Brazilin Actuates Ferroptosis in Breast Cancer Cells via p53/SLC7A11/GPX4 Signaling Pathway.

OBJECTIVE: To investigate the mechanism of induction of ferroptosis by brazilin in breast cancer cells. METHODS: Breast cancer 4T1 cells were divided into 6 groups: control, brazilin 1/2 half maximal inhibitory concentration (IC50), IC50, 2×IC50, erastin (10 µg/mL) and capecitabine (10 µg/mL) groups. The effect of brazilin on the proliferation of 4T1 cells was detected by cell counting kit-8 assay, and the treatment dose of brazilin was screened. The effect of brazilin on the mitochondrial morphology of 4T1 cells, and the mitochondrial damage was evaluated under electron microscopy. The levels of Fe2+, reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH) and glutathione peroxidase 4 (GPX4) were estimated using various detection kits. The invasion and migration abilities of 4T1 cells were detected by scratch assay and transwell assay. The expressions levels of tumor protein p53, solute carrier family 7 member 11 (SLC7A11), GPX4 and acyl-CoA synthetase long-chain family member 4 (ACSL4) proteins were quantified by Western blot assay. RESULTS: Compared to the control group, the 10 (1/2 IC50), 20 (IC50) and 40 (2×IC50) µg/mL brazilin, erastin, and capecitabine groups showed a significant decrease in the cell survival rate, invasion and migration abilities, GSH, SLC7A11 and GPX4 protein expression levels, and mitochondrial volume and ridge (P<0.05), and a significant increase in the mitochondria membrane density, Fe2+, ROS and MDA levels, and p53 and ACSL4 protein expression levels (P<0.05). CONCLUSIONS: Brazilin actuated ferroptosis in breast cancer cells, and the underlying mechanism is mainly associated with the p53/SLC7A11/GPX4 signaling pathway.

Penthorum chinense Pursh leaf tea debittering mechanisms via green tea manufacturing process and its influence on NAFLD-alleviation activities.

The green-tea manufacturing process showed good effect of flavor improving, debittering and shaping in making Penthorum chinensePursh leaf (PL) tea (PLT), which serves as a polyphenol dietary supplement and beverage raw material. GC-MS results showed that its unpleasant grassy odor decreased by 42.8% due to dodecanal, geranylacetone, and (E)-2-nonenal reduction, coupled with 1-hexadecanol increasing. UPLC-ESI-TOF-MS identified 95 compounds and showed that the debittering effect of green-tea manufacturing process was attributed to decreasing of flavonols and lignans, especially quercetins, kaempferols and luteolins, and increasing of dihydrochalcones which act as sweeteners bitterness-masking agents, while astringency was weakened by reducing delphinidin-3,5-O-diglucoside chloride, kaempferol-7-O-ß-d-glucopyranoside, and tannins. The increase of pinocembrins and catechins in aqueous extracts of PLT, maintained its hepatoprotective, NAFLD-alleviation, and hepatofibrosis-prevention activities similar to PL in high fat-diet C57BL/6 mice, with flavonoids, tannins, tannic acids, and some newfound chemicals, including norbergenin, gomisin K2, pseudolaric acid B, tanshinol B, as functional ingredients.

Unraveling the evolutionary dynamics of the TPS gene family in land plants.

Terpenes and terpenoids are key natural compounds for plant defense, development, and composition of plant oil. The synthesis and accumulation of a myriad of volatile terpenoid compounds in these plants may dramatically alter the quality and flavor of the oils, which provide great commercial utilization value for oil-producing plants. Terpene synthases (TPSs) are important enzymes responsible for terpenic diversity. Investigating the differentiation of the TPS gene family could provide valuable theoretical support for the genetic improvement of oil-producing plants. While the origin and function of TPS genes have been extensively studied, the exact origin of the initial gene fusion event - it occurred in plants or microbes - remains uncertain. Furthermore, a comprehensive exploration of the TPS gene differentiation is still pending. Here, phylogenetic analysis revealed that the fusion of the TPS gene likely occurred in the ancestor of land plants, following the acquisition of individual C- and N- terminal domains. Potential mutual transfer of TPS genes was observed among microbes and plants. Gene synteny analysis disclosed a differential divergence pattern between TPS-c and TPS-e/f subfamilies involved in primary metabolism and those (TPS-a/b/d/g/h subfamilies) crucial for secondary metabolites. Biosynthetic gene clusters (BGCs) analysis suggested a correlation between lineage divergence and potential natural selection in structuring terpene diversities. This study provides fresh perspectives on the origin and evolution of the TPS gene family.

[Effects of Xihuang Pills on proliferation and apoptosis of prostate cancer LNCaP cells based on AR/m TOR signaling pathway].

