oxaliplatin in nude mice.

Inhibitory Effect of Sesquiterpenoid M36 from Myrrha on Growth of Human Hepatoma Cells / 中国实验方剂学杂志

ObjectiveThe antitumor activity of sesquiterpenoid M36 isolated from Myrrha against human hepatoma HepG2 cells was investigated in this study. MethodHepG2 cells were treated with M36 at different concentrations （0， 2， 4， 6， 8， 10 μmol·L-1）. Firstly， the effects of M36 on the proliferation of human hepatoma HepG2 cells were detected by methyl thiazolyl tetrazolium （MTT）， colony formation assay， and EdU proliferation assay. Hoechst staining， flow cytometry analysis， and Western blot were used to explore the effect of M36 on the apoptosis of human hepatoma HepG2 cells. Acridine orange staining and western blotting were used to examine the effect of M36 on autophagy in HepG2 cells. Finally， Western blot was used to detect protein expression of cancer-related signaling pathways. ResultCompared with the blank group， M36 treatment significantly inhibited the proliferation of human hepatoma HepG2 cells （P<0.01）， and the half inhibitory concentration （IC50） value of M36 for 48 h was 5.03 μmol·L-1， in a dose- and time-dependent manner. M36 was also able to induce apoptosis and autophagy in human hepatoma HepG2 cells. After treatment with 8 μmol·L-1 M36 for 48 hours， the apoptosis rate of HepG2 cells was （42.03±9.65）% （P<0.01）. Compared with the blank group， HepG2 cells treated with 4 and 8 μmol·L-1 M36 for 48 h had a significant increase in cleaved poly ADP-ribose polymerase （cleaved-PARP） protein levels （P<0.01）. Acridine orange staining showed that autophagy was significantly activated in HepG2 cells treated with 4 and 8 μmol·L-1 M36 for 48 h compared with the blank group （P<0.01）， which was further verified by the up-regulation of microtubule-associated protein 1 light chain 3 Ⅱ （LC3 Ⅱ）. Western blot results showed that compared with the blank group， the levels of phosphorylated extracellular regulated protein kinase （p-ERK）， phosphorylated p38 mitogen-activated protein kinase （p-p38 MAPK）， phosphorylated c-Jun N-terminal kinase （p-JNK）， and its downstream nuclear transcription factors c-Jun and p-c-Jun protein were significantly increased in M36 group （P<0.05， P<0.01）. The mechanism may be related to the up-regulation of MAPK signaling pathway. ConclusionThe sesquiterpenoid M36 isolated from Myrrha inhibits the proliferation of human hepatoma HepG2 cells and promotes apoptosis and autophagy， which may be related to the activation of the MAPK signaling pathway.

Role of Autophagy in Ulcerative Colitis and Chinese Medicine Intervention： A Review / 中国实验方剂学杂志

Ulcerative colitis （UC） is a chronic inflammatory bowel disease with complex etiology. The pathogenesis of this disease， due to a combination of factors， is complex and has not yet been elucidated. Among them， intestinal mucosal barrier damage is the basic pathological change of UC. As a non-destructive response of cells， autophagy regulates intestinal mucosal immunity， inflammation， oxidative stress， and bacterial homeostasis through degradation and reabsorption to actively repair damaged intestinal mucosal barrier， exerting a key role in the occurrence and development of UC. The disease is mainly treated clinically with aminosalicylic acid preparations， glucocorticoids， and immunosuppressants. Western medicine treatment of the disease has a fast onset of effect， and the short-term efficacy is definite， but the long-term application is easy to be accompanied by more adverse reactions. Moreover， some drugs are expensive， bringing great physical and mental pain and economic burden to patients. Therefore， it is urgent to explore new therapies with stable efficacy and mild adverse effects. In recent years， a large number of studies have shown that Chinese medicine can regulate autophagy of the intestinal mucosa with multiple targets and effects and repair the intestinal mucosal barrier function， thereby inhibiting the development of UC. Many experiments have shown that the active ingredient or monomers and compound formulas of Chinese medicine can improve the immunity of the intestinal mucosa， inflammation， oxidative stress， and flora by regulating the level of autophagy to maintain the normal function of the intestinal mucosal barrier to effectively intervene in UC， providing a new measure for the prevention and treatment of UC. However， there is a lack of systematic review of Chinese medicine in regulating the level of autophagy in the intestinal mucosa for the prevention and treatment of UC. Therefore， based on the current research on UC， autophagy process， and Chinese medicine treatment， this article reviewed the relationship of autophagy and its key target proteins with UC to clarify the key role of autophagy in UC production and systematically summarized Chinese medicines targeting the regulation of autophagy to treat UC in recent years to provide new ideas for the treatment and drug development of UC.

