Artesunate-Nanoliposome-TPP, a Novel Drug Delivery System That Targets the Mitochondria, Attenuates Cisplatin-Induced Acute Kidney Injury by Suppressing Oxidative Stress and Inflammatory Effects.

Background: Acute kidney injury (AKI) is a syndrome, posing a substantial healthcare burden. The pathological basis of AKI is associated with inflammation and oxidative stress which cause additional damage to mitochondria. Artesunate (ATS) is a derivative of artemisinin isolated from Artemisia annua L. that is an effective treatment for malaria and favored for the prevention and treatment of kidney diseases. However, there are still challenges related to its efficacy, including poor water solubility, limited oral bioavailability and short half-life. Liposome-based nanoparticles are used for drug delivery due to their ideal biocompatibility and their ability to improve the bioavailability of specific drugs and enhance drug efficacy. Methods: In this study, a novel TPP-based natural ATS-nanoliposome, namely T-A-Ls, was applied for the treatment of AKI. ATS was encapsulated with or without triphenylphosphonium (TPP)-modified nanoliposomes. AKI was induced by cisplatin in C57BL/6J mice and a cisplatin-induced injury model was generated in HK-2 cells in vitro. Blood urea nitrogen (BUN), serum creatinine (Scr) measurements and section staining were utilized to assess renal protective effect of T-A-Ls. Inflammatory-related factors and proteins were quantified via Elisa, Immunofluorescence and Western Blot (WB). The anti-mitochondrial oxidative stress effect of T-A-Ls was determined by ROS, JC-1 and oxygen consumption rate (OCR) kits. Immunohistochemistry and WB were conducted to measure associated protein expressions. In vivo biodistribution and the concentration of T-A-Ls in kidney were also explored. Results: T-A-Ls exhibited good oxidative resistance, preferential renal uptake, mitochondrial targeting, and it ameliorated kidney injury in cisplatin-induced AKI mice. Mitochondrial dysfunction, ATP production and respiratory capacity were improved in damaged HK-2 cells; ROS content decreased while mitochondrial membrane potential recovered. T-A-Ls exerted renal protection by inhibiting inflammation and reducing oxidative stress; these effects were mediated by a downregulation in the expression of RAGE and iNOS and an upregulation in both Nrf2 and HO-1. Conclusion: T-A-Ls could improve the delivery of ATS to the kidney, offering a promising avenue to treat AKI.

Dihydroartemisinin abolishes cisplatin-induced nephrotoxicity in vivo.

Dihydroartemisinin (DHA), a derivative of artemisinin which is primarily used to treat malaria in clinic, also confers protective effect on lipopolysaccharide-induced nephrotoxicity. While, the activities of DHA in cisplatin (CDDP)-caused nephrotoxicity are elusive. To investigate the role and underlying mechanism of DHA in CDDP-induced nephrotoxicity. Mice were randomly separated into four groups: normal, CDDP, and DHA (25 and 50 mg/kg were orally injected 1 h before CDDP for consecutive 10 days). All mice except the normal were single injected intraperitoneally with CDDP (22 mg/kg) for once on the 7th day. Combined with quantitative proteomics and bioinformatics analysis, the impact of DHA on renal cell apoptosis, oxidative stress, biochemical indexes, and inflammation in mice were investigated. Moreover, a human hepatocellular carcinoma cells xenograft model was established to elucidate the impact of DHA on tumor-related effects of CDDP. DHA reduced the levels of creatinine (CREA) (p < 0.01) and blood urea nitrogen (BUN) (p < 0.01), reversed CDDP-induced oxidative, inflammatory, and apoptosis indexes (p < 0.01). Mechanistically, DHA attenuated CDDP-induced inflammation by inhibiting nuclear factor κB p65 (NFκB p65) expression, and suppressed CDDP-induced renal cell apoptosis by inhibiting p63-mediated endogenous and exogenous apoptosis pathways. Additionally, DHA alone significantly decreased the tumor weight and did not destroy the antitumor effect of CDDP, and did not impact AST and ALT. In conclusion, DHA prevents CDDP-triggered nephrotoxicity via reducing inflammation, oxidative stress, and apoptosis. The mechanisms refer to inhibiting NFκB p65-regulated inflammation and alleviating p63-mediated mitochondrial endogenous and Fas death receptor exogenous apoptosis pathway.