Based on the androgen receptor(AR)/mammalian target of rapamycin(mTOR)signaling pathway, the effects of Xihuang Pills-medicated serum on the proliferation and apoptosis of prostate cancer LNCaP cells were investigated. The drug-containing serum of SD rats was prepared by intragastric administration of Xihuang Pills suspension. The effects of low-, medium-, and high-dose Xihuang Pills-containing serum on the in vitro proliferation of LNCaP cells were detected by cell counting kit-8(CCK-8). Flow cytometry was used to detect the apoptosis level of LNCaP cells after intervention with different concentrations of Xihuang Pills. Protein expression of cleaved cysteinyl aspartate-specific proteinase caspase-3(cleaved caspase-3), B-cell lymphoma-2(Bcl-2), and AR as well as the phosphorylation level of mTOR protein were detected by Western blot. The results showed that compared with the blank serum, the drug-medicated serum could blunt the activity of LNCaP cells. Low-, medium-, and high-dose Xihuang Pills-containing serum could significantly increase the cell apoptosis rate, increase the expression of cleaved caspase-3 protein, decrease the expression of Bcl-2 protein, reduce the expression of AR protein, and down-regulate the level of phosphorylated mTOR(p-mTOR). To study the effect of Xihuang Pills on the growth of LNCaP cells in vivo, different doses of Xihuang Pills were used to intervene in the subcutaneous graft model in nude mice inoculated with LNCaP cells. The expression levels of AR, mTOR, p-mTOR, Bcl-2, and cleaved caspase-3 were detected by Western blot. The results showed that the volumes of subcutaneous graft tumor in the low-dose, medium-dose, and high-dose Xihuang Pills groups significantly decreased compared with that in the model group. The weight of subcutaneous transplanted tumor in each group with drug intervention was significantly lower than that in the model group. Compared with the model group, the low-dose, medium-dose, and high-dose Xihuang Pills groups showed increased cleaved caspase-3 protein expression, decreased Bcl-2 and AR protein expression, and reduced p-mTOR protein expression. Further experiments showed that AR agonist R1881 could block the anti-proliferation and pro-apoptotic effects of Xihuang Pills. The mechanism of Xihuang Pills against prostate cancer is related to the inhibition of the AR/mTOR signaling pathway, inhibition of LNCaP cell proliferation, and induction of apoptosis in cancer cells.

[Preliminary observation on the clinical application of rehabilitation support for lumbar disc herniation based on 3D printing technology].

OBJECTIVE: To analyze the important effect of 3D printing personalized lumbar support on lumbar pain and lumbar function in patients with lumbar disc herniation. METHODS: From October 2018 to May 2021, 60 patients initially diagnosed with lumbar disc herniation were selected and divided into an observation group and a control group, with 30 patients in each group. Among them, there were 18 males and 12 females in the observation group;the age ranged from 24 to 56 years old, with an average of (45.23±6.07) years old. The course of disease ranged from 1 to 24 months, with an average of(6.25±0.82) months, and rehabilitation treatment was carried out by wearing 3D printed personalized lumbar support. There were 19 males and 11 females in the control group;the age ranged from 25 to 57 years old, with an average of (42.78±7.58) years old. The course of disease ranged from 1 to 24 months, with an average of (6.72±1.36) months, and rehabilitation treatment is carried out by wearing traditional lumbar protective equipment. The Japanese Orthopaedic Association (JOA) scores, lumbar Oswestry dysfunction index (ODI) and visual analogue scale (VAS) were evaluated and compared between the two groups before and 1 course after treatment (3 weeks). RESULTS: There was no statistically significant difference in JOA, ODI, and VAS between two groups before treatment (P>0.05). After one course of treatment (3 weeks), JOA scores of both groups was increased compared to before treatment (P<0.05), while ODI and VAS decreased compared to before treatment (P<0.05). After treatment, JOA score of observation group was higher than that of control group (P<0.05), while ODI and VAS scores were lower than those of control group. No adverse events occurred in both groups. CONCLUSION: The application of 3D printing personalized lumbar support can effectively alleviate the pain of patients with lumbar disc herniation and improve their lumbar function of patients.

Molecular characterization and potential function of Rxrγ in gonadal differentiation of Chinese soft-shelled turtle (Pelodiscus sinensis).

Retinoid X receptor (RXR) is a member of the ligand-dependent nuclear receptor family. Previous studies revealed that RXRs are involved in reproduction in vertebrates. However, information on the function of RXRs in turtles is scarce. In this study, the Rxrγ cDNA sequence of Pelodiscus sinensis was cloned and analyzed, and a polyclonal antibody was constructed. RXRγ protein showed a positive signal in both mature and differentiated gonads of the turtle. Subsequently, the function of the Rxrγ gene in gonadal differentiation was confirmed using short interfering RNA (RNAi). The full-length cDNA sequence of the Rxrγ gene in P. sinensis was 2152 bp, encoding 407 amino acids and containing typical nuclear receptor family domains, including the DNA-binding domain (DBD), ligand-binding domain (LBD), and activation function 1 (AF1). Moreover, gonadal Ps-Rxrγ showed sexual dimorphism expression patterns in differentiated gonads. Real-time quantitative PCR results revealed that the Rxrγ gene was highly expressed in the turtle ovary. RNAi treatment increased the number of Sertoli cells in ZZ embryonic gonads. Furthermore, RNA interference upregulated Dmrt1 and Sox9 in ZZ and ZW embryonic gonads. However, Foxl2, Cyp19a1, Stra8, and Cyp26b1 were downregulated in embryonic gonads. The results indicated that Rxrγ participated in gonadal differentiation and development in P. sinensis.