Research Progress on Dual Role of Autophagy in Cancer and Intervention of Traditional Chinese Medicine / 中国实验方剂学杂志

With the change in environmental pollution and lifestyle， the incidence and mortality of cancer are increasing year by year， which is a serious threat to human life and health. Autophagy is a process in which eukaryotic cells use lysosomes to degrade cytoplasmic proteins and damaged organelles under the regulation of autophagy-related genes. It plays a dynamic inhibiting or promoting role in the occurrence and development of cancer and is involved in the regulation of tumor formation， proliferation， metastasis， and response to anticancer therapy. With the deepening of the research on the mechanism of cancer， a variety of cancer treatment methods have been established， such as chemotherapy， radiotherapy， surgery， immunotherapy， and gene therapy. Pharmacological or genetic inhibition of autophagy has been shown to enhance the lethal effect of various anticancer treatments on tumor cells， suggesting that inhibition of autophagy is an effective sensitization strategy in cancer therapy. Meanwhile， over-stimulation of autophagy may also provide a new method for the treatment of drug-resistant cancer cells with high apoptotic thresholds. As a treasure of Chinese culture， traditional Chinese medicine plays an important role in the adjuvant treatment of cancer with its advantages of multi-target， multi-pathway， and small side effects. In recent years， many positive results have been achieved in the study of natural autophagy regulatory factors of traditional Chinese medicine in cancer， and they have been widely verified in different autophagy regulatory models. This article outlines the mechanism of autophagy， summarizes the dual regulatory role of autophagy in tumor biology， and collects relevant studies published in the databases of China National Knowledge Infrastructure (CNKI） and Wanfang Data for nearly 10 years that affect the role of tumorigenesis and development by regulating autophagy. The article also collates autophagy rules of tumor cells induced by traditional Chinese medicine and its active ingredients， so as to provide a certain reference for the research on the development and application of anti-tumor drugs.

Screening and bioinformatics analysis of key autophagy-related genes in alcoholic hepatitis / 器官移植

Objective To screen key autophagy-related genes in alcoholic hepatitis (AH) and investigate potential biomarkers and therapeutic targets for AH. Methods Two AH gene chips in Gene Expression Omnibus (GEO) and autophagy-related data sets obtained from MSigDB and GeneCards databases were used, and the key genes were verified and obtained by weighted gene co-expression network analysis (WGCNA). The screened key genes were subject to gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein-protein interaction (PPI) and immune infiltration analyses. Messenger RNA (mRNA)- microRNA (miRNA) network was constructed to analyze the expression differences of key autophagy-related genes during different stages of AH, which were further validated by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) in the liver tissues of AH patients and mice. Results Eleven autophagy-related genes were screened in AH (EEF1A2, CFTR, SOX4, TREM2, CTHRC1, HSPB8, TUBB3, PRKAA2, RNASE1, MTCL1 and HGF), all of which were up-regulated. In the liver tissues of AH patients and mice, the relative expression levels of SOX4, TREM2, HSPB8 and PRKAA2 in the AH group were higher than those in the control group. Conclusions SOX4, TREM2, HSPB8 and PRKAA2 may be potential biomarkers and therapeutic targets for AH.

