Dual Regulation of Nicotine on NLRP3 Inflammasome in Macrophages with the Involvement of Lysosomal Destabilization, ROS and α7nAChR.

Nicotine, the primary alkaloid in tobacco products, has been shown to have immunoregulatory function in at least 20 diseases. The biological mechanism of action of nicotine immunoregulation is complex, resulting in an improvement of some disease states and exacerbation of others. Given the central role of the NLRP3 inflammasome in macrophages among multiple inflammatory diseases, this study examined how nicotine alters NLRP3 inflammasome activation in macrophages. NLRP3 inflammasome activation was examined mechanistically in the context of different nicotine dosages. We show NLRP3 inflammasome activation, apoptosis-associated speck-like protein (ASC) expression, caspase-1 activity and subsequent IL-1ß secretion were positively correlated with nicotine in a dose-dependent relationship, and destabilization of lysosomes and ROS production were also involved. At high concentrations of nicotine surpassing 0.25 mM, NLRP3 inflammasome activity declined, along with increased expression of the anti-inflammatory Alpha7 nicotinic acetylcholine receptor (α7nAChR) and the inhibition of TLR4/NF-κB signaling. Consequently, high doses of nicotine also reduced ASC expression, caspase-1 activity and IL-1ß secretion in macrophages. Collectively, these results suggest a dual regulatory function of nicotine on NLRP3 inflammasome activation in macrophages, that is involved with the pro-inflammatory effects of lysosomal destabilization and ROS production. We also show nicotine mediates anti-inflammatory effects by activating α7nAChR at high doses.

Construction of N-doped carbon encapsulated Mn2O3/MnO heterojunction for enhanced lithium storage performance.

Owing to high theoretical capacity, low cost and abundant availability, manganese oxides are widely viewed as promising anodes for lithium-ion batteries (LIBs). Nonetheless, their practical application is significantly hindered by poor electrical conductivity, sluggish reaction kinetics and substantial volume change. In this work, an ingenious polypyrrole encapsulation followed by pyrolysis strategy is proposed to produce N-doped carbon encapsulated Mn2O3/MnO heterojunction (Mn2O3/MnO@NC) by using mechanically ground Mn3O4/C3N4 mixture as the precursor. The results show that the selection of precursor plays a pivotal role in the successful preparation of Mn2O3/MnO@NC hybrid. It is revealed that the uniform encapsulation by N-doped carbon significantly enhances the conductivity and structural stability of the final product. Concurrently, the Mn2O3/MnO heterojunction within the resultant hybrid exhibits a unique quantum-dot size, which effectively shortens ion transport pathways and exposes the active sites for lithium storage. Additionally, experimental observations and theoretical calculations demonstrate that the built-in electric fields generated at the interfaces of Mn2O3/MnO heterojunction accelerate the charge transfer and ion diffusion, thereby enhancing the electrochemical reaction kinetics. As a result, the Mn2O3/MnO@NC hybrid displays much enhanced lithium storage performance. Evidently, our work offers a good guidance for the design and synthesis of advanced transition metal oxide/carbon anodes for LIBs.

Assessment of cellular senescence potential of PM2.5 using 3D human lung fibroblast spheroids in vitro model.

Background: Epidemiological studies demonstrate that particulate matter 2.5 (PM2.5) exposure closely related to chronic respiratory diseases. Cellular senescence plays an important role in many diseases. However, it is not fully clear whether PM2.5 exposure could induce cellular senescence in the human lung. In this study, we generated a three-dimensional (3D) spheroid model using isolated primary human lung fibroblasts (HLFs) to investigate the effects of PM2.5 on cellular senescence at the 3D level. Methods: 3D spheroids were exposed to 25-100 µg/ml of PM2.5 in order to evaluate the impact on cellular senescence. SA-ß-galactosidase activity, cell proliferation, and the expression of key genes and proteins were detected. Results: Exposure of the HLF spheroids to PM2.5 yielded a more sensitive cytotoxicity than 2D HLF cell culture. Importantly, PM2.5 exposure induced the rapid progression of cellular senescence in 3D HLF spheroids, with a dramatically increased SA-ß-Gal activity. In exploiting the mechanism underlying the effect of PM2.5 on senescence, we found a significant increase of DNA damage, upregulation of p21 protein levels, and suppression of cell proliferation in PM2.5-treated HLF spheroids. Moreover, PM2.5 exposure created a significant inflammatory response, which may be at least partially associated with the activation of TGF-ß1/Smad3 axis and HMGB1 pathway. Conclusions: Our results indicate that PM2.5 could induce DNA damage, inflammation, and cellular senescence in 3D HLF spheroids, which may provide a new evidence for PM2.5 toxicity based on a 3D model which has been shown to be more in vivo-like in their phenotype and physiology than 2D cultures.

