Induction of autophagy-dependent and caspase- and microtubule-acetylation-independent cell death by phytochemical-stabilized gold nanopolygons in colorectal adenocarcinoma cells.

Collective functionalization of the phytochemicals of medicinal herbs on nanoparticles is emerging as a potential cancer therapeutic strategy. This study presents the facile synthesis of surface-functionalized gold nanoparticles using Bacopa monnieri (Brahmi; Bm) phytochemicals and their therapeutically relevant mechanism of action in the colorectal cancer cell line, HT29. The nanoparticles were characterized using UV-visible spectroscopy, TEM-EDAX, zeta potential analysis, TGA, FTIR and 1H NMR spectroscopy, and HR-LC-MS. The particles (Bm-GNPs) were of polygonal shape and were stable against aggregation. They entered the target cells and inhibited the viability and clonogenicity of the cells with eight times more antiproliferative efficacy (25 ± 1.5 µg mL-1) than Bm extract (Bm-EX). In vitro studies revealed that Bm-GNPs bind tubulin (a protein crucial in cell division and a target of anticancer drugs) and disrupt its helical structure without grossly altering its tertiary conformation. Like other antitubulin agents, Bm-GNPs induced G2/M arrest and ultimately killed the cells, as confirmed using flow cytometry analyses. ZVAD-FMK-mediated global pan-caspase inhibition and the apparent absence of cleaved caspase-3 in treated cells indicated that the death did not involve the classic apoptosis pathway. Cellular ultrastructure analyses, western immunoblots, and in situ immunofluorescence visualization of cellular microtubules revealed microtubule-acetylation-independent induction of autophagy as the facilitator of cell death. Together, the data indicate strong antiproliferative efficacy and a possible mechanism of action for these designer nanoparticles. Bm-GNPs, therefore, merit further investigations, including preclinical evaluations, for their therapeutic potential as inducers of non-apoptotic cell death.

Drug repurposing-based nanoplatform via modulating autophagy to enhance chemo-phototherapy against colorectal cancer.

Multi-modal combination therapy is regarded as a promising approach to cancer treatment. Combining chemotherapy and phototherapy is an essential multi-modal combination therapy endeavor. Ivermectin (IVM) is a potent antiparasitic agent identified as having potential antitumor properties. However, the fact that it induces protective autophagy while killing tumor cells poses a challenge to its further application. IR780 iodide (IR780) is a near-infrared (NIR) dye with outstanding photothermal therapy (PTT) and photodynamic therapy (PDT) effects. However, the hydrophobicity, instability, and low tumor uptake of IR780 limit its clinical applications. Here, we have structurally modified IR780 with hydroxychloroquine, an autophagy inhibitor, to synthesize a novel compound H780. H780 and IVM can form H780-IVM nanoparticles (H-I NPs) via self-assembly. Using hyaluronic acid (HA) to modify the H-I NPs, a novel nano-delivery system HA/H780-IVM nanoparticles (HA/H-I NPs) was synthesized for chemotherapy-phototherapy of colorectal cancer (CRC). Under NIR laser irradiation, HA/H-I NPs effectively overcame the limitations of IR780 and IVM and exhibited potent cytotoxicity. In vitro and in vivo experiment results showed that HA/H-I NPs exhibited excellent anti-CRC effects. Therefore, our study provides a novel strategy for CRC treatment that could enhance chemo-phototherapy by modulating autophagy.

Autophagy plays a pro-apoptotic role in arsenic trioxide-induced cell death of liver cancer.

OBJECTIVE: The effects of arsenic trioxide (As2O3) on hepatocellular carcinoma have been documented widely. Autophagy plays dual roles in the survival and death of cancer cells. Therefore, we investigated the exact role of autophagy in As2O3-induced apoptosis in liver cancer cells. METHODS: The viability of hepatoma cells was determined using the MTT assay with or without fetal bovine serum. The rate of apoptosis in liver cancer cells treated with As2O3 was evaluated using flow cytometry, Hoechst 33258 staining, and TUNEL assays. The rate of autophagy among liver cancer cells treated with As2O3 was detected using immunofluorescence, Western blot assay and transmission electron microscopy. RESULTS: Upon treatment with As2O3, the viability of HepG2 and SMMC-7721 cells was decreased in a time- and dose-dependent manner. The apoptosis rates of both liver cancer cell lines increased with the concentration of As2O3, as shown by flow cytometry. Apoptosis in liver cancer cells treated with As2O3 was also shown by the activation of the caspase cascade and the regulation of Bcl-2/Bax expression. Furthermore, As2O3 treatment induced autophagy in liver cancer cells; this finding was supported by Western blot, immunofluorescence of LC3-II and beclin 1, and transmission electron microscopy. In liver cancer cells, As2O3 inhibited the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signal pathway that plays a vital role in both apoptosis and autophagy. The PI3K activator SC-79 partially reversed As2O3-induced autophagy and apoptosis. Furthermore, inhibiting autophagy with 3-methyladenine partially reversed the negative effects of As2O3 on cell viability. Serum starvation increased autophagy and amplified the effect of As2O3 on cell death. CONCLUSION: As2O3 induces apoptosis and autophagy in liver cancer cells. Autophagy induced by As2O3 may have a proapoptotic effect that helps to reduce the viability of liver cancer cells. This study provides novel insights into the effects of As2O3 against liver cancer. Please cite this article as: Deng ZT, Liang SF, Huang GK, Wang YQ, Tu XY, Zhang YN, Li S, Liu T, Cheng BB. Autophagy plays a pro-apoptotic role in arsenic trioxide-induced cell death of liver cancer. J Integr Med. 2024; 22(3): 295-302.