[Heat-tonifying acupuncture relieves pain and synovial inflammatory injury by regulating Keap1-Nrf2/ARE/ HO-1 signaling pathway in rabbits with cold syndrome type rheumatoid arthritis].

OBJECTIVE: To observe the effect of heat-tonifying needling on Keap1-Nrf2/ARE/HO-1 signal transduction pathway in knee synovium in rabbits with cold syndrome type rheumatoid arthritis (RA), so as to explore its mechanisms underl-ying improvement of RA. METHODS: New Zealand rabbits were randomly divided into normal control, RA model, uniform reinforcing-reducing acupuncture, twisting reinforcing acupuncture and heat-tonifying acupuncture groups, with 6 rabbits in each group. The cold syndrome type RA model was established by subcutaneous injection of mixture fluid of ovalbumin and Freund's complete adjuvant at the shoulder-back as well as injection of mixture of ovalbumin and normal saline into knee-joint cavity combined with ice-compress freezing. Acupuncture stimulation (uniform reinforcing-reducing, or twisting reinforcing or heat-tonifying) was applied to bilateral "Zusanli"(ST36) for 1 min with the needle retained for 30 min, once a day for 7 consecutive days. The general conditions of rabbits in each group were recorded, the thermal pain threshold (TPT) and perimeter of knee joints was measured. Conditions of the synovium in the knee cavity, hydrops, blood flow signal, articular surface, and related muscles were observed by using a color Doppler ultrasonic diagnostic apparatus, and the blood flow signals inside the synovium (image scores) were divided into 0 (no signals), I (1 or 2 dot-like signal), II (less than half) ad III (more than half). After H.E. staining, the pathological changes (0-3 points) were assessed according to the state of inflammatory cell infiltration, and hyperplasia of synovial matrix and coating cells. The expression levels of Keap1, Nrf2, HO-1 and GSH-PX1 mRNAs in the knee synovium were detected by quantitative real-time PCR, and the expression of knee synovial HO-1 protein was measured by Western blot. RESULTS: In comparison with the normal control group, the model group had a significant increase in the perimeter, pathological score, expression of Nrf2, HO-1 mRNAs and HO-1 protein (P<0.05), and an obvious decrease in the TPT, expression levels of Keap1 and GSH-PX1 mRNAs (P<0.05). Relevant to the model group, all the three acupuncture maneuvers reversed modeling-induced increase of perimeter and pathological score (P<0.05), decrease of TPT and expression of GSH-PX1 mRNA(P<0.05), further down-regulated expression of Keap1 mRNA (P<0.05), further up-regulated the expression of Nrf2, HO-1 mRNAs and HO-1 protein (P<0.05). The heat-reinforcing manipulation was significantly superior to uniform reinforcing-reducing and twirling reinforcing manipulations in up-regulating TPT, and expression of Nrf2 mRNA, GSH-PX1 mRNA, HO-1 mRNA and protein (P<0.05), and in down-regulating pathological score and Keap1 mRNA expression (P<0.05). CONCLUSION: Heat-tonifying, uniform reinforcing-reducing and twirling reinforcing needling manipulations may relieve pain and improve pathological state in RA rabbits, which may be associated with their functions in raising the ability of anti-oxidative stress by regulating Keap1-Nrf2/ARE/ HO-1 signaling pathway, the therapeutic effect of heat-tonifying needling is superior to that of uniform reinforcing-reducing and twirling reinforcing needling.

Evaluating the effects of vitamin D Level on airway obstruction in two asthma endotypes in humans and in two mouse models with different intake of vitamin D during early-life.

Introduction: Asthma is primarily divided into two categories: type 2 (T2-high) and non-type 2 (T2-low). A relationship between asthma severity and vitamin D deficiency has been identified, but its impact on each asthma endotype remains unknown. Methods: We clinically examined the influence of vitamin D on patients with T2-high (n = 60) or T2-low asthma (n = 36) compared with controls (n = 40). Serum 25(OH)D levels, inflammatory cytokines and spirometry were measured. Mouse models were then used to further analyze the effects of vitamin D on both asthmatic endotypes. BALB/c mice were fed with vitamin D-deficient (LVD), -sufficient (NVD), or -supplemented diets (HVD) throughout lactation and offspring followed the same diet after weaning. Offspring were sensitized/challenged with ovalbumin (OVA) to establish "T2-high" asthma or OVA combined with ozone exposure (OVA + ozone) to induce "T2-low" asthma. Spirometry and serum, bronchoalveolar lavage fluid (BALF), and lung tissues were analyzed. Results: Serum 25(OH)D levels were decreased in asthmatic patients compared with controls. Patients with vitamin D deficiency (Lo) had varying degrees of elevation of the pro-inflammatory cytokines IL-5, IL-6, and IL-17A, decreased expression of the anti-inflammatory cytokine IL-10, and altered forced expiratory volume in the first second as a percentage of predicted value (FEV1%pred) in both asthmatic endotypes. Vitamin D status had a stronger correlation with FEV1%pred in T2-low asthma than T2-high asthma, and 25(OH)D level was only positively linked to maximal mid-expiratory flow as a percentage of predicted value (MMEF%pred) in the T2-low group. Inflammation, hyperresponsiveness, and airway resistance (RL) was increased in both asthma models compared with controls while vitamin D deficiency further increased airway inflammation and airway obstruction. These findings were particularly prominent in T2-low asthma. Discussion: The potential function and mechanisms of vitamin D and both asthma endotypes should be studied individually, and further analysis of the potential signaling pathways involved with vitamin D on T2-low asthma is warranted.