Andrographolide anti-proliferation and metastasis of hepatocellular carcinoma through LncRNA MIR22HG regulation.

Hepatocellular carcinoma (HCC) treatment is a major challenge. Although andrographolide (Andro) has an anti-proliferation effect on HCC, its underlying mechanism is not yet elucidated, and whether Andro can inhibit HCC metastasis has not been reported. The present study aimed to clarify whether Andro inhibits SK-Hep-1 cell proliferation and HCC metastasis, and the mechanisms. The results showed that Andro significantly reduced the survival of HCC cells and tumor weight and volume in tumor-bearing nude mice. Andro also triggered apoptosis of HCC cells and upregulated MIR22HG, Cleaved Caspase 9/7/3 expression levels, and downregulated BCL-2 mRNA, BCL-2 expression levels. Knockdown of MIR22HG or overexpression of HuR attenuated the effects of Andro on the signal transduction of mitochondrial apoptotic pathway and proliferation ability in HCC cells. Moreover, Andro significantly reduced the invasive ability of the cells and the level of HCC cell lung metastasis, upregulated miR-22-3p expression level and downregulated HMGB1 and MMP-9 expression levels. MIR22HG or miR-22-3p knockdown attenuated the effects of Andro on the signaling of HMGB1/MMP-9 pathway and invasive ability in HCC cells, while the overexpression of HMGB1 attenuated the inhibitory effects of Andro on the MMP-9 expression level and invasive ability in HCC cells. Thus, the regulation of MIR22HG-HuR/BCL-2 and MIR22HG/HMGB1 signaling pathways is involved in the anti-HCC proliferation and metastasis effects of Andro. This study provided a new pharmacological basis for Andro in HCC treatment and, for the first time, identified a natural product molecule capable of positively regulating MIR22HG, which has a robust biological function.

PI3K/AKT1 Signaling Pathway Mediates Sinomenine-Induced Hepatocellular Carcinoma Cells Apoptosis: An in Vitro and in Vivo Study.

Hepatocellular carcinoma (HCC) is one of the most frequent cancers. Sinomenine (SIN) is a compound derived from Sinomenium acutum. Our previous investigations have found that SIN inhibited protein kinase B (AKT) signaling to induce autophagic death of tumor cells. However, whether inhibition of this pathway by SIN could impact the proliferation of HCC cells is unknown. Thus, we applied SIN to SK-Hep-1 cells and used cell counting kit 8 (CCK8), lactate dehydrogenase (LDH), colony formation and 5-ethynyl-20-deoxyuridine (EdU) incorporation experiments to detect cell viability. Then, staining with annexin V/propidium iodide (PI) coupled with terminal deoxynucleotidyl transferase-mediated biotinylated uridine 5'-triphosphate (UTP) nick end labeling (TUNEL) staining were utilized to monitor apoptosis. Changes in cell mitochondrial membrane capacity were explored via 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining, whilst Western blot or immunohistochemistry was applied to evaluate the expression levels of key proteins, consisting of Cleaved Caspase 3, AKT1, B-cell leukemia/lymphoma 2 (BCL-2), phosphatidylinositol 3-kinase (PI3K) p85α, and Cleaved Caspase 9 etc. The Balb/c nude mice were utilized to establish HCC xenograft tumor model, administered by SIN. After treatments, the tumor volume along with weight were measured. The results illustrated that SIN suppressed SK-Hep-1 HCC cells' proliferation, enhanced the collapse of potential of the mitochondrial membrane, triggered cell apoptosis, down-regulated PI3K p85α, AKT1, BCL-2, Pro-Caspase 9, Pro-Caspase 3 expressions, and up-regulated Cleaved Caspase 9 and Cleaved Caspase 3 expressions in vitro and in vivo. Meanwhile, SIN reduced the tumor volume along with weight of mice. In addition, insulin-like growth factor-1 (IGF-1), a powerful activator of the PI3K/AKT pathway, could reverse the high apoptosis of SK-Hep-1 HCC cells induced by SIN. Overall, inhibition of PI3K/AKT1 signaling cascade by SIN induced HCC cells apoptosis.