Prediction of sulfate concentrations in groundwater in areas with complex hydrogeological conditions based on machine learning.

The persistent and increasing levels of sulfate due to a variety of human activities over the last decades present a widely concerning environmental issue. Understanding the controlling factors of groundwater sulfate and predicting sulfate concentration is critical for governments or managers to provide information on groundwater protection. In this study, the integration of self-organizing map (SOM) approach and machine learning (ML) modeling offers the potential to determine the factors and predict sulfate concentrations in the Huaibei Plain, where groundwater is enriched with sulfate and the areas have complex hydrogeological conditions. The SOM calculation was used to illustrate groundwater hydrochemistry and analyze the correlations among the hydrochemical parameters. Three ML algorithms including random forest (RF), support vector machine (SVM), and back propagation neural network (BPNN) were adopted to predict sulfate levels in groundwater by using 501 groundwater samples and 8 predictor variables. The prediction performance was evaluated through statistical metrics (R2, MSE and MAE). Mine drainage mainly facilitated increase in groundwater SO42- while gypsum dissolution and pyrite oxidation were found another two potential sources. The major water chemistry type was Ca-HCO3. The dominant cation was Na+ while the dominant anion was HCO3-. There was an intuitive correlation between groundwater sulfate and total dissolved solids (TDS), Cl-, and Na+. By using input variables identified by the SOM method, the evaluation results of ML algorithms showed that the R2, MSE and MAE of RF, SVM, BPNN were 0.43-0.70, 0.16-0.49 and 0.25-0.44. Overall, BPNN showed the best prediction performance and had higher R2 values and lower error indices. TDS and Na+ had a high contribution to the prediction accuracy. These findings are crucial for developing groundwater protection and remediation policies, enabling more sustainable management.

Liver toxicity in rats after subchronic exposure to HTP aerosol and cigarette smoke.

Background: Heated tobacco product (HTP) considered to be a novel tobacco product which was reported safer than traditional cigarettes evidenced by lower potential harmful components released. Liver is an important detoxification organ of the body, the chemical components in aerosols are metabolized in the liver after absorbed, so it is necessary to explore the effect of HTP on the liver. Materials and Methods: The potential effect of HTP and cigarette smoke (CS) on SD rats was explored according to OECD 413 subchronic inhalation. The rats were randomly divided into Sham (air), different dosage of HTP groups (HTP_10, 23 and 50 µg nicotine/L aerosol) and Cig_23 (23 µg nicotine/L aerosol) group. After exposure, the clinical pathology, inflammation and oxidative stress were measured. Results: The clinical pathology results showed that both HTP_50 and Cig_23 led to abnormality of ALT for male rats. CS and HTP exposure reduced the expression of IL-1ß, IL-6 and TNF-α and mitochondrial medicated oxidative stress. In addition, the ATP production was reduced in Cig_23 group. Although inflammation and oxidative stress were displayed, no apoptosis were observed by TUNEL assay and these existed obvious pathological changes only in HTP_50 group, while in CS group with equivalent nicotine, hepatocytes swelling were observed in liver. Conclusion: CS exposure induced liver damage through mitochondrial mediated oxidative stress and inflammation, which was also observed in high concentration of HTP exposure group. For the same equivalent nicotine, HTP may show lower toxic effect on liver than CS.