Integrated network pharmacology and experimental verification to explore the molecular mechanism of Jingxin Zhidong formula for treating Tic disorder.

ETHNOPHARMACOLOGICAL RELEVANCE: As a traditional Chinese medicine formula, Jingxin Zhidong Formula (JXZDF) based on ancient amber powder has been prescribed to alleviate tic disorders (TD) according to our clinical practice for many years. However, the underlying molecular mechanisms remain largely unknown. AIM OF STUDY: To explore the potential mechanism of JXZDF in the treatment of TD by using network pharmacology and experimental validation. MATERIALS AND METHODS: The chemical components of JXZDF were detected and the potential pathway enrichment analyses were conducted based on network pharmacology. Finally, we performed cell viability assays and Western blotting on LPS-induced BV-2 cells, and subsequently performed behavioral tests and Western blotting in SD rats model for TD to explore the mechanism of JXZDF on TD. RESULTS: By LC-ESI-MS/MS system and searching the databases, we identified 5 key compounds and 29 hub targets of JXZDF on TD. KEGG enrichment analysis showed that PI3K/AKT signaling pathway may be the key pathway for JXZDF on TD. The vitro experimental results proved that JXZDF can inhibit the phosphorylation of PI3K and AKT proteins on LPS-induced BV-2 cells. The animal experimental results indicated that JXZDF can effectively alleviate the stereotypic behavior and hyperactivity of the TD rats, and downregulated PI3K/AKT pathway to inhibit microglia activation in the hippocampus tissue. CONCLUSION: This study indicated that JXZDF can change microglial activation and expression of proinflammatory mediators through the inactivation of PI3K/AKT signaling pathway, which may be one of the mechanisms of JXZDF in treating TD.

Spatial Metallomics Reveals Preferable Accumulation of Methylated Selenium in a Single Seed of the Hyperaccumulator Cardamine violifolia†.

Cardamine violifolia is a Se hyperaccumulator found in Enshi, China. In this study, spatial metallomics was applied to visualize the distribution and speciation of Se in a single seed of C. violifolia. It was found that Se reached 1729.89 ± 28.14 mg/kg and the main Se species were SeCys and SeMet in bulk seeds. Further in situ study on a single seed found that the methylated Se species located mostly in the episperm. This is the first visualized evidence of the in situ distribution of methylated Se species in the seeds of C. violifolia. In all, spatial metallomics finds a preferable accumulation of methylated Se species in the seed coat, which deepens the understanding of the tolerance of Se by C. violifolia. The protocol applied in this study may also be used for the understanding of the tolerance of heavy metals/metalloids in other hyperaccumulators.

Cardamine violifolia as a potential Hg hyperaccumulator and the cellular responses.

Cardamine violifolia belongs to the Brassicaceae family and is a selenium (Se) hyperaccumulator found in Enshi, China. In this study, C. violifolia was found to accumulate mercury (Hg) in its roots and aboveground parts at concentrations up to 6000 µg/g. In the seedling and mature stages, the bioaccumulation factors (BAFS) of Hg reached 1.8-223, while the translocation factor (TF) for Hg reached 1.5. We observed a significant positive correlation between THg concentrations in plant tissues and those in the soil (r2 = 0.71-0.84). Synchrotron radiation X-ray fluorescence with focused X-ray (µ-SRXRF) showed that Hg was translocated from the roots to shoots through the vascular bundle and was transported through the leaf veins in leaves. Transmission electron microscopy showed that root cells were more tolerant to Hg than leaf cells. These findings provide insights into the mechanisms of Hg hyperaccumulation in C. violifolia. Overall, we demonstrated that C. violifolia is a promising Hg hyperaccumulator that may be used for phytoremediating Hg-contaminated farmlands.

Nano mercury selenide as a source of mercury for rice.