Protective Mechanism of Paeoniflorin on Mice with Ulcerative Colitis Based on AMPK/mTOR Autophagy Pathway / 中国实验方剂学杂志

ObjectiveTo explore the protective mechanism of paeoniflorin on mice with ulcerative colitis （UC） through the adenosine monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） autophagy pathway. MethodUC mouse model was established by allowing mice freely drink 4% DSS， and 56 BALB/c male mice were randomly divided into model group， AMPK inhibitor group （20 mg·kg-1）， paeoniflorin （50 mg·kg-1） + inhibitor （20 mg·kg-1） group， and high dose （50 mg·kg-1）， medium dose （25 mg·kg-1）， and low dose （12.5 mg·kg-1） paeoniflorin groups. After seven days of drug intervention， the protective effect of paeoniflorin on mice with UC was determined by comparing the body weight， disease activity index （DAI） changes， and Hematoxylin-eosin （HE） staining results. Enzyme linked immunosorbent assay （ELISA） was used to detect the levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in the serum of mice in each group， and immunofluorescence was utilized to detect microtubule-associated protein 1 light chain 3 （LC3） content in the colon， AMPK， mTOR proteins， and their phosphorylated proteins including p-AMPK and p-mTOR in the colon tissue were detected by Western blot， and the mRNA expression levels of AMPK， mTOR， Beclin1， LC3， and p62 were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultCompared with the blank group， the model group showed a decrease in body mass， an increase in DAI score， and severe pathological damage to the colon. The levels of inflammatory factors including TNF-α and IL-6 increased in serum （P<0.01）， while the protein levels of LC3 and p-AMPK/AMPK were down-regulated in colon tissue， and those of p-mTOR/mTOR were up-regulated （P<0.01）. The mRNA expression levels of AMPK and LC3 were down-regulated， while the mRNA expression levels of mTOR and p62 were up-regulated （P<0.01）. Compared with the model group and the paeoniflorin + inhibitor group， the mice treated with paeoniflorin showed an increase in body mass， a decrease in DAI score， a reduction in pathological damage to colon tissue， and a reduction in the levels of inflammatory factors of TNF-α and IL-6 in serum （P<0.05）. The protein levels of LC3 and p-AMPK/AMPK in colon tissue were up-regulated， while the protein levels of p-mTOR/mTOR were down-regulated （P<0.01）. The mRNA expression levels of AMPK， Beclin1， and LC3 were up-regulated， while the mRNA expression of mTOR and p62 were down-regulated （P<0.01）. The colon tissue of the inhibitor group was severely damaged， and the trend of various indicators was completely opposite to that of the high dose paeoniflorin group. ConclusionPaeoniflorin can enhance autophagy and reduce inflammatory damage in mice with UC by activating the AMPK/mTOR signaling pathway and thus play a protective role.

Traditional Chinese Medicine Regulates Autophagy to Prevent and Treat Osteoarthritis： A Review / 中国实验方剂学杂志

Osteoarthritis （OA） is a common chronic， highly prevalent， painful， and disabling degenerative joint disease. It has imposed a heavy burden on social healthcare and patients' psychology and economy due to its clinical symptoms such as impaired joint mobility and severe joint pain and the immature therapies. Studies have shown that OA is closely associated with articular cartilage dysfunction， synthesis and degradation disorders of chondrocyte extracellular matrix （ECM）， and joint inflammation. Moderate autophagy can restore the function of damaged chondrocytes， regulate chondrocyte apoptosis， and promote the synthesis and metabolism of ECM to alleviate the inflammation of joints and delay the onset and progression of OA. According to the clinical symptoms， OA can be classified into the category of impediment in traditional Chinese medicine. With the theories of holistic conception， treatment based on syndrome differentiation， and individualised diagnosis and treatment， traditional Chinese medicine has demonstrated definite effects in the treatment of OA in thousands of years of practice. Moreover， traditional Chinese medicine causes mild adverse reactions， and the patients have high tolerance and acceptance. This paper briefly explains the roles of autophagy and the related regulatory proteins， such as Unc-51-like autophagy-activated kinase 1 （ULK1）， Beclin-1， and microtubule-associated protein light chain 3 （LC3）， and details the latest research achievements in the prevention and control of OA by traditional Chinese medicines and its related markers via the regulation of autophagy， so as to provide a idea for the in-depth research in this field and the clinical application of traditional Chinese medicine in preventing and treating OA.