[Research Progress on the Role of Berberine in Hematologic Malignancies and Its Related Mechanisms --Review].

Berberine, a traditional Chinese medicine, is an isoquinoline alkaloid extracted from the rhizome of Coptis chinensis. It has anti-inflammatory and antidiarrheal effects and is commonly used in the treatment of infections and gastrointestinal diseases. In recent years, studies have found that berberine can play a wide range of anti-cancer effects in the treatment of leukemia, lymphoma, multiple myeloma, etc. In hematologic malignancies, berberine can induce autophagy, promote apoptosis, regulate cell cycle, inhibit inflammatory response, cause oxidative damage to cancer cells and interact with miRNA to inhibit the proliferation, migration and colony formation of cancer cells. This paper will review the role and related mechanisms of berberine in hematological malignancies.

Sishen Wan enhances intestinal barrier function via regulating endoplasmic reticulum stress to improve mice with diarrheal irritable bowel syndrome.

BACKGROUND: Diarrheal irritable bowel syndrome (IBS-D), characterized primarily by the presence of diarrhea and abdominal pain, is a clinical manifestation resulting from a multitude of causative factors. Furthermore, Sishen Wan (SSW) has demonstrated efficacy in treating IBS-D. Nevertheless, its mechanism of action remains unclear. METHODS: A model of IBS-D was induced by a diet containing 45 % lactose and chronic unpredictable mild stress. Additionally, the impact of SSW was assessed by measuring body weight, visceral sensitivity, defecation parameters, intestinal transport velocity, intestinal neurotransmitter levels, immunohistochemistry, and transmission electron microscopy analysis. Immunofluorescent staining was used to detect the expression of Mucin 2 (MUC2) and Occludin in the colon. Western blotting was used to detect changes in proteins related to tight junction (TJ), autophagy, and endoplasmic reticulum (ER) stress in the colon. Finally, 16S rRNA amplicon sequencing was used to monitor the alteration of gut microbiota after SSW treatment. RESULTS: Our study revealed that SSW administration resulted in reduced visceral sensitivity, improved defecation parameters, decreased intestinal transport velocity, and reduced intestinal permeability in IBS-D mice. Furthermore, SSW promotes the secretion of colonic mucus by enhancing autophagy and inhibiting ER stress. SSW treatment caused remodeling of the gut microbiome by increasing the abundance of Blautia, Muribaculum and Ruminococcus torques group. CONCLUSION: SSW can improve intestinal barrier function by promoting autophagy and inhibiting ER stress, thus exerting a therapeutic effect on IBS-D.

Fangji Huangqi Decoction alleviates rheumatoid arthritis through regulating HIF-1α mediated the angiogenesis and the balance between autophagy and apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Fangji Huangqi Decoction (FHD) is frequently prescribed for the clinical treatment of wind-cold and wind-dampness pathogenic superficial deficiency syndrome. It also has a notable curative effect on rheumatoid arthritis (RA). AIM OF THE STUDY: The study aimed to explore the possible mechanism of FHD against RA and provided a theoretical basis for alternative therapies for RA. MATERIALS AND METHODS: We used UPLC-Q-TOF-MS to analysis the ingredients and absorbed blood components of FHD. At the same time, the collagen-induced arthritis (CIA) rat model was established to estimate the therapeutic effects on FHD by considering body weight, arthritis score, paw swelling, autonomous movement ability, and synovial microvessel counts. Subsequently, immunofluorescence, immunohistochemistry, and Western blot were employed to detect the anti-angiogenic capacity of FHD in vivo, as well as the levels of apoptosis and autophagy in the synovial tissue. In addition, flow cytometry and Western blot were used to assess the effects of FHD on apoptosis and autophagy in MH7A cells. The effects of FHD on the proliferation and migration of MH7A cells were measured by CCK8 assay, cell migration and, invasion experiments. Finally, a tube formation assay was performed to evaluate the angiogenic capacity of FHD in co-cultures of MH7A cells and HUVEC cells. RESULTS: Through testing of FHD's original formula, a total of 26 active ingredients have been identified, with 17 of them being absorbed into the bloodstream. FHD significantly improved the pathological symptoms and synovial hyperplasia of CIA rats. FHD could suppress the expression of HIF-1α, promote apoptosis in CIA rat synovial tissue, and suppress autophagy and angiogenesis. In vitro experiments showed that serum containing FHD inhibited the proliferation, migration, and invasion of MH7A cells, and also suppressed the expression of autophagy-related proteins while promoting apoptosis. FHD markedly repressed the expression of HIF-1α protein in TNF-α-stimulated MH7A cells and inhibited the tube formation capacity induced by MH7A cells in HUVEC cells. CONCLUSIONS: The study had proven that FHD played an excellent anti-RA role, which may be attributed to its potential mechanism of regulating the balance between autophagy and apoptosis in RA FLS by suppressing the HIF-1α, thus contributing to its anti-angiogenic activities.