Mercury (Hg) is a persistent and toxic metal while mercury selenide (HgSe) is generally considered as the environmental sink of Hg in its biogeochemical cycle. Recent studies found nano-sized HgSe (nano-HgSe) could be transformed by certain bacteria. This raises safety concerns about the application of selenium (Se) to curb Hg contamination in farmlands. Therefore, hydroponic experiments were performed in which rice plants were cultured with different concentrations of nano-HgSe and micro-sized HgSe (micro-HgSe) to explore their bioavailability and toxicity. It was found that both nano-HgSe and micro-HgSe did not affect the germination of rice seeds but affected the growth of rice seedlings. However, nano-HgSe could be more readily absorbed by roots and transferred to the aboveground parts compared to micro-HgSe. The highest Hg and Se levels were found to be 5255.67 ± 2496.14 µg/g and 1743.75 ± 61.87 µg/g, respectively in roots when exposed to 5000 mg/L nano-HgSe. Besides, small portion (1.2%) of methylmercury (MeHg) to total Hg was found accumulated in rice stem when exposed to 100 mg/L nano-HgSe, suggesting that nano-HgSe could be decomposed. Furthermore, nano-HgSe exposure brought oxidative damage to rice with decreased chlorophyll content and GSH-Px activity. In all, nano-HgSe was found to be more absorbable, transportable and methylated in rice plant compared to micro-HgSe. This suggests that although Se application in Hg contaminated farmland is an effective way to reduce the bioavailability of Hg, the risk of the possible remobilization of HgSe should not be neglected. Besides, the finding that nano-HgSe can act as an environmental source of Hg for plants deepens the understanding of biogeochemical cycle of Hg. More works are required to study the factors affecting the formation of nano-HgSe in the environment and the mechanisms of Hg methylation in rice plants after exposure to nano-HgSe.

Identification, characterization and differential expression analysis of a pteridine synthesis related gene, Ccptps, in koi carp (Cyprinus carpio L.).

6-pyruvoyl-tetrahydropterin synthase (PTPS) is the second key enzyme of the pteridine biosynthetic pathway and it plays vital roles in fish body color formation. In this study, Ccptps of koi carp (Cyprinus carpio L.) was cloned, identified and characterized. The full-length cDNA of Ccptps was 1140 bp and encodes for 139 amino acids. Multiple alignments revealed that the amino acids sequence of CcPTPS shared the highest identity to that of C. carpio, and Ccptps was clustered with cyprinid fishes in phylogenetic tree. Liver tissues of koi carp exhibited the highest expression of Ccptps, followed by muscle and skin tissues. During early developmental stages, the expression of Ccptps declined from 2 dph to 4 dph, and increased from 4 dph to 12 dph. The expressions of Ccptps in three color-related tissues (skin, scale and caudal fin) of whole red (WR) koi carp were significantly higher than that of whole while (WW) koi carp. Immunohistochemistry results of skin tissues showed that CcPTPS was mainly located in epidermis, stratum compactum of dermis and muscle layer, with the signal intensities in stratum compactum and muscle layer were stronger in WR koi carp compared to WW koi carp. Co-expressions of CcPTPS, CcSPR and CcXDH were detected in skin tissues of WW and WR koi carps, with CcPTPS exhibited stronger signal intensity compared to CcSPR and CcXDH. These findings imply that Ccptps is potentially involved in koi carp body color formation through the pteridine synthesis pathway.

[Material basis of Rhei Radix et Rhizoma-Coptidis Rhizoma combination in alleviating "bitter-cold" properties based on supramolecular chemistry of Chinese medicine].

The present study aimed to explore the material basis of Rhei Radix et Rhizoma-Coptidis Rhizoma combination in alleviating "bitter-cold" properties based on the supramolecular chemistry of Chinese medicine.Dynamic light scattering and scanning/transmission electron microscopy were used to characterize the morphological characteristics of supramolecules in the decoction of Rhei Radix et Rhizoma and Coptidis Rhizoma.The chemical composition of supramolecules, as well as the dissolution and release processes of supramolecules and the medicinal components of Coptidis Rhizoma decoction, was determined by the high-performance liquid chromatography-mass spectrometry.The differences in "bitter-cold" medicinal properties between Rhei Radix et Rhizoma decoction, Coptidis Rhizoma decoction, and co-decoction were analyzed by sensory evaluation, electronic tongue, mouse diarrhea model, and pathological indicators.The anthraquinones/tannins and alkaloids interacted to form supramolecules with a scale of about 400 nm when Rhei Radix et Rhizoma and Coptidis Rhizoma were decocted together, which delayed the dissolution and release of the active components represented by berberine. Compared with the consequence of single drug administration at 4 g·kg~(-1), the combination of the two drugs at 8 g·kg~(-1) significantly alleviated the "bitter-cold" properties.The effective components interacted to form supramolecules in the co-decoction of Rhei Radix et Rhizoma and Coptidis Rhizoma, which affected the dissolution and release of the effective components of Chinese medicinal decoction, thereby alleviating the "bitter-cold" properties.The findings of this study provide a new idea for revealing the scientific compatibility of Rhei Radix et Rhizoma and Coptidis Rhizoma.

Nano-WSe2 Is Absorbable and Transformable by Rice Plants.