Quickly Identifying High-Risk Variables of Ultrasonic Extraction Oil from Multi-Dimensional Risk Variable Patterns and a Comparative Evaluation of Different Extraction Methods on the Quality of Forsythia suspensa Seed Oil.

Generally, essential oils and components of interest are extracted from plants using organic solvent, distillation, ultrasound and supercritical extraction methods. Ultrasonic extraction (UE) has the advantage of high efficiency, but its process is complicated and it has numerous variables. In this study, an L18-Hunter experimental design was applied for the first time to investigate the practicability of applying UE to Forsythia suspensa seed oil. Six potential high-risk variables, including numerical and non-numeric types, were obtained from the risk analysis and their impacts on global yield and antioxidant activity were screened. Furthermore, oils obtained by different extraction processes (i.e., UE, supercritical fluid extraction (SFE), soxhlet extraction (SE) and hydrodistillation extraction (HD)) were analyzed. A comparative study of these oils was characterized and compared by FT-IR, GC-MS and antioxidant activity. The obtained results show that the type of solvent, solvent-to-solid ratio, extraction power and time were the significant variables affecting the extraction yield, whereas antioxidant activity was only affected by the type of solvent. The regression coefficients of the yield and antioxidant activity models were 0.79 and 0.91, and the ANOVA of the models were 0.013 and <0.0001, respectively. Beta-Pinene was the main abundant component in the oils for the UE, SFE, SE and HD methods and the content was about 46%~52.4%. In conclusion, the L18-Hunter design could be used as an effective experimental design method for rapid screening of high-risk variables. Regarding extraction efficiency, chemical composition and biological activity, UE not only offered a robust Forsythia suspensa seed oil extraction process, but also provided a time- and cost-effective advantage to the food and pharmaceutical industry when compared to the SFE, SE and HD extraction processes.

Sinomenine Hydrochloride Inhibits the Metastasis of Human Glioblastoma Cells by Suppressing the Expression of Matrix Metalloproteinase-2/-9 and Reversing the Endogenous and Exogenous Epithelial-Mesenchymal Transition.

As shown in our previous study, sinomenine hydrochloride (SH), the major bioactive alkaloid isolated from Sinomenium acutum Rehd. et Wils. (Fam. Menispermaceae), initiates the autophagy-mediated death of human glioblastoma cells by generating reactive oxygen species and activating the autophagy-lysosome pathway. However, its effects on the migration and invasion of human glioblastoma cells have not yet been elucidated. Therefore, human glioblastoma U87 and SF767 cells were treated with SH (0.125 and 0.25 mM) for 24 h, and cell migration and invasion were assessed using scratch wound healing, migration and invasion assays. SH promoted G0/G1 phase arrest, inhibited the migration and invasion of the two cell lines, suppressed the activation of nuclear factor kappa B (NFκB) and the expression of matrix metalloproteinase (MMP)-2/-9, triggered endoplasmic reticulum (ER) stress, reversed the exogenous epithelial-mesenchymal transition (EMT) induced by the inflammatory microenvironment and the endogenous EMT. Additionally, NFκB p65 overexpression blocked the SH-mediated inhibitory effects on MMP-2/-9 expression and cell invasion. SH-induced autophagy was reduced in CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) or autophagy-related 5 (ATG5)-silenced human glioblastoma cells and cells treated with 4-phenylbutyric acid (4-PBA) or 3-methyladenine (3-MA), as shown by the decreased levels of the microtubule-associated protein light chain 3B (LC3B)-II and autophagic vacuoles (AVs) stained with monodansylcadaverine (MDC), respectively. Moreover, knockdown of CHOP or ATG5 and treatment with 4-PBA or 3-MA abolished the SH-mediated inhibition of mesenchymal markers (vimentin, Snail and Slug) expression and cell invasion, respectively. Importantly, SH also regulated the above related pathways in nude mice. Based on these findings, SH inhibited cell proliferation by inducing cell cycle arrest, and attenuated the metastasis of U87 and SF767 cells by suppressing MMP-2/-9 expression and reversing the endogenous and exogenous EMT in vitro and/or in vivo. Thus, SH might be a new potential anti-metastasis agent for the treatment of human glioblastoma.