Kai-Xin-San ameliorates Alzheimer's disease-related neuropathology and cognitive impairment in APP/PS1 mice via the mitochondrial autophagy-NLRP3 inflammasome pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Kai-Xin-San (KXS) is a classic famous prescription that has been utilized for centuries to address dementia. New investigations have shown that the anti-dementia effect of KXS is connected with improved neuroinflammation. Nevertheless, the underlying mechanism is not well elucidated. AIM OF THE STUDY: We propose to discover the ameliorative impact of KXS on Alzheimer's disease (AD) and its regulatory role on the mitochondrial autophagy-nod-like receptor protein 3 (NLRP3) inflammasome pathway. MATERIALS AND METHODS: The Y maze, Morris water maze, and new objection recognition tests were applied to ascertain the spatial learning and memory capacities of amyloid precursor protein/presenilin 1 (APP/PS1) mice after KXS-treatment. Meanwhile, the biochemical indexes of the hippocampus were detected by reagent kits. The pathological alterations and mitochondrial autophagy in the mice' hippocampus were detected utilizing hematoxylin and eosin (H&E), immunohistochemistry, immunofluorescence staining, and transmission electron microscopy. Besides, the PTEN-induced putative kinase 1 (PINK1)/Parkin and NLRP3 inflammasome pathways protein expressions were determined employing the immunoblot analysis. RESULTS: The results of behavioral tests showed that KXS significantly enhanced the AD mice' spatial learning and memory capacities. Furthermore, KXS reversed the biochemical index levels and reduced amyloid-ß protein deposition in AD mice brains. Besides, H&E staining showed that KXS remarkably ameliorated the neuronal damage in AD mice. Concurrently, the results of transmission electron microscopy suggest that KXS ameliorated the mitochondrial damage in microglia and promoted mitochondrial autophagy. Moreover, the immunofluorescence outcomes exhibited that KXS promoted the expression of protein 1 light chain 3B (LC3B) associated with microtubule and the generation of autophagic flux. Notably, the immunofluorescence co-localization results confirmed the presence of mitochondrial autophagy in microglia. Finally, KXS promoted the protein expressions of the PINK1/Parkin pathway and reduced the activation of NLRP3 inflammasome. Most importantly, these beneficial effects of KXS were attenuated by the mitochondrial autophagy inhibitor chloroquine. CONCLUSION: KXS ameliorates AD-related neuropathology and cognitive impairment in APP/PS1 mice by enhancing the mitochondrial autophagy and suppressing the NLRP3 inflammasome pathway.

Geniposide ameliorates atherosclerosis by restoring lipophagy via suppressing PARP1/PI3K/AKT signaling pathway.