Evaluation of toxicity of heated tobacco products aerosol and cigarette smoke to BEAS-2B cells based on 3D biomimetic chip model.

It is still a controversial topic about evaluating whether heated tobacco products (HTP) really reduce harm, which involves the choice of an experimental model. Here, a three-dimensional (3D) biomimetic chip model was used to evaluate the toxicity of aerosols came from HTP and smoke produced by cigarettes (Cig). Based on cell-related experiments, we found that the toxicity of Cig smoke extract diluted four times was also much higher than that of undiluted HTP, showing higher oxidative stress response and cause mitochondrial dysfunction. Meanwhile, both tobacco products all affect the tricarboxylic acid cycle (TCA), which is manifested by a significant decrease in the mRNA expression of TCA key rate-limiting enzymes. Summarily, 3D Biomimetic chip technology can be used as an ideal model to evaluate HTP. It can provide important data for tobacco risk assessment when 3D chip model was used. Our experimental results showed that HTP may be less harmful than tobacco cigarettes, but it does show significant cytotoxicity with the increase of dose. Therefore, the potential clinical effects of HTP on targeted organs such as lung should be further studied.

Inhibition of demethylase by IOX1 modulates chromatin accessibility to enhance NSCLC radiation sensitivity through attenuated PIF1.

Chromatin accessibility is a critical determinant of gene transcriptional expression and regulated by histones modification. However, the potential for manipulating chromatin accessibility to regulate radiation sensitivity remains unclear. Our findings demonstrated that the histone demethylase inhibitor, 5-carboxy-8-hydroxyquinoline (IOX1), could enhance the radiosensitivity of non-small cell lung cancer (NSCLC) in vitro and in vivo. Mechanistically, IOX1 treatment reduced chromatin accessibility in the promoter region of DNA damage repair genes, leading to decreased DNA repair efficiency and elevated DNA damage induced by γ irradiation. Notably, IOX1 treatment significantly reduced both chromatin accessibility and the transcription of phytochrome interacting factor 1 (PIF1), a key player in telomere maintenance. Inhibition of PIF1 delayed radiation-induced DNA and telomeric DNA damage repair, as well as increased radiosensitivity of NSCLC in vitro and in vivo. Further study indicated that the above process was regulated by a reduction of transcription factor myc-associated zinc finger protein (MAZ) binding to the distal intergenic region of the PIF1. Taken together, IOX1-mediated demethylase inactivation reduced chromatin accessibility, leading to elevated telomere damage which is partly due to PIF1 inhibition, thereby enhancing NSCLC radiosensitivity.

Testicular tissue response following a 90-day subchronic exposure to HTP aerosols and cigarette smoke in rats.

Background: Researches have shown that chronic inhalation of cigarette smoke (CS) disrupts male reproductive system, but it is unclear about the mechanisms behind reproductive damages by tobacco toxicants in male rats. This study was designed to explore the effects of heated tobacco products (HTP) aerosols and CS exposure on the testicular health of rats. Materials and Methods: Experiments were performed on male SD rats exposed to filtered air, HTP aerosols at 10 µg/L, 23 µg/L, and 50 µg/L nicotine-equivalent contents, and also CS at 23 µg/L nicotine-equivalent content for 90 days in five exposure groups (coded as sham, HTP_10, HTP_23, HTP_50 and Cig_23). The expression of serum testosterone, testicular tissue inflammatory cytokines (IL-1ß, IL-6, IL-10, TNF-α), reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA), NLRP3 inflammasome-related mRNAs and proteins (NLRP3, ASC, and Caspase-1), the degree of pyroptosis and histopathology were investigated. Results: The results demonstrated that HTP_50 and Cig_23 caused varying degrees of oxidative damage to rat testis, resulting in a decrease of sperm quantity and serum testosterone contents, an increase in the deformity rate, expression levels of proinflammatory cytokines, and NLRP3 inflammasome-related mRNA, and an increase in the NLRP3, ASC, and Caspase-1-immunopositive cells, pyroptosis cell indices, and histopathological damage in the testes of rats. Responses from the HTP_10 and HTP_23 groups were less than those found in the above two exposure groups. Conclusion: These findings indicate that HTP_50 and Cig_23 induced oxidative stress in rat testes, induced inflammation and pyroptosis through the ROS/NLRP3/Caspase-1 pathway, and destroyed the integrity of thetesticular tissue structure.