As typical transition metal dichalcogenides (TMDC), tungsten selenide (WSe2) nanosheets (nano-WSe2) are widely used in various fields due to their layered structures and highly tunable electronic and magnetic properties, which results in the unwanted release of tungsten (W) and selenium (Se) into the environment. However, the environmental effects of nano-WSe2 in plants are still unclear. Herein, we evaluated the impacts and fate of nano-WSe2 and micro-WSe2 in rice plants (Oryza sativa L.). It was found that both nano-WSe2 and micro-WSe2 did not affect the germination of rice seeds up to 5000 mg/L but nano-WSe2 affected the growth of rice seedlings with shortened root lengths. The uptake and transportation of WSe2 was found to be size-dependent. Moreover, W in WSe2 was oxidized to tungstate while Se was transformed to selenocysteine, selenomethionine, SeIV and SeVI in the roots of rice when exposed to nano-WSe2, suggesting the transformation of nano-WSe2 in rice plants. The exposure to nano-WSe2 brought lipid peroxidative damage to rice seedlings. However, Se in nano-WSe2 did not contribute to the synthesis of glutathione peroxidase (GSH-Px) since the latter did not change when exposed to nano-WSe2. This is the first report on the impacts and fate of nano-WSe2 in rice plants, which has raised environmental safety concerns about the wide application of TMDCs, such as WSe2 nanosheets.

Tanshinone I inhibits doxorubicin-induced cardiotoxicity by regulating Nrf2 signaling pathway.

BACKGROUND: Doxorubicin (DOX) is a powerful anti-tumor anthracycline drug. However, its clinical use is limited due to the side effect of cardiotoxicity. Tanshinone I (Tan I) is one of the major tanshinones isolated from Salvia miltiorrhiza. Studies have shown that Tan I is effective in the treatment of cardiovascular diseases. However, the potential effects of Tan I against DOX-induced cardiotoxicity (DIC) have yet to be explored. PURPOSE: This study aimed to explore whether Tan I can protect against DIC and to reveal whether Tan I can exert anti-oxidative effect by regulating nuclear erythroid factor 2-related factor 2 (Nrf2) pathway. METHODS: DIC models were established in vivo by intravenous injection of DOX. Echocardiography was used to monitor the cardiac function of mice. Transmission electron microscopy was used to assess mitochondrial damage. Oxidative stress was measured by dihydroethidium (DHE) staining and western blotting. The accumulation and nuclear translocation of Nrf2 was detected by immunofluorescence. H9C2 cellular DIC model was established in vitro to explore the pharmacological mechanism. Nrf2 small interfering (si)-RNA was applied to H9C2 cells to explore whether Tan I exerted protective effect against DIC through Nrf2 signaling pathway. The protective effects of Tan I on mitochondrial function and mitochondrial membrane permeability were measured by MitoSOX™ Red and JC-1 staining assays, respectively. RESULTS: In vivo experiments revealed that Tan I could improve cardiac function and protect against DOX-induced myocardial structural damages in mice models. The oxidative stress induced by DOX was suppressed and apoptosis was mitigated by Tan I treatment. Tan I protected against DOX-induced mitochondrial structural damage. Meanwhile, key proteins in Nrf2 pathways were upregulated by Tan I treatment. In vitro studies showed that Tan I attenuated DOX-induced generation of reactive oxygen species (ROS) in cultured H9C2 cells, reduced apoptotic rates, protected mitochondrial functions and up-regulated Nrf2 signaling pathway. Tan I promoted accumulation and nuclear translocation of Nrf2 protein. In addition, interference of Nrf2 abrogated the anti-oxidative effects of Tan I and reversed the expressions of key proteins in Nrf2 pathway. The protective effects of Tan I on mitochondrial integrity was also mitigated by Nrf2 interference. CONCLUSION: Tan I could reduce oxidative stress and protect against DIC through regulating Nrf2 signaling pathway. Nrf2 is a potential target and Tan I is a novel candidate agent for the treatment of DIC.

[Molecular mechanisms of Gupi Xiaoji decoction inducing apoptosis of human hepatoma HepG2 cells].

Objective: To investigate the molecular mechanisms of Gupi Xiaoji decoction on apoptosis of human hepatoma cells HepG2. Methods: HepG2 cells were divided into 4 groups: control group (Control), blank serum group (Blank), Gupi Xiaoji Yin serum group (GPXJY) and cisplatin group (Positive). Eight duplicate holes were set in each group. After treated with Gupi Xiaoji Decoction-containing serum or cisplatin for 24 hours, the cell viability, the number of viable cells, the state of apoptosis, the cell cycle and the mitochondrial membrane potential were detected, and the level of lipid peroxidation (MDA) and glycolysis rate of the cells were detected. The expressions of apoptotic Bax, Bcl-2, and Caspase-3 proteins, and the contents of triacylglycerol (TG), cholesterol (TC), pyruvate and glucose in the cell supernatant were detected. Results: Compared with the control group, in the GPXJY group, the inhibition rate was increased (P＜0.05), the number of cells was decreased, the number of apoptosis-positive cells was increased (P＜0.01), the number of cells in the G1 phase was increased significantly (P＜0.05), and the cell membrane potential was decreased (P＜0.05,P＜0.01), the glycolytic function was inhibited significantly, the MDA level was increased, the expressions of Bax and Caspase-3 in the GPXJY group were increased, and the expression of Bcl-2 was decreased (P＜0.05, P＜0.01). In cell supernatant, the TC, TG and glucose contents were decreased significantly, and the pyruvate content was increased significantly (P＜0.05,P＜0.01). Conclusion: Gupi Xiaoji Decoction can induce apoptosis of HepG2 cells and may play a role in energy metabolism.

Xihuang pills induce apoptosis in hepatocellular carcinoma by suppressing phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin pathway.