BACKGROUND: Atherosclerosis (AS) is the leading cause of global death, which manifests as arterial lipid stack and plaque formation. Geniposide is an iridoid glycoside extract from Gardenia jasminoides J.Ellis that ameliorates AS by mediating autophagy. However, how Geniposide regulates autophagy and treats AS remains unclear. PURPOSE: To evaluate the efficacy and mechanism of Geniposide in treating AS. STUDY DESIGN AND METHODS: Geniposide was administered to high-fat diet-fed ApoE-/- mice and oxidized low-density lipoprotein-incubated primary vascular smooth muscle cells (VSMCs). AS was evaluated with arterial lipid stack, plaque progression, and collagen loss in the artery. Foam cell formation was detected by lipid accumulation, inflammation, apoptosis, and the expression of foam cell markers. The mechanism of Geniposide in treating AS was assessed using network pharmacology. Lipophagy was measured by lysosomal activity, expression of lipophagy markers, and the co-localization of lipids and lipophagy markers. The effects of lipophagy were blocked using Chloroquine. The role of PARP1 was assessed by Olaparib (a PARP1 inhibitor) intervention and PARP1 overexpression. RESULTS: In vivo, Geniposide reversed high-fat diet-induced hyperlipidemia, plaque progression, and inflammation. In vitro, Geniposide inhibited VSMC-derived foam cell formation by suppressing lipid stack, apoptosis, and the expressions of foam cell markers. Network pharmacological analysis and in vitro validation suggested that Geniposide treated AS by enhancing lipophagy via suppressing the PI3K/AKT signaling pathway. The benefits of Geniposide in alleviating AS were offset by Chloroquine in vivo and in vitro. Inhibiting PARP1 using Olaparib promoted lipophagy and alleviated AS progression, while PARP1 overexpression exacerbated foam cell formation and lipophagy blockage. The above effects of PARP1 were weakened by PI3K inhibitor LY294002. PARP1 also inhibited the combination of the ABCG1 and PLIN1. CONCLUSION: Geniposide alleviated AS by restoring PARP1/PI3K/AKT signaling pathway-suppressed lipophagy. This study is the first to present the lipophagy-inducing effect of Geniposide and the binding of ABCG1 and PLIN1 inhibited by PARP1.

Isoprenoid flavonoids isolated from Sophora davidii and their activities induces apoptosis and autophagy in HT29 cells.

Four previously undescribed isoprenoid flavonoids (2-5) were isolated from Sophora davidii, along with five known analogues. The structures of the compounds were established through comprehensive analysis of spectroscopic data, including HRESIMS, 1D and 2D NMR, and absolute configurations determined by theoretical calculations, including ECD and NMR calculation. The cytotoxic effects of the isolated compounds on human HT29 colon cancer cells were evaluated using the MTT assay, compound 1 exhibited cytotoxicity against human HT29 colon cancer cells with an IC50 value of 8.39 ± 0.09 µM. Studies conducted with compound 1 in HT29 cells demonstrated that it may induce apoptosis and autophagy in HT29 by promoting the phosphorylation of P38 MAPK and inhibiting the phosphorylation of Erk MAPK.

Ginsenoside Rb1 attenuates doxorubicin induced cardiotoxicity by suppressing autophagy and ferroptosis.

Ginsenoside Rb1 (Rb1), an active component isolated from traditional Chinese medicine Ginseng, is beneficial to many cardiovascular diseases. However, whether it can protect against doxorubicin induced cardiotoxicity (DIC) is not clear yet. In this study, we aimed to investigate the role of Rb1 in DIC. Mice were injected with a single dose of doxorubicin (20 mg/kg) to induce acute cardiotoxicity. Rb1 was given daily gavage to mice for 7 days. Changes in cardiac function, myocardium histopathology, oxidative stress, cardiomyocyte mitochondrion morphology were studied to evaluate Rb1's function on DIC. Meanwhile, RNA-seq analysis was performed to explore the potential underline molecular mechanism involved in Rb1's function on DIC. We found that Rb1 treatment can improve survival rate and body weight in Dox treated mice group. Rb1 can attenuate Dox induced cardiac dysfunction and myocardium hypertrophy and interstitial fibrosis. The oxidative stress increase and cardiomyocyte mitochondrion injury were improved by Rb1 treatment. Mechanism study found that Rb1's beneficial role in DIC is through suppressing of autophagy and ferroptosis. This study shown that Ginsenoside Rb1 can protect against DIC by regulating autophagy and ferroptosis.

High-dose Vitamin C injection ameliorates against sepsis-induced myocardial injury by anti-apoptosis, anti-inflammatory and pro-autophagy through regulating MAPK, NF-κB and PI3K/AKT/mTOR signaling pathways in rats.

AIMS: This study aimed to evaluate the effects of VC on SIMI in rats. METHODS: In this study, the survival rate of high dose VC for SIMI was evaluated within 7 days. Rats were randomly assigned to three groups: Sham group, CLP group, and high dose VC (500 mg/kg i.v.) group. The animals in each group were treated with drugs for 1 day, 3 days or 5 days, respectively. Echocardiography, myocardial enzymes and HE were used to detect cardiac function. IL-1ß, IL-6, IL-10 and TNF-α) in serum were measured using ELISA kits. Western blot was used to detect proteins related to apoptosis, inflammation, autophagy, MAPK, NF-κB and PI3K/Akt/mTOR signaling pathways. RESULTS: High dose VC improved the survival rate of SIMI within 7 days. Echocardiography, HE staining and myocardial enzymes showed that high-dose VC relieved SIMI in rats in a time-dependent manner. And compared with CLP group, high-dose VC decreased the expressions of pro-apoptotic proteins, while increased the expression of anti-apoptotic protein. And compared with CLP group, high dose VC decreased phosphorylation levels of Erk1/2, P38, JNK, NF-κB and IKK α/ß in SIMI rats. High dose VC increased the expression of the protein Beclin-1 and LC3-II/LC3-I ratio, whereas decreased the expression of P62 in SIMI rats. Finally, high dose VC attenuated phosphorylation of PI3K, AKT and mTOR compared with the CLP group. SIGNIFICANCE: Our results showed that high dose VC has a good protective effect on SIMI after continuous treatment, which may be mediated by inhibiting apoptosis and inflammatory, and promoting autophagy through regulating MAPK, NF-κB and PI3K/AKT/mTOR pathway.