Pharmacokinetics of freebase nicotine and nicotine salts following subcutaneous administration in male rats.

Nicotine lactate, nicotine tartrate, nicotine benzoate, and freebase nicotine (FBN) are four forms of nicotine salt systems that are present in tobacco products. However, few in vivo studies have compared their pharmacological (pK) efficacies, which are important for understanding their roles in the addiction and abuse of tobacco and nicotine products. In this work, the pK of the above nicotine salt systems was studied by subcutaneously injecting their aqueous solutions in rats and obtaining blood samples from the jugular vein. Nicotine levels in the blood were analyzed by LC-MS/MS. The results demonstrated that rapid nicotine absorption occurred in all nicotine systems. Of them, NB had the smallest Tmax , while FBN had the largest Tmax . The nicotine metabolic rate and clearance decreased for FBN, indicating that nicotine retention in the body was higher than for the other three salt-based systems. Compared with nicotine salts, FBN could reach and maintain a higher concentration in the animal model. Additionally, as the benzoic acid ratios increased, the Cmax of the nicotine benzoate (NB) in the plasma decreased. This indicates that the lower the pH, the lower the Cmax . When different concentrations of NB were used, the higher the NB concentration, the greater the Cmax and AUC(0-t) . These results demonstrate that nicotine adsorption by NB in the animal model depended on both pH and concentration. This baseline information could be used to explain different clinical pharmacological observations in humans, though this study only considered the effects of nicotine on pharmacokinetics in vivo.

A self-administered questionnaire to measure Chinese smokers' cigarette dependence.

A self-administered questionnaire for screening cigarette dependence was developed based on a set of 6335 Chinese adult smokers (termed the China Cigarette Dependence Test, CCDT). Both a 20-item version (CCDT-20) and a 7-item of the questionnaire (CCDT-7) were developed following 2-round of tests on their construct validity, test-retest reliability and internal consistency, covering seven dimensions (cigarettes per day, tolerance, withdrawal symptoms, craving, loss of control, regularity, and stereotypy). The results showed that the CCDT-20 and CCDT-7 scores were higher in daily smokers than in occasional smokers, and both were associated with self-rated nicotine dependence, exhaled carbon monoxide (CO), saliva cotinine, and DSM-V. The CCDT-20 and CCDT-7 scales were found to be easier to use by smokers in China and provided a more reliable measure of their cigarette dependence.

Simultaneous quantification of nicotine salts in e-liquids by LC-MS/MS and GC-MS.

Nicotine salts, formed by nicotine and organic acids, are commonly added to electronic cigarette liquids for their ability to provide desirable sensory effects. Analytical strategies have been developed to detect the types of organic acids and nicotine levels, but methods for directly measuring nicotine salts are still desirable. Herein, a novel approach is presented for the simultaneous quantification of non-volatile and volatile nicotine salts via liquid chromatography/tandem mass spectroscopy (LC-MS/MS) and gas chromatography/mass spectroscopy (GC-MS). This approach was validated with recovery experiments, which yielded recovery values between 92.0% and 110.8%. This method is the first technique for quantifying multiple nicotine salts that could be present in commercial e-liquids. Without using derivatization steps, different nicotine salts could be detected rapidly and conveniently. This new method was demonstrated with 10 e-cigarette liquid samples, providing satisfactory outcomes. It could be used to study organic acids and protonated nicotine in e-liquids and the release behaviour of nicotine salts in electronic cigarettes.

Effects of Smoking on Inflammatory-Related Cytokine Levels in Human Serum.