Antitumor Effect of Fagopyri Dibotryis Rhizoma Extract on Colorectal Cancer Cells Through ROS/MAPK Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effects and underlying mechanisms of Fagopyri Dibotryis Rhizoma extract on the proliferation， apoptosis， and autophagy of human colorectal cancer HCT-116 cells. MethodFirstly， cellular activity was detected by the cell proliferation and activity-8 （CCK-8） assay， and the half inhibition rate （IC50） was calculated. Blank group and Fagopyri Dibotryis Rhizoma group （2， 4， 8 mg·L-1） were set. The effect of Fagopyri Dibotryis Rhizoma on the proliferation of HCT-116 cells was observed by cloning experiments， and that of Fagopyri Dibotryis Rhizoma on apoptosis was observed by flow cytometry. The expressions of autophagy-related proteins， p38 mitogen-activated protein kinase （p38 MAPK）， extracellular signal-regulated kinase （ERK）， c-Jun amino-terminal kinase （JNK）， phosphorylated （p）-p38， p-ERK， p-JNK， and other proteins were detected by Western blot. Finally， flow cytometry instrumentation and fluorescence microscopy were used to analyze the changes in reactive oxygen species （ROS）， and a ROS scavenger （NAC） was adopted for verification. ResultCompared with the blank group， the activity of HCT-116 cells was significantly decreased in the Fagopyri Dibotryis Rhizoma group （P<0.05， P<0.01）. The apoptosis rate of HCT-116 cells in the Fagopyri Dibotryis Rhizoma group was significantly increased （P<0.01）. The expression of autophagy-related protein ubiquitin-binding protein （p62） was decreased， but that of autophagy-specific genes （Beclin1） and autophagic microtubule-associated protein 1 light-chain 3B （LC3B） was enhanced （P<0.05， P<0.01）. Compared with the Fagopyri Dibotryis Rhizoma group， the apoptosis rate of HCT-116 cells in the Fagopyri Dibotryis Rhizoma + NAC group was significantly reduced （P<0.01）. The expression of related autophagy protein Beclin1 was significantly reduced （P<0.01）， and that of LC3B protein was reduced （P<0.01）. In addition， the expression of MAPK pathway-related proteins ERK and JNK was decreased in the Fagopyri Dibotryis Rhizoma group （P<0.05， P<0.01）， and that of p-ERK and p-JNK was enhanced （P<0.05， P<0.01）. ConclusionFagopyri Dibotryis Rhizoma can inhibit the proliferation of HCT-116 cells and induce apoptosis and autophagy through the ROS/MAPK signaling pathway.

Effect of Gandou Fumu Decoction on Autophagy in Mice with Liver Fibrosis in Wilson's Disease by Regulating Expression of miR-29b-3p/ULK1 / 中国实验方剂学杂志