Sinomenine Hydrochloride Inhibits Human Glioblastoma Cell Growth through Reactive Oxygen Species Generation and Autophagy-Lysosome Pathway Activation: An In Vitro and In Vivo Study.

Glioblastoma is the most common malignant primary brain tumor, and it is one of the causes of cancer fatality in both adult and pediatric populations. Patients with glioblastoma require chemotherapy after surgical resection and radiotherapy. Therefore, chemotherapy constitutes a viable approach for the eradication of glioblastoma cells. In this study, the anti-tumor activity of sinomenine hydrochloride (SH) was evaluated in U87 and SF767 cells in vitro and in vivo. The results showed that SH potently inhibited U87 and SF767 cell viability and did not cause caspase-dependent cell death, as demonstrated by the absence of significant early apoptosis and caspase-3 cleavage. Instead, SH activated an autophagy-mediated cell death pathway, as indicated by the accumulated microtubule-associated protein light chain 3B (LC3B)-II, triggered autophagic flux and enhanced cell viability after pretreatment with autophagy inhibitors. SH-mediated autophagy in the two cell lines was implicated in reactive oxygen species (ROS) generation, protein kinase B (Akt)-mammalian target of rapamycin (mTOR) pathway suppression and c-Jun NH2-terminal kinase (JNK) pathway activation. The ROS antioxidant N-acetylcysteine (NAC), the Akt-specific activator insulin-like growth factor-1 (IGF-1) and the JNK-specific inhibitor SP600125 attenuated SH-induced autophagy. Moreover, ROS activated autophagy via the Akt-mTOR and JNK pathways. Additionally, SH treatment may promote lysosome biogenesis through activating transcription factor EB (TFEB). The in vivo study found that SH effectively suppressed glioblastoma growth without exhibiting significant toxicity. In conclusion, our findings reveal a novel mechanism of action of SH in cancer cells via the induction of autophagy through ROS generation and autophagy-lysosome pathway activation; these findings also supply a new potential therapeutic agent for the treatment of human glioblastoma.

[Analgesic effect and central mechanisms of CQ prescription on cancer invasion induced mirror image pain in model mice].