BACKGROUND: The phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin (PI3K/Akt/mTOR) signalling pathway is crucial for cell survival, differentiation, apoptosis and metabolism. Xihuang pills (XHP) are a traditional Chinese preparation with antitumour properties. They inhibit the growth of breast cancer, glioma, and other tumours by regulating the PI3K/Akt/mTOR signalling pathway. However, the effects and mechanisms of action of XHP in hepatocellular carcinoma (HCC) remain unclear. Regulation of the PI3K/Akt/mTOR signalling pathway effectively inhibits the progression of HCC. However, no study has focused on the XHP-associated PI3K/Akt/mTOR signalling pathway. Therefore, we hypothesized that XHP might play a role in inhibiting HCC through the PI3K/Akt/mTOR signalling pathway. AIM: To confirm the effect of XHP on HCC and the possible mechanisms involved. METHODS: The chemical constituents and active components of XHP were analysed using ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS). Cell-based experiments and in vivo xenograft tumour experiments were utilized to evaluate the effect of XHP on HCC tumorigenesis. First, SMMC-7721 cells were incubated with different concentrations of XHP (0, 0.3125, 0.625, 1.25, and 2.5 mg/mL) for 12 h, 24 h and 48 h. Cell viability was assessed using the CCK-8 assay, followed by an assessment of cell migration using a wound healing assay. Second, the effect of XHP on the apoptosis of SMMC-7721 cells was evaluated. SMMC-7721 cells were stained with fluorescein isothiocyanate and annexin V/propidium iodide. The number of apoptotic cells and cell cycle distribution were measured using flow cytometry. The cleaved protein and mRNA expression levels of caspase-3 and caspase-9 were detected using Western blotting and quantitative reverse-transcription polymerase chain reaction (RT-qPCR), respectively. Third, Western blotting and RT-qPCR were performed to confirm the effects of XHP on the protein and mRNA expression of components of the PI3K/Akt/mTOR signalling pathway. Finally, the effects of XHP on the tumorigenesis of subcutaneous hepatocellular tumours in nude mice were assessed. RESULTS: The following 12 compounds were identified in XHP using high-resolution mass spectrometry: Valine, 4-gingerol, myrrhone, ricinoleic acid, glycocholic acid, curzerenone, 11-keto-ß-boswellic acid, oleic acid, germacrone, 3-acetyl-9,11-dehydro-ß-boswellic acid, 5ß-androstane-3,17-dione, and 3-acetyl-11-keto-ß-boswellic acid. The cell viability assay results showed that treatment with 0.625 mg/mL XHP extract decreased HCC cell viability after 12 h, and the effects were dose- and time-dependent. The results of the cell scratch assay showed that the migration of HCC cells was significantly inhibited in a time-dependent manner by the administration of XHP extract (0.625 mg/mL). Moreover, XHP significantly inhibited cell migration and resulted in cell cycle arrest and apoptosis. Furthermore, XHP downregulated the PI3K/Akt/mTOR signalling pathway, which activated apoptosis executioner proteins (e.g., caspase-9 and caspase-3). The inhibitory effects of XHP on HCC cell growth were determined in vivo by analysing the tumour xenograft volumes and weights. CONCLUSION: XHP inhibited HCC cell growth and migration by stimulating apoptosis via the downregulation of the PI3K/Akt/mTOR signalling pathway, followed by the activation of caspase-9 and caspase-3. Our findings clarified that the antitumour effects of XHP on HCC cells are mediated by the PI3K/Akt/mTOR signalling pathway, revealing that XHP may be a potential complementary therapy for HCC.

Notoginsenoside R1 Ameliorates Cardiac Lipotoxicity Through AMPK Signaling Pathway.

Aims: Cardiac lipotoxicity is the common consequence of lipid metabolism disorders in cardiomyocytes during development of heart failure (HF). Adenosine 5'monophosphate-activated protein kinase (AMPK) acts as an energy sensor and has a beneficial effect in reducing lipotoxicity. Notoginsenoside R1 (NGR1) is extracted from the traditional Chinese medicine Panax notoginseng (Burkill) F.H.Chen (P. notoginseng) and has definite cardioprotective effects. However, whether NGR1 can attenuate HF by mitigating lipotoxicity has not been elucidated yet. This study aimed to explore whether NGR1 plays a protective role against HF by ameliorating cardiac lipotoxicity via the AMPK pathway. Methods: In this study, HF mice model was established by left anterior descending (LAD) ligation. palmitic acid (PA) stimulated H9C2 cell model was applied to clarify the effects and potential mechanism of NGR1 on lipotoxicity. In vivo, NGR1 (7.14 mg/kg/days) and positive drug (simvastatin: 2.9 mg/kg/days) were orally administered for 14 days. Echocardiography was applied to assess heart functions. Lipid levels were measured by Enzyme-linked immunosorbent assay (ELISA) and key proteins in the AMPK pathway were detected by western blots. In vitro, NGR1 (40 µmol/L) or Compound C (an inhibitor of AMPK, 10 µmol/L) was co-cultured with PA stimulation for 24 h in H9C2 cells. CCK-8 assay was used to detect cell viability. Key lipotoxicity-related proteins were detected by western blots and the LipidTOX™ neutral lipid stains were used to assess lipid accumulation. In addition, Apoptosis was assessed by Hoechst/PI staining. Results: NGR1 could significantly improve the cardiac function and myocardial injury in mice with HF and up-regulate the expression of p-AMPK. Impressively, NGR1 inhibited the synthesis of diacylglycerol (DAG) and ceramide and promoted fatty acid oxidation (FAO) in vivo. Moreover, NGR1 significantly promoted expression of CPT-1A, the key enzyme in FAO pathway, and down-regulated the expression of GPAT and SPT, which were the key enzymes catalyzing production of DAG and ceramide. In vitro experiments showed that NGR1 could significantly attenuate lipid accumulation in PA-induced H9C2 cells and the Hoechst/PI staining results showed that NGR1 ameliorated lipotoxicity-induced apoptosis in PA-stimulated H9C2 cell model. Furthermore, co-treatment with inhibitor of AMPK abrogated the protective effects of NGR1. The regulative effects of NGR1 on lipid metabolism were also reversed by AMPK inhibitor. Conclusion: NGR1 could significantly improve the heart function of mice with HF and reduce cardiac lipotoxicity. The cardio-protective effects of NGR1 are mediated by the activation of AMPK pathway.