ObjectiveTo explore the mechanism and pathway of Gandou Fumu decoction （GDFMD） in the development of liver fibrosis in Wilson's disease （WD）. MethodFirst， 30 TX-j mice were randomly divided into the model group， high-dose， medium-dose， and low-dose GDFMD groups， and penicillamine group， with six mice in each group， and another six wild-type mice were used as the normal group. The high-dose， medium-dose， and low-dose GDFMD groups were intragastrically administered drugs of 13.92， 6.96， 3.48 g·kg-1. In the penicillamine group， 0.1 g·kg-1 of penicillamine was given by intragastric administration. The model group and the normal group were given equal volume of normal saline， once a day， for four consecutive weeks. Samples were collected four weeks after gavage， and enzyme-linked immunosorbent assay （ELISA） was used to detect type Ⅲ procollagen peptide （PCⅢ）， collagen type Ⅳ （Col Ⅳ）， hyaluronic acid （HA）， and laminin （LN）. Hematoxylin-eosin （HE）， Masson， and picric acid-Sirus red collagen （Sirus Red） staining were used to observe the histopathological changes of liver fibrosis. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）， immunohistochemistry， and Western blot were used to observe the expressions of α-smooth muscle actin （α-SMA） and collagen type Ⅰ （Col Ⅰ）， which were related to the activation of hepatic stellate cells （HSCs）. The expression of miR-29b-3p was observed by Real-time PCR. The expression of Unc-51-like kinase 1 （ULK1） and its downstream-related factors were observed by Western blot. The downstream genes of miR-29b-3p were verified by the dual luciferase reporter gene detection method. ResultCompared with the normal group， the four items of liver fibrosis （PCⅢ， Col Ⅳ， HA， and LN） in the model group were significantly abnormal （P<0.01）， and the pathology was significantly abnormal. The expression of HSC activation-related indicators including α-SMA and Col Ⅰ， as well as α-SMA mRNA and Col Ⅰ mRNA was up-regulated （P<0.05， P<0.01）， and miR-29b-3p expression was down-regulated （P<0.01）. ULK1， p-ULK1， autophagy-related gene 13 （Atg13）， p-Atg13， Beclin-1， FAK family kinase-interacting protein of 200 kDa （FIP200）， activating molecule in BECN1-regulated autophagy protein 1 （AMBKA1）， and microtubule-associated protein 1 light chain 3Ⅱ/Ⅰ（LC3Ⅱ/Ⅰ） were up-regulated （P<0.05， P<0.01）. p62 protein expression was down-regulated （P<0.01）. Compared with the model group， the four items of liver fibrosis in the high-dose， medium-dose， and low-dose GDFMD groups and the penicillamine group were significantly improve （P<0.01）， and the pathological conditions were improved. The expression of HSC activation-related indicators including α-SMA and Col Ⅰ， as well as α-SMA mRNA and Col Ⅰ mRNA was down-regulated （P<0.05， P<0.01）， and the expression of miR-29b-3p was up-regulated （P<0.01）. ULK1， p-ULK1， Atg13， p-Atg13， Beclin-1， FIP200， AMBKA1， and LC3Ⅱ/Ⅰ were down-regulated （P<0.05， P<0.01）， and p62 protein expression was up-regulated （P<0.01）. The prediction software predicted that there was a binding site between miR-29b-3p and ULK1. The dual-luciferase reporter gene detection method indicated that the luciferase activity of the ULK1-WT plasmid-transfected cell group was reduced when miR-29b-3p mimics were co-cultured （P<0.01）. ConclusionGDFMD can regulate ULK1-mediated autophagy by up-regulating miR-29b-3p and further exert its anti-hepatic fibrosis effect in Wilson's disease.

Danggui Shaoyaosan Regulates Autophagy via AMPK/mTOR/ULK1 Signaling Pathway in Rat Model of Metabolism-associated Fatty Liver Disease / 中国实验方剂学杂志