Cardiovascular and respiratory diseases, and several cancers resulting from tobacco smoking, are initially characterized by chronic systemic inflammation. Cytokine imbalances can result in inflammation, making it important to understand the pathological mechanisms behind cytokine production. In this study, we collected blood samples from 78 healthy male volunteers, including non-smokers (n = 30), current smokers (n = 30), and ex-smokers (n = 18), and utilized the liquid suspension chip technique to investigate and compare the expression levels of 17 cytokines and chemokines in the human serum of these volunteers. The results demonstrated that the expression levels of CXCL9/MIG and sIL-6R significantly increased after smoking, and continued to increase after quitting smoking. The expression levels of TARC, ITAC, and sVEGFR-3 increased after smoking but decreased after quitting smoking; the expression level of SAA significantly decreased after smoking and showed an upward trend after quitting smoking. Seven cytokines (IL-1ß, BCA-1, TNF-α, CRP, ENA-78, MDC, and TNFRII) did not vary between the three groups, while four cytokines (IL-1α, IL-6, IL-8, and SCF) were not detected in any serum sample. In conclusion, this study assessed the physiological production of cytokines and chemokines, highlighting the differences in each due to smoking status. Our results could help evaluate the early development of smoking-related chronic diseases and cancers.

Evaluation of inhalation toxicology after a 90-day xylitol aerosol exposure in Sprague-Dawley rats.

Xylitol is a hygroscopic compound known to protect nasal cavity against bacteria. It has also been developed into nasal spray and evaluated as a potential candidate drug for respiratory diseases. Consequently, it is necessary to study its inhalation toxicity. Based on our previous study on its subacute inhalation toxicity, this study aimed to investigate the safety of xylitol inhalation for long-term use. According to the OECD Test Guideline 413, Sprague-Dawley rats were randomly divided into six groups and exposed with different concentrations of xylitol aerosol or air. After exposure for 90-day, the recovery groups were continued to observe for a recovery period of 28-day. No significant changes in body weight were observed between sham and xylitol groups. Several significant differences in hematological, clinical chemistry, bronchoalveolar lavage fluid were observed, which either had no dose-effect relationship for both male and female rats or were restored during the recovery period. Finally, except for high dose group of xylitol, two rats showed a small amount of inflammatory exudate in alveolar and bronchial cavities, which was restored in the recovery period. The rest of rats showed no obvious difference. For the recovery groups, no significant difference was observed between these two groups. In conclusion, the no observable adverse effect level (NOAEL) of xylitol in our subchronic inhalation toxicological experiments was 2.9 mg/L, which indicated that xylitol for rats' long-time inhalation is tolerant and safe.

In Vitro Toxicological Investigation and Risk Assessment of E-Cigarette Aerosols Based on a Novel Solvent-Free Extraction Method.

Cigarettes, potentially safer alternatives to combustible cigarettes, have been reported to increase the health risk for long-term users, so accumulating information about their potential toxicity is of great concern. However, toxicological evaluations of e-cigarette aerosols are limited, which may be attributed to the lack of a simple and efficient extraction method. Here, we developed a high-speed centrifugal method for extracting e-cigarette aerosol collected mass (ACM) and prepared ACM samples of 26 representative e-cigarettes, and 10 samples were further selected based on their cytotoxicity for systematic toxicological assessments. The average extraction efficiency of ACM, primary aerosol components, and typical carbonyls exceeded 85%. The toxicological evaluation showed that the IC50 value range of e-cigarettes for cytotoxicity was 2-52 mg/mL ACM, all e-cigarettes can induce the risk of DNA damage, mitochondrial depolarization, and c-Jun-related signal disturbances; most e-cigarettes significantly caused disturbance of oxidative stress balance. E-cigarettes with higher cytotoxicity appeared to cause a higher degree of damage, while no e-cigarette promoted mutagenicity and cytochrome c release. The toxicity difference among e-cigarettes using nicotine equivalent was significantly lower than that of ACM. This study provides a novel extraction method and a comprehensive in vitro toxicity risk profile of e-cigarette aerosols.

Investigation of the effective components inhibited macrophage foam cell formation in Ophiopogonis Radix.