Frankincense myrrh attenuates hepatocellular carcinoma by regulating tumor blood vessel development through multiple epidermal growth factor receptor-mediated signaling pathways.

BACKGROUND: In traditional Chinese medicine (TCM), frankincense and myrrh are the main components of the antitumor drug Xihuang Pill. These compounds show anticancer activity in other biological systems. However, whether frankincense and/or myrrh can inhibit the occurrence of hepatocellular carcinoma (HCC) is unknown, and the potential molecular mechanism(s) has not yet been determined. AIM: To predict and determine latent anti-HCC therapeutic targets and molecular mechanisms of frankincense and myrrh in vivo. METHODS: In the present study, which was based on the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (http://tcmspw.com/tcmsp.php), Universal Protein database (http://www.uniprot.org), GeneCards: The Human Gene Database (http://www.genecards.org/) and Comparative Toxicogenomics Database (http://www.ctdbase.org/), the efficacy of and mechanism by which frankincense and myrrh act as anti-HCC compounds were predicted. The core prediction targets were screened by molecular docking. In vivo, SMMC-7721 human liver cancer cells were transplanted as xenografts into nude mice to establish a subcutaneous tumor model, and two doses of frankincense plus myrrh or one dose of an EGFR inhibitor was administered to these mice continuously for 14 d. The tumors were collected and evaluated: the tumor volume and growth rate were gauged to evaluate tumor growth; hematoxylin-eosin staining was performed to estimate histopathological changes; immunofluorescence (IF) was performed to detect the expression of CD31, α-SMA and collagen IV; transmission electron microscopy (TEM) was conducted to observe the morphological structure of vascular cells; enzyme-linked immunosorbent assay (ELISA) was performed to measure the levels of secreted HIF-1α and TNF-α; reverse transcription-polymerase chain reaction (RT-qPCR) was performed to measure the mRNA expression of HIF-1α, TNF-α, VEGF and MMP-9; and Western blot (WB) was performed to determine the levels of proteins expressed in the EGFR-mediated PI3K/Akt and MAPK signaling pathways. RESULTS: The results of the network pharmacology analysis showed that there were 35 active components in the frankincense and myrrh extracts targeting 151 key targets. The molecular docking analysis showed that both boswellic acid and stigmasterol showed strong affinity for the targets, with the greatest affinity for EGFR. Frankincense and myrrh treatment may play a role in the treatment of HCC by regulating hypoxia responses and vascular system-related pathological processes, such as cytokine-receptor binding, and pathways, such as those involving serine/threonine protein kinase complexes and MAPK, HIF-1 and ErbB signaling cascades. The animal experiment results were verified. First, we found that, through frankincense and/or myrrh treatment, the volume of subcutaneously transplanted HCC tumors was significantly reduced, and the pathological morphology was attenuated. Then, IF and TEM showed that frankincense and/or myrrh treatment reduced CD31 and collagen IV expression, increased the coverage of perivascular cells, tightened the connection between cells, and improved the shape of blood vessels. In addition, ELISA, RT-qPCR and WB analyses showed that frankincense and/or myrrh treatment inhibited the levels of hypoxia-inducible factors, inflammatory factors and angiogenesis-related factors, namely, HIF-1α, TNF-α, VEGF and MMP-9. Furthermore, mechanistic experiments illustrated that the effect of frankincense plus myrrh treatment was similar to that of an EGFR inhibitor with regard to controlling EGFR activation, thereby inhibiting the phosphorylation activity of its downstream targets: the PI3K/Akt and MAPK (ERK, p38 and JNK) pathways. CONCLUSION: In summary, frankincense and myrrh treatment targets tumor blood vessels to exert anti-HCC effects via EGFR-activated PI3K/Akt and MAPK signaling pathways, highlighting the potential of this dual TCM compound as an anti-HCC candidate.