ObjectiveTo investigate the regulatory effect of Danggui Shaoyaosan on adenosine monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR）/Unc-51-like kinase-1 （ULK1） signaling pathway in the rat model of metabolism-associated fatty liver disease （MAFLD）. MethodSixty SD rats were randomized into control， model， western medicine （polyene phosphatidylcholine capsules，0.144 g·kg-1）， and low-， medium-， and high-dose （2.44， 4.88， 9.76 g·kg-1， respectively） Danggui Shaoyaosan groups. After being fed with a high-fat diet for 8 weeks， the rats in each group were administrated with corresponding drugs for 4 weeks. At the end of drug treatment， serum and liver tissue were collected for subsequent determination of related indicators. ResultCompared with the control group， the model group showed increased contents of total cholesterol （TC）， triglyceride （TG）， low-density lipoprotein cholesterol （LDL-C） and activities of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in the serum， increased contents of TC， TG， and free fatty acids （FFAs） in the liver （P<0.01）， and decreased content of high-density lipoprotein cholesterol （HDL-C） in the serum （P<0.01）. Furthermore， the model group showed down-regulated protein levels of p-AMPK， microtubule-associated protein 1 light chain 3B （LC3B） Ⅱ， Beclin1， and ULK1 （P<0.01） and up-regulated protein levels of p-mTOR and ubiquitin-binding protein p62 in the liver （P<0.01）. The hepatic steatosis was obvious and the NAFLD activity score （NAS） and oil red O staining area increased in the model group， （P<0.05， P<0.01）. Compared with the model group， Danggui Shaoyaosan reduced the contents of TC and TG and the activities of ALT and AST in the serum， lowered the levels of TC， TG， and FFA in the liver， down-regulated the protein levels of p-mTOR and p62 （P<0.01）， elevated the serum HDL-C level， and up-regulated the protein levels of p-AMPK， LCBⅡ， Beclin1， and ULK1 in the liver （P<0.05， P<0.01）. Moreover， it alleviated hepatic steatosis and decreased the NAS and oil red O staining area （P<0.05， P<0.01）. ConclusionDanggui Shaoyaosan has therapeutic effect on MAFLD rats by regulating AMPK/mTOR/ULK1 signaling pathway to enhance autophagy.

Regulatory effect of autophagy on the resistance of human liver cancer cell Huh7 to lenvatinib / 中国药房

OBJECTIVE To investigate the regulatory effect of autophagy on the resistance of human liver cancer cell Huh7 to lenvatinib. METHODS Using human liver cancer cell Huh7 as subject， the lenvatinib-resist cell model （Huh7-LR） was generated by the low-dose gradient method combined with long-term administration. The sensitivity of parental cell Huh7 and drug-resistant cell Huh7-LR to lenvatinib was detected by using CCK-8 assay and flow cytometry. Western blot assay and GFP-mCherry-LC3 plasmid transfection were performed to detect the expression levels of autophagic protein Beclin-1， autophagic adapter protein sequestosome 1 （p62）， microtubule-associated protein 1 light chain 3 （LC3） and autophagic level. Furthermore， an autophagy activation model was constructed by cell starvation， the protein expression of p62 and autophagy level were detected by using Western blot assay and GFP-mCherry-LC3 plasmid transfection， and the effect of autophagy activation on the sensitivity of Huh7-LR cells to lenvatinib was detected by flow cytometry. RESULTS Compared with parental cells， the drug resistance index of Huh7-LR cells was 6.2； protein expression of p62 was increased significantly， while apoptotic rate， protein expression of Beclin-1 and LC3Ⅱ/ LC3Ⅰ ratio were all reduced significantly （P＜0.05 or P＜0.01）； the level of autophagy was decreased to some extent. Autophagy activation could significantly increase the protein expression of p62 in Huh7-LR cells （P＜0.05） and autophagy level， and significantly increase its apoptotic rate （P＜0.05）. CONCLUSIONS Autophagy is involved in lenvatinib resistance， and activating autophagy can reverse the resistance of liver cancer cells to lenvatinib to some extent.