ETHNOPHARMACOLOGICAL RELEVANCE: Ophiopogonis Radix, the commonly used traditional Chinese medicine in clinic for treating cardiovascular diseases, is returned to the stomach, lung and heart meridian. It is reported to nourish yin, moisten lung and is used to treat heart yin deficiency syndromes and asthenia of heart and lung, which indicated that Ophiopogonis Radix may have a protective effect on heart disorders. Atherosclerosisis is an important process in the development of cardiovascular diseases and abnormal lipid deposition induced macrophage foam cells is its crucial foundation. Our previous study showed the extract of Ophiopogonis Radix (EOR) ameliorates atherosclerosis in vitro. However, it may protect against cardiovascular diseases through inhibiting macrophage foam cell formation and its potential effective components and mechanisms are still unclear. AIM OF THE STUDY: Our study aimed to investigate the effect of Ophiopogonis Radix on macrophage foam cell formation and its potential active constituents and mechanisms. MATERIALS AND METHODS: Ox-LDL induced macrophage cells were employed to evaluate the effect of Ophiopogonis Radix on macrophage foam cell formation. Then the potential active constituents inhibited formation of macrophage foam cells were screened by biospecific cell extraction and its underlying mechanisms were also explored by Western blot. RESULTS: The extract of Ophiopogonis Radix was found to significantly inhibit macrophage foam cell formation, evidenced by the decrease of TG and TC and Oil Red O staining analysis in macrophage cells, which indicated that EOR reduced the formation of macrophage foam cells. At the same time, EOR was showed to increase antioxidant capacity in macrophage cells. After treatment with EOR, two potential active components interacted with macrophage foam cells specifically were identified to inhibit macrophage foam cell formation including methylophiopogonanone A and methylophiopogonanone B. Methylophiopogonanone A was then proved to decrease the expression of CD36, Lox-1 and SREBP2, increase the expression of ABCA1 obviously, while the expression of ABCG1 and SREBP1 had no changes. CONCLUSIONS: In our study, Ophiopogonis Radix was found to protect against atherosclerosis through suppressing ox-LDL induced macrophage foam cell formation and two potential compounds were identified by biospecific cell extraction including methylophiopogonanone A and methylophiopogonanone B. Moreover, methylophiopogonanone A was proved to inhibit foam cells through reducing uptake, synthesis and increasing efflux, which may provide guidance and reference for application of Ophiopogonis Radix and investigation of the effective components of TCMs.

Toxicity evaluation in rats following 28 days of inhalation exposure to xylitol aerosol.

Xylitol has reported to decrease gingival inflammation and nasopharyngeal pneumonia, which indicated that xylitol may have potential application in respiratory diseases. Although some studies have reported the inhalation toxicity of xylitol, however, the longest period tested was only for 14 days. The inhalation toxicity of xylitol is insufficient. This work investigated the potential subacute toxicity of xylitol according to the OECD TG 412. Rats were randomly divided into a control group and different dosage groups (2 g/m3, 3 g/m3, 5 g/m3), and exposed for 6 hours/day, 5 days/week for 28 days. At the end of the exposure or recovery period, clinical signs, mortality, body weight, food consumption, hematology, blood biochemistry, gross pathology, organ weight, and histopathology were examined. Compared with the control group, rats of both sexes in the exposure groups exhibited no significant changes in body weight, organ mass, and food uptake. After the xylitol exposure, aspartate aminotransferase activity in the xylitol group (3 g/m3) was significantly higher than that in the control group, while other blood indicators and pathological changes of liver and the analysis of the recovery group showed no changes, suggesting that xylitol exerted no observable toxic effect on the liver. Finally, other observations including the histopathology of target organs and hematology also showed no alterations. These results indicated that xylitol had no significant inhalation toxicity at doses up to 5 g/m3. These subacute inhalation toxicity results of xylitol showed that its no-observed-adverse-effect concentration (NOAEC) in rats was determined to 5 g/m3.

Novel Solvent-Free Extraction Method for Analyzing Tobacco Heating Product Aerosols: An Analytical and In Vitro Toxicological Five-Way Product Comparison.