The pathological mechanisms and potential therapeutic drugs for myocardial ischemia reperfusion injury.

BACKGROUND: Cardiovascular disease is the main cause of death and disability, with myocardial ischemia being the predominant type that poses a significant threat to humans. Reperfusion, an essential therapeutic approach, promptly reinstates blood circulation to the ischemic myocardium and stands as the most efficacious clinical method for myocardial preservation. Nevertheless, the restoration of blood flow associated with this process can potentially induce myocardial ischemia-reperfusion injury (MIRI), thereby diminishing the effectiveness of reperfusion and impacting patient prognosis. Therefore, it is of great significance to prevent and treat MIRI. PURPOSE: MIRI is an important factor affecting the prognosis of patients, and there is no specific in-clinic treatment plan. In this review, we have endeavored to summarize its pathological mechanisms and therapeutic drugs to provide more powerful evidence for clinical application. METHODS: A comprehensive literature review was conducted using PubMed, Web of Science, Embase, Medline and Google Scholar with a core focus on the pathological mechanisms and potential therapeutic drugs of MIRI. RESULTS: Accumulated evidence revealed that oxidative stress, calcium overload, mitochondrial dysfunction, energy metabolism disorder, ferroptosis, inflammatory reaction, endoplasmic reticulum stress, pyroptosis and autophagy regulation have been shown to participate in the process, and that the occurrence and development of MIRI are related to plenty of signaling pathways. Currently, a range of chemical drugs, natural products, and traditional Chinese medicine (TCM) preparations have demonstrated the ability to mitigate MIRI by targeting various mechanisms. CONCLUSIONS: At present, most of the research focuses on animal and cell experiments, and the regulatory mechanisms of each signaling pathway are still unclear. The translation of experimental findings into clinical practice remains incomplete, necessitating further exploration through large-scale, multi-center randomized controlled trials. Given the absence of a specific drug for MIRI, the identification of therapeutic agents to reduce myocardial ischemia is of utmost significance. For the future, it is imperative to enhance our understanding of the pathological mechanism underlying MIRI, continuously investigate and develop novel pharmaceutical agents, expedite the clinical translation of these drugs, and foster innovative approaches that integrate TCM with Western medicine. These efforts will facilitate the emergence of fresh perspectives for the clinical management of MIRI.

Rosmarinic acid alleviates toosendanin-induced liver injury through restoration of autophagic flux and lysosomal function by activating JAK2/STAT3/CTSC pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Rosmarinic acid (RA), a natural polyphenol abundant in numerous herbal remedies, has been attracting growing interest owing to its exceptional ability to protect the liver. Toosendanin (TSN), a prominent bioactive compound derived from Melia toosendan Siebold & Zucc., boasts diverse pharmacological properties. Nevertheless, TSN possesses remarkable hepatotoxicity. Intriguingly, the potential of RA to counteract TSN-induced liver damage and its probable mechanisms remain unexplored. AIM OF THE STUDY: This study is aimed at exploring whether RA can alleviate TSN-induced liver injury and the potential mechanisms involved autophagy. MATERIALS AND METHODS: CCK-8 and LDH leakage rate assay were used to evaluate cytotoxicity. Balb/c mice were intraperitoneally administered TSN (20 mg/kg) for 24 h after pretreatment with RA (0, 40, 80 mg/kg) by gavage for 5 days. The autophagic proteins P62 and LC3B expressions were detected using western blot and immunohistochemistry. RFP-GFP-LC3B and transmission electron microscopy were applied to observe the accumulation levels of autophagosomes and autolysosomes. LysoTracker Red and DQ-BSA staining were used to evaluate the lysosomal acidity and degradation ability respectively. Western blot, immunohistochemistry and immunofluorescence staining were employed to measure the expressions of JAK2/STAT3/CTSC pathway proteins. Dual-luciferase reporter gene was used to measure the transcriptional activity of CTSC and RT-PCR was used to detect its mRNA level. H&E staining and serum biochemical assay were employed to determine the degree of damage to the liver. RESULTS: TSN-induced damage to hepatocytes and livers was significantly alleviated by RA. RA markedly diminished the autophagic flux blockade and lysosomal dysfunction caused by TSN. Mechanically, RA alleviated TSN-induced down-regulation of CTSC by activating JAK2/STAT3 signaling pathway. CONCLUSION: RA could protect against TSN-induced liver injury by activating the JAK2/STAT3/CTSC pathway-mediated autophagy and lysosomal function.