Effect and mechanism of gracillin-induced autophagy in lung cancer A549 cells / 中国药房

OBJECTIVE To investigate the effect and mechanism of gracillin from Reineckia carnea on autophagy in non- small cell lung cancer A549 cells. METHODS Using A549 cells as subjects， the effects of different concentrations of gracillin （0.25， 0.5， 1， 2， 4 μmol/L） on the proliferation of cells were detected by CCK-8 after being treated for different time （12， 24， 48 h）. Compared with the control group without medication， the effect of gracillin （2 μmol/L） on the formation of autophagosomes in cells was observed by transmission electron microscope after 24 h of exposure. The aggregation of GFP-LC3 on autophagosome membrane was detected by GFP-LC3 plasmid transfection after being treated with gracillin （0.25， 0.5， 1， 2 μmol/L） for 24 h. Quantitative real-time PCR and Western blot assay were used to detect the mRNA and protein expressions of family with sequence similarity 102 member A（FAM102A）， the expressions of autophagy-related proteins [p62， Beclin-1， microtubule-associated protein 1 light chain 3B （LC3B）]， and the expressions of phosphoinositide 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway-related proteins in A549 cells after being treated with gracillin （0.25， 0.5， 1 and 2 μmol/L） for 24 h. RESULTS Gracillin significantly inhibited the proliferation of A549 cells in a concentration- and time-dependent manner. The IC50 was 2.55 μmol/L at 24 h. After 24 h of gracillin treatment， autophagosomes with bilayer membrane structure were found in the cell cytoplasm， and GFP-LC3 green fluorescent spots on autophagosome membrane were obvious， representing an increasing trend as drug concentration. Compared with the control group， mRNA and protein expressions of FAM102A （0.5， 1， 2 μmol/L groups）， protein expression of Beclin-1 （1， 2 μmol/L groups） and LC3B-Ⅱ/LC3B-Ⅰ ratio （2 μmol/L group） were significantly increased in different concentrations of gracillin groups， while the protein expression of p62 （1， 2 μmol/L groups）， and the protein phosphorylations of Akt （1， 2 μmol/L groups） and PI3K （2 μmol/L group） were all decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS Gracillin can promote excessive autophagy in A549 cells by up-regulating mRNA and protein expressions of FAM102A and inhibiting PI3K/Akt signaling pathway， thus inhibiting cell proliferation.

Pathogenesis of Idiopathic Pulmonary Fibrosis and Modulating Effect of Chinese Medicine： A Review / 中国实验方剂学杂志

Idiopathic pulmonary fibrosis （IPF）， as a progressive lung disease， has a poor prognosis and no reliable and effective therapies. IPF is mainly treated by organ transplantation and administration of chemical drugs， which are ineffective and induce side effects， failing to meet the clinical needs. Therefore， developing safer and more effective drugs has become an urgent task， which necessitates clear understanding of the pathogenesis of IPF. The available studies about the pathogenesis of IPF mainly focus on macrophage polarization， epithelial-mesenchymal transition （EMT）， oxidative stress， and autophagy， while few studies systematically explain the principles and links of the pathogeneses. According to the traditional Chinese medicine theory， Qi deficiency and blood stasis and Qi-Yang deficiency are the key pathogeneses of IPF. Therefore， the Chinese medicines or compound prescriptions with the effects of replenishing Qi and activating blood， warming Yang and tonifying Qi， and eliminating stasis and resolving phlegm are often used to treat IPF. Modern pharmacological studies have shown that such medicines play a positive role in inhibiting macrophage polarization， restoring redox balance， inhibiting EMT， and regulating cell autophagy. However， few studies report how Chinese medicines regulate the pathways in the treatment of IPF. By reviewing the latest articles in this field， we elaborate on the pathogenesis of IPF and provide a comprehensive overview of the mechanism of the active ingredients or compound prescriptions of Chinese medicines in regulating IPF. Combining the pathogenesis of IPF with the modulating effects of Chinese medicines， we focus on exploring systemic treatment options for IPF， with a view to providing new ideas for the in-depth study of IPF and the research and development of related drugs.