Harmful and potentially harmful constituents (HPHCs) in tobacco smoke are thought to be responsible for the increased health risks. Tobacco heating products (THPs) heat tobacco instead of burning it to achieve significantly fewer toxicants than conventional cigarettes. To assess the toxicity of THP aerosols, it is often desirable to extract the main constituents using a solvent method. In this study, we developed a high-speed centrifugal method for extracting the total particulate matter (TPM) from THPs to quantitatively compare the toxicity of different THPs and conventional cigarettes. Its TPM extraction efficiency exceeded 85%, and the primary aerosol components and typical HPHCs were comparable to those of the solvent method. The TPMs extracted from five THPs were subjected to 14 in vitro toxicology assessments, and the results were compared with those of a 3R4F reference cigarette. Physical separation can improve biases from solvent selectivity and potential interactions between solvent and aerosol constituents. By eliminating solvent influence, the extraction method could achieve high-dose exposures, enabling the toxicity comparison of different THPs. The relative toxicity of the THPs differed under different dosage units, including the TPM concentration, nicotine equivalent, and puff number.

[Study on effect of gypenosides on insulin sensitivity of rats with diabetes mellitus via regulating NF-κB signaling pathway].

This study focused on the ameliorative effects of gypenosides(GPS) on insulin sensitivity and inflammatory factors in rats with type 2 diabetes mellitus(T2 DM) and explored their possible molecular mechanisms. After the successful establishment of T2 DM model, diabetic rats were randomly divided into four groups, including model group, GPS groups(200, 100 mg·kg~(-1)) and metformin group(100 mg·kg~(-1)), with healthy rats serving as the control. After 6-week intragastric administration, fasting blood glucose(FBG) and oral glucose tolerance were examined. The levels of insulin, C-peptide, tumor necrosis factor-α(TNF-α), interleukin-1ß(IL-1ß), interleukin-6(IL-6) and C-reactive protein(CRP) in serum were examined. Then the homeostasis model assessment of insulin resistance(HOMA-IR) and insulin sensitivity index(ISI) were calculated. The protein expression levels of phosphorylated insulin receptor substrate-1(p-IRS-1) and phosphorylated protein kinase B(p-Akt) in skeletal muscle were measured by Western blot, as well as those of phosphorylated inhibitor of nuclear factor-κB(NF-κB) kinase ß(p-IKKß), phosphorylated alpha inhibitor of NF-κB(p-IκBα) and phosphorylated p65 subunit of NF-κB(p-p65) in adipose tissue. The relative expression levels of glucose transporter 4(GLUT4) mRNA in skeletal muscle and NF-κB mRNA in adipose tissue were measured by qRT-PCR, and the morphological changes of pancreatic tissue were observed. Compared with the model group, the GPS groups witnessed significant decrease in FBG, marked amelioration of impaired oral glucose tolerance and significant increase in ISI. Further, the high-dose GPS group saw significantly reduced HOMA-IR, TNF-α, IL-1ß and CRP, significantly increased expression levels of p-IRS-1(Tyr), p-Akt and GLUT4, and markedly inhibited p-IRS-1(Ser), p-IKKß, p-IκBα, p-p65 and NF-κB. The concentration of CRP and the expression levels of p-IRS-1(Ser), p-IKKß, p-IκBα and NF-κB were remarkably reduced in the low-dose GPS group. However, GPS was found less effective in the regulation of serum insulin, C-peptide and IL-6 levels and the alleviation of pancreatic islet injury. The results indicated that GPS can reduce FBG and improve insulin sensitivity in diabetic rats possibly by regulating the NF-κB signaling pathway, inhibiting inflammation, and thereby regulating the expression of key proteins in the insulin signaling pathway.

Decreased mitochondrial DNA copy number in nerve cells and the hippocampus during nicotine exposure is mediated by autophagy.