This study aimed to analyze the analgesic effect and related central mechanisms of CQ prescription on cancer invasion induced mirror image pain (CIIMIP)in model mice.In the study, male BALB/c mice were randomly divided into normal group, operation control group (injected with 0.2 mL inactivated S180 sarcoma cell sap), model group (injected with 0.2 mL S180 sarcoma cell sap on the right leg near the greater trochanter of femur) and CQ prescription low dose group (intraperitoneally injected with CQ prescription 100 mg•kg⁻¹ on the basis of model mice), CQ prescription middle dose group (intraperitoneally injected with CQ prescription 150 mg•kg⁻¹ on the basis of model mice), and CQ prescription high dose group (intraperitoneally injected with CQ prescription 200 mg•kg⁻¹ on the basis of model mice). Mechanical withdraw threshold (MWT) of the mirror image lateral hind paws were evaluated by Von Frey hairs before modeling and after surgery. The levels of glutamate (Glu), gamma aminobutyric acid (GABA), glycine (Gly), and taurine (Tau) in the L3-L5 spinal cord were measured by the high performance liquid chromatography-fluorescence detector (HPLC-FLD); AimPlex detection technology with multiple factors was used to detect the levels of regulated on activation in normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein (MCP-3) in the L3-L5 spinal cord. Then we observed the influence of GABAa receptor antagonist (Bicuculline) on analgesic effect of CQ prescription.The results indicated that CQ prescription could remarkably increase MWT of model mice(P<0.01, P<0.05), decrease the level of Glu(P<0.01, P<0.05), improve the levels of GABA, Gly, Tau(P<0.01, P<0.05), lower the ratio of Glu/GABA(P<0.01, P<0.05), and reduce the levels of RANTES, MCP-3(P<0.05) in the L3-L5 spinal cord, and GABAa receptor antagonist significantly blocked the analgesic effect of CQ prescription at two time points(P<0.05).This study showed that CQ prescription had significant analgesic effect on CIIMIP model mice, and its mechanism was associated with regulating the balance between excitability amino acid(EAA) and inhibitory amino acid (IAA) transmitters in central nervous system, partially activating GABAa receptor, and reducing the release of RANTES and MCP-3 in the spinal cord.

Neuroprotective effect of water extract of Panax ginseng on corticosterone-induced apoptosis in PC12 cells and its underlying molecule mechanisms.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Panax ginseng C.A. Meyer (Family Araliaceae) is an important medicinal plant which has been employed as a panacea for more than 2,000 years in China. It has the actions of invigorating primordial qi, recovering pulse and desertion, engendering liquid, and calming spirit. The water extract of Panax ginseng (WEG) has been used to treat kinds of central nervous system disorders, such as depression, insomnia, Alzheimer׳s disease and Parkinson׳s disease. Our previous work has demonstrated that WEG possessed antidepressant-like activities in both acute and chronic stress models of depression. Nevertheless, there are no studies on the cytoprotection and potential mechanisms of WEG on corticosterone-induced apoptosis. The present study focuses on cytoprotection against corticosterone-induced neurotoxicity in PC12 cells and its underlying molecule mechanisms of the antidepressant-like effect of WEG. MATERIALS AND METHODS: The PC12 cells were treated with 250 µmol/L corticosterone in the absence or presence of WEG for 24h, then 3-(4,5-dimethy thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) detection, Hoechst33342 staining and TUNEL staining were investigated to confirm the neuroprotection of WEG. Then, mitochondrial permeability transition pore (mPTP), mitochondrial membrane potential (MMP), intracellular Ca(2+) ([Ca(2+)]i), reactive oxygen species (ROS) concentration, and the expression level of glucocorticoid receptor (GR), heat shock protein 90 (Hsp90), histone deactylase 6 (HDAC6), glucose-regulated protein 78 (GRP78), growth arrest and DNA damage inducible protein 153 (GADD153), X-box DNA-binding protein-1 (XBP-1), caspase-12, cytochrome C, inhibitor of caspase-activated deoxyribonuclease (ICAD), caspase-3 and caspase-9 were assessed by Western Blot analysis to understand the molecule mechanisms of neuroprotection of WEG. RESULTS: WEG partly reversed corticosterone-induced damage in PC12 cells, which increased cell viability, decreased LDH release, and attenuated corticosterone-induced apoptosis as compared with the corticosterone-treated group. Mechanistically, compared with the corticosterone-treated group, WEG strongly attenuated [Ca(2+)]i overload and ROS level, and restored mitochondrial function, including mPTP and MMP. Furthermore, WEG strongly up-regulated the expression of GR and HDAC6, and down-regulated the expression of Hsp90, cytochrome C, ICAD, caspase-3, caspase-9 as well as endoplasmic reticulum (ER) stress-related proteins, such as GADD153, GRP78, XBP-1, and caspase-12. CONCLUSION: WEG possessed neuroprotection against corticosterone-induced damage in PC12 cells, and the underlying molecule mechanisms was depended on the intervening of HDAC6 and HSP90 of the GR-related function proteins, and subsequent restoration of ER and mitochondria functions.

