Sinomenine inhibits macrophage M1 polarization by downregulating α7nAChR via a feedback pathway of α7nAChR/ERK/Egr-1.

BACKGROUND: Sinomenine (SIN) is an anti-inflammatory drug that has been used for decades in China to treat arthritis. In a previous study, SIN acted on α7 nicotinic acetylcholine receptor (α7nAChR) to inhibit inflammatory responses in macrophages, which indicates a new anti-inflammatory mechanism of SIN. However, the level of α7nAChR was increased in the inflammatory responses and was downregulated by SIN in vitro, so the underlying mechanisms of SIN acting on α7nAChR remain unclear. PURPOSE: To analyze the role of α7nAChR in inflammation and the effect and mechanism of SIN regulation of α7nAChR. METHODS: The effects of SIN on α7nAChR in endotoxemic mice and LPS-stimulated macrophages were observed. Nicotine (Nic) was used as a positive control, and berberine (Ber) was used as a negative control targeting α7nAChR. The antagonists of α7nAChR, α-bungarotoxin (BTX) and mecamylamine (Me), were used to block α7nAChR. In RAW264.7 macrophage cells in vitro, α7nAChR short hairpin RNA (shRNA) was used to knock down α7nAChR. Macrophage polarization was analyzed by the detection of TNF-α, IL-6, iNOS, IL-10, Arg-1, and Fizz1. U0126 was used to block ERK phosphorylation. The cytokines α7nAChR, ERK1/2, p-ERK1/2 and Egr-1 were detected. RESULTS: SIN decreased the levels of TNF-α, IL-6 and the expression of α7nAChR increased by LPS in endotoxemic mice. The above effects of SIN were attenuated by BTX. In the α7nAChR shRNA transfected RAW264.7 cells, compared with the control, α7nAChR was knocked down, and M1 phenotype markers (including TNF-α, IL-6, and iNOS) were significantly downregulated, whereas M2 phenotype markers (including IL-10, Arg-1, and Fizz1) were significantly upregulated when stimulated by LPS. SIN inhibited the expression of p-ERK1/2 and the transcription factor Egr-1 induced by LPS in RAW264.7 cells, and the above effects of SIN were attenuated by BTX. The expression of α7nAChR was suppressed by U0126, which lessened the expression of p-ERK1/2 and Egr-1. CONCLUSIONS: SIN acts on α7nAChR to inhibit inflammatory responses and downregulates high expression of α7nAChR in vivo and in vitro. The increase of α7nAChR expression is correlated with inflammatory responses and participates in macrophage M1 polarization. SIN downregulates α7nAChR via a feedback pathway of α7nAChR/ERK/Egr-1, which contributes