Quercetin and tanshinone prevent mitochondria from oxidation and autophagy to inhibit KGN cell apoptosis through the SIRT1/SIRT3-FOXO3a axis.

Granulosa cells are somatic cells located inside follicles that play a crucial role in the growth and development of follicles. Quercetin and tanshinone are two key monomers in traditional Chinese medicine that have antioxidant and anti-aging properties. The KGN cell apoptosis model caused by triptolide (TP) was employed in this work to investigate granulosa cell death and medication rescue. Quercetin and tanshinone therapy suppressed KGN cell death and oxidation while also regulating the expression of critical apoptosis and oxidation-related markers such as B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax). Further research revealed that the effects of Quercetin and Tanshinone were accomplished via deacetylation of FOXO3A in the cytoplasm and mitochondria via the SIRT1/SIRT3-FOXO3a axis. In summary, Quercetin and tanshinone protect KGN cells from apoptosis by reducing mitochondrial apoptosis and oxidation via the SIRT1/SIRT3-FOXO3a axis.

Shikonin suppresses rheumatoid arthritis by inducing apoptosis and autophagy via modulation of the AMPK/mTOR/ULK-1 signaling pathway.

BACKGROUND: The overproliferation of fibroblast-like synoviocytes (FLS) contributes to synovial hyperplasia, a pivotal pathological feature of rheumatoid arthritis (RA). Shikonin (SKN), the active compound from Lithospermum erythrorhizon, exerts anti-RA effects by diverse means. However, further research is needed to confirm SKN's in vitro and in vivo anti-proliferative functions and reveal the underlying specific molecular mechanisms. PURPOSE: This study revealed SKN's anti-proliferative effects by inducing both apoptosis and autophagic cell death in RA FLS and adjuvant-induced arthritis (AIA) rat synovium, with involvement of regulating the AMPK/mTOR/ULK-1 pathway. METHODS: SKN's influences on RA FLS were assessed for proliferation, apoptosis, and autophagy with immunofluorescence staining (Ki67, LC3B, P62), EdU incorporation assay, staining assays of Hoechst, Annexin V-FITC/PI, and JC-1, transmission electron microscopy, mCherry-GFP-LC3B puncta assay, and western blot. In AIA rats, SKN's anti-arthritic effects were assessed, and its impacts on synovial proliferation, apoptosis, and autophagy were studied using Ki67 immunohistochemistry, TUNEL, and western blot. The involvement of AMPK/mTOR/ULK-1 pathway was examined via western blot. RESULTS: SKN suppressed RA FLS proliferation with reduced cell viability and decreased Ki67-positive and EdU-positive cells. SKN promoted RA FLS apoptosis, as evidenced by apoptotic nuclear fragmentation, increased Annexin V-FITC/PI-stained cells, reduced mitochondrial potential, elevated Bax/Bcl-2 ratio, and increased cleaved-caspase 3 and cleaved-PARP protein levels. SKN also enhanced RA FLS autophagy, featuring increased LC3B, reduced P62, autophagosome formation, and activated autophagic flux. Autophagy inhibition by 3-MA attenuated SKN's anti-proliferative roles, implying that SKN-induced autophagy contributes to cell death. In vivo, SKN mitigated the severity of rat AIA while also reducing Ki67 expression, inducing apoptosis, and enhancing autophagy within AIA rat synovium. Mechanistically, SKN modulated the AMPK/mTOR/ULK-1 pathway in RA FLS and AIA rat synovium, as shown by elevated P-AMPK and P-ULK-1 expression and decreased P-mTOR expression. This regulation was supported by the reversal of SKN's in vitro and in vivo effects upon co-administration with the AMPK inhibitor compound C. CONCLUSION: SKN exerted in vitro and in vivo anti-proliferative properties by inducing apoptosis and autophagic cell death via modulating the AMPK/mTOR/ULK-1 pathway. Our study revealed novel molecular mechanisms underlying SKN's anti-RA effects.

Xijiao Dihuang decoction combined with Yinqiao powder promotes autophagy-dependent ROS decrease to inhibit ROS/NLRP3/pyroptosis regulation axis in influenza virus infection.