Cajaninstilbene acid protects corticosterone-induced injury in PC12 cells by inhibiting oxidative and endoplasmic reticulum stress-mediated apoptosis.

It has been reported that high corticosterone level could damage the normal hippocampal neurons both in vitro and in vivo. Furthermore, high concentration of corticosterone induced impair in PC12 cells has been widely used as in vitro model to screen neuroprotective agents. Cajaninstilbene acid (CSA), a natural stilbene isolated from Cajanus cajan leaves, has various activities. In present study, we investigated the effect of CSA on corticosterone-induced cell apoptosis and explored its possible signaling pathways in PC12 cells. We demonstrated that pretreatment with CSA at the concentrations of 1-8 µmol/L remarkably reduced the cytotoxicity induced by 200 µmol/L of corticosterone in PC12 cells by MTT, and further confirmed the neuroprotection by Hoechst 33342 and PI double staining and lactate dehydrogenase release (LDH) assay at the concentration of 8 µmol/L. Moreover, the cytoprotection of CSA was proved to be associated with the homeostasis of intracellular Ca(2+), relieving corticosterone-induced oxidative stress by decreasing the contents of ROS and malondialdehyde (MDA), increasing the activities of superoxide dismutase (SOD) and catalase (CAT), and the stabilization of ER stress via down-regulating the expression of ER chaperone protein glucose-regulated protein 78 (GRP78), ER stress associated transcription factor C/EBP homologous protein (CHOP/GADD153), and the X box-binding protein-1 (XBP-1), as well as the expression of ER stress-specific protein caspase-12 and its downstream protein caspase-9. Considering all the findings, it is suggested that the neuroprotective activity of CSA against the impairment induced by corticosterone in PC12 cells was through the inhibition of oxidative stress and ER stress-mediated pathway.

Saikosaponin D acts against corticosterone-induced apoptosis via regulation of mitochondrial GR translocation and a GR-dependent pathway.

Saikosaponin D is an agonist of the glucocorticoid receptor (GR), and our preliminary study showed that it possesses neuroprotective effects in corticosterone-treated PC12 cells. However, further proof is required, and the molecular mechanisms of this neuroprotection remain unclear. This study sought to further examine the cytoprotective efficiency and potential mechanisms of action of Saikosaponin D in corticosterone-treated PC12 cells. The cells were treated with 250 µM corticosterone in the absence or presence of Saikosaponin D for 24 h; cell viability was then determined, and Hoechst 33342/propidium iodide (PI) and annexin/PI double staining, and TUNEL staining were performed. Next, mPTP, MMP, [Ca(2+)]i, translocation of the GR to the nucleus and Western blot analyses for caspase-3, caspase-9, cytochrome C, GR, GILZ, SGK-1, NF-Κb (P65), IκB-α, Bad, Akt, Hsp90 and HDAC-6 were investigated. The neuroprotective effects of Saikosaponin D were further confirmed by Hoechst 33342/PI, annexin/PI and TUNEL staining assays. These additional data suggested that Saikosaponin D partially reversed the physiological changes induced by corticosterone by inhibiting the translocation of the GR to the mitochondria, restoring mitochondrial function, down-regulating the expression of pro-apoptotic-related signalling events and up-regulating anti-apoptotic-related signalling events. These findings suggest that SSD exhibited its anti-apoptotic effects via differential regulation of mitochondrial and nuclear GR translocation, partial reversal of mitochondrial dysfunction, inhibition of the mitochondrial apoptotic pathway, and selective activation of the GR-dependent survival pathway.