Cigarette smoke is a harmful air pollutant and nicotine dependence is the essential cause of the tobacco epidemic. Since mitochondrial abnormalities are associated with substance addiction, in this work we used mitochondrial DNA (mtDNA) copy number as an indicator of mitochondrial function to investigate whether nicotine addicts also exhibit mitochondrial abnormalities. We found significantly lower mtDNA copy number in the peripheral blood of healthy nicotine addicts than in non-smokers, indicating that long-term nicotine exposure through smoking has detrimental effects on mitochondria. We also examined the effects of nicotine on mtDNA levels in a rat conditioned place preference (CPP) model of addiction and in cultured neuron cells, which revealed that the mtDNA copy number was significantly reduced in the hippocampus of CPP rats, in human neuroblastoma SH-SY5Y cells, and in rat pheochromocytoma PC12 cells, suggesting that significantly reduced mtDNA copy number is a potential biomarker of nicotine addiction. In SH-SY5Y cells, nicotine treatment induced several mitochondrial defects, such as increased mtDNA damage, increased reactive oxygen species (ROS) levels, decreased mitochondrial membrane potential (△Ψm), and stimulation of autophagic flux via transcriptional up-regulation of several autophagy-related genes and elevated marker protein accumulation, although genes controlling mtDNA replication were unaffected. In addition, pretreatment with the autophagy inhibitor Bafilomycin A1 led to accumulation of microtubule-associated protein 1 light chain 3b-II (LC3B-II) and counteracted the nicotine-induced decrease in mtDNA copy number. These results were recapitulated in PC12 cells, which also showed significant down-regulation of the marker SQSTM1/P62, suggesting that the decrease in mtDNA copy number is mediated by autophagy. This study shows that prolonged nicotine exposure, such as that in nicotine addicts, leads to a decrease of mtDNA copy number in neurons due to enhanced induction of autophagy. CAPSULE: It was found that smoking or nicotine exposure decreased mtDNA copy number based on population, animal, and cell models, and these effects appear to be mediated by autophagy.

Reverse tracing anti-thrombotic active ingredients from dried Rehmannia Radix based on multidimensional spectrum-effect relationship analysis of steaming and drying for nine cycles.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine (TCM) and modern pharmacodynamics, dried Rehmannia Radix (DRR) possesses prominent anti-thrombotic activity that decreases after processing by nine steaming and drying cycles to develop processed Rehmannia Radix (PRR). Due to the complexity of the DRR components, the chemical mechanism leading to efficacy changes of DRR caused by processing is still unclear. AIM OF STUDY: This study aimed to trace the anti-thrombotic active compounds of DRR and different degrees of processed RR (PRR) and to evaluate the synergistic effects among different active components. MATERIALS AND METHODS: The anti-thrombotic active chemical fraction of DRR extracts was evaluated. Targeted fractions of the processed products of RR were prepared at different processing stages. The changes in monosaccharides, oligosaccharides and secondary metabolites during processing were characterized by multidimensional high-performance liquid chromatography (HPLC). The anti-thrombotic effects of targeted fractions of different RR samples were evaluated by analyzing the length of tail thrombus (LT) and serum biochemical indicators in carrageenan-induced tail-thrombus mice. The spectrum-effect relationships were investigated by partial least squares regression (PLSR) analysis and gray correlation analysis (GRA). Finally, the active compounds were screened by spectrum-effect relationship analysis and validated in vivo, and their synergistic effects were determined by Webb's fraction multiplication method. RESULTS: Six ingredients highly associated with anti-thrombotic activities were screened out by the spectrum-effect relationship analysis, of which oligosaccharides (stachyose, sucrose and raffinose) and iridoid glycosides (catalpol, leonuride and melitoside) possessed a synergistic effect on tumor necrosis factors (TNF-α), interleukin 1ß (IL-1ß) and plasminogen activator inhibitor 1 (PAI-1)/tissue-type plasminogen activator (t-PA) ratio in vivo with synergistic coefficient (SC) > 1. CONCLUSION: The main material basis of the anti-thrombotic activities of DRR is oligosaccharide components of stachyose, raffinose and sucrose, iridoid glycosides components of catalpol, leonuride and melittoside. The two kinds of components exert synergistic anti-thrombotic effects by inhibiting the expression of inflammatory factors and regulating the balance of the fibrinolysis system.