BACKGROUND: Influenza viral pneumonia is a common complication after influenza virus infection. Xijiao Dihuang Decoction combined with Yinqiao Powder (XDY) is effective on improving influenza viral pneumonia. PURPOSE: This study further explores the anti-inflammatory mechanism of XDY in the treatment of influenza viral pneumonia. STUDY DESIGN: The effects of XDY on inflammation, autophagy, NACHT-LRR-PYD-containing protein 3 (NLRP3) inflammasome and pyroptosis were assessed in the mice with influenza viral pneumonia. In addition, the mouse macrophage cell line (J774A.1) infected with influenza virus was adopted to decode the in vitro effects of XDY on autophagy, reactive oxygen species (ROS), NLRP3 inflammasome and pyroptosis. We analyzed the XDY-induced autophagy, especially the mitophagy-related ROS clearance, and the subsequent inhibition of ROS/NLRP3 inflammasome/pyroptosis signaling in the infected macrophages by different assays based on quantitative polymerase chain reaction, western blot, flow cytometry, immunofluorescence and enzyme-linked immunosorbent assay. RESULTS: In vivo, XDY could effectively improve the lung inflammatory response in the mice with influenza virus pneumonia, due to an intact autophagy flux-promoting effect and the inhibiting roles on NLRP3 inflammasome and pyroptosis. Notably, in vitro, compared with the infected macrophages treated by the NLRP3 inflammasome agonist (Monosodium urate) or the mitochondrial-targeted antioxidant agent, the XDY-dependent treating could inhibit pyroptosis by negatively regulating the signaling axis of ROS/NLRP3 inflammasome/pyroptosis in the influenza virus-infected macrophages. More interestingly, XDY could promote an intact autophagy flux, inducing mitophagy eliminating the damaged mitochondria to reduce the intracellular ROS accumulation, and thus decrease the oxidative stress in the infected macrophages. Especially, the inhibitor of autophagy inition, 3-Methyladenine, could reverse the inhibitory effect of XDY on ROS-NLRP3 inflammasome-mediated pyroptosis, indicating an XDY-promoted mitophagy-dependent ROS scavenging. CONCLUSION: XDY can promote an intact autophagy flux to eliminate damaged mitochondria, namely mitophagy, which reduces the intracellular ROS accumulation contributing to NLRP3 inflammasome activation, restricting pyroptosis and eventually alleviating the influenza virus-induced inflammatory lesions. The obtained results provide new insights into the mechanism of action of XDY in alleviating influenza virus pneumonia, especially the roles of XDY in anti-oxidation, anti-inflammation and anti-pyroptosis, with potential therapeutic targets for future application in integrative medicine.

Yi-Qi-Huo-Xue decoction alleviates intracerebral hemorrhage injury through inhibiting neuronal autophagy of ipsilateral cortex via BDNF/TrkB pathway.

BACKGROUND: Yi-Qi-Huo-Xue Decoction (YQHXD), a traditional Chinese medicine formula, has demonstrated efficacy in the clinical treatment of intracerebral hemorrhage (ICH) for over a decade. Nevertheless, the precise pharmacotherapeutic compounds of YQHXD capable of penetrating into cerebral tissue and the pharmacological underpinnings of YQHXD remain ambiguous. METHODS: The active components of YQHXD in rat brains was analyzed by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. The potential targets, pathways and biological progresses of YQHXD ameliorating ICH induced injury was predicted by network pharmacology. Moreover, collagenase-induced ICH rat model, primary cortex neurons exposed to hemin and molecular docking were applied to validate the molecular mechanisms of YQHXD. RESULTS: Eleven active components of YQHXD were identified within the brains. Employing the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, our investigation concentrated on the roles of autophagy and the BDNF/TrkB signaling pathway in the pharmacological context. The pharmacological results revealed that YQHXD alleviated neurological dysfunction, brain water content, brain swelling, and pathological injury caused by ICH. Meanwhile, YQHXD inhibited autophagy influx and autophagosome in vivo, and regulated cortex neuronal autophagy and TrkB/BDNF pathway both in vivo and in vitro. Subsequently, N-acetyl serotonin (NAS), a selective TrkB agonist, was employed to corroborate the significance of the BDNF/TrkB pathway in this process. The combination of NAS and YQHXD did not further enhance the protective efficacy of YQHXD in ICH rats. Additionally, outcomes of molecular docking analysis revealed that nine compounds of YQHXD exhibited potential regulatory effects on TrkB. CONCLUSIONS: Ipsilateral neuronal autophagy and BDNF/TrkB pathway were activated 72 h after ICH. YQHXD effectively resisted injury induced by ICH, which was related with suppression of ipsilateral neuronal autophagy via BDNF/TrkB pathway. This study provides novel insights into the therapeutic mechanisms of traditional Chinese medicine in the context of ICH treatment.

Nobiletin targets SREBP1/ACLY to induce autophagy-dependent cell death of gastric cancer cells through PI3K/Akt/mTOR signaling pathway.

BACKGROUND: Autophagy could sense metabolic conditions and safeguard cells against nutrient deprivation, ultimately supporting the survival of cancer cells. Nobiletin (NOB) is a kind of bioactive component of the traditional Chinese medicine Citri Reticulatae Pericarpium and has been proven to induce GC cell death by reducing de novo fatty acid synthesis in our previous study. Nevertheless, the precise mechanisms by which NOB induces cell death in GC cells still need further elucidation. OBJECTIVES: To examine the mechanism by which NOB inhibits gastric cancer progression through the regulation of autophagy under the condition of lipid metabolism inhibition. METHODS/ STUDY DESIGN: Proliferation was detected by the CCK-8 assay. RNA sequencing (RNA-seq) was used to examine signaling pathway changes. Electron microscopy and mRFP-GFP-LC3 lentiviral transfection were performed to observe autophagy in vitro. Western blot, plasmid transfection, immunofluorescence staining, and CUT & Tag-qPCR techniques were utilized to explore the mechanisms by which NOB affects GC cells. Molecular docking and molecular dynamics simulations were conducted to predict the binding mode of NOB and SREBP1. CETSA was adopted to verify the predicted of binding model. A patient-derived xenograft (PDX) model was employed to verify the therapeutic efficacy of NOB in vivo. RESULTS: We conducted functional studies and discovered that NOB inhibited the protective effect of autophagy via the PI3K/Akt/mTOR axis in GC cells. Based on previous research, we found that the overexpression of ACLY abrogated the NOB-induced autophagy-dependent cell death. In silico analysis predicted the formation of a stable complex between NOB and SREBP1. In vitro assays confirmed that NOB treatment increased the thermal stability of SREBP1 at the same temperature conditions. Moreover, CUT&TAG-qPCR analysis revealed that NOB could inhibit SREBP1 binding to the ACLY promoter. In the PDX model, NOB suppressed tumor growth, causing SREBP1 nuclear translocation inhibition, PI3K/Akt/mTOR inactivation, and autophagy-dependent cell death. CONCLUSION: NOB demonstrated the ability to directly bind to SREBP1, inhibiting its nuclear translocation and binding to the ACLY promoter, thereby inducing autophagy-dependent cell death via PI3K/Akt/mTOR pathway.

SIRT1: Harnessing multiple pathways to hinder NAFLD.

Non-alcoholic fatty liver disease (NAFLD) encompasses hepatic steatosis, non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma. It is the primary cause of chronic liver disorders, with a high prevalence but no approved treatment. Therefore, it is indispensable to find a trustworthy therapy for NAFLD. Recently, mounting evidence illustrates that Sirtuin 1 (SIRT1) is strongly associated with NAFLD. SIRT1 activation or overexpression attenuate NAFLD, while SIRT1 deficiency aggravates NAFLD. Besides, an array of therapeutic agents, including natural compounds, synthetic compounds, traditional Chinese medicine formula, and stem cell transplantation, alleviates NALFD via SIRT1 activation or upregulation. Mechanically, SIRT1 alleviates NAFLD by reestablishing autophagy, enhancing mitochondrial function, suppressing oxidative stress, and coordinating lipid metabolism, as well as reducing hepatocyte apoptosis and inflammation. In this review, we introduced the structure and function of SIRT1 briefly, and summarized the effect of SIRT1 on NAFLD and its mechanism, along with the application of SIRT1 agonists in treating NAFLD.

Protective effect of phenylpropionamides in the seed of Cannabis Sativa L. on Parkinson's disease through autophagy.

Parkinson's disease (PD) is the second most common neurodegenerative disease in the world. As one of the major degradation pathways, autophagy plays a pivotal role in maintaining the effective turnover of proteins and damaged organelles in cells. Lewy bodies composed of α-synuclein (α-syn) abnormally aggregated in the substantia nigra are important pathological features of PD, and autophagy dysfunction is considered to be an important factor leading to abnormal aggregation of α-syn. Phenylpropionamides (PHS) in the seed of Cannabis sativa L. have a protective effect on neuroinflammation and antioxidant activity. However, the therapeutic role of PHS in PD is unclear. In this study, the seeds of Cannabis sativa L. were extracted under reflux with 60% EtOH-H2O, and the 60% EtOH-H2O elution fraction was identified as PHS with the UPLC-QTOF-MS. The 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP)-induced PD model in C57BL/6 J mice was used for behavioral and pharmacodynamic experiments. Behavioral symptoms were improved, Nissl-stained and TH-positive neurons in the substantia nigra were significantly increased in PHS-treated MPTP-induced PD model mice. Compared with the model group, PHS treatment reduced the expression level of α-syn, and the expression of TH increased significantly by western blotting, compared with the model group, the PHS group suppressed Caspase 3 and Bax expression and promoted Bcl-2 expression and levels of p62 decreased significantly, the ratio of LC3-II/I and p-mTOR/mTOR in the PHS group had a downward trend, suggesting that the therapeutic effect of PHS on MPTP-induced PD model mice may be triggered by the regulation of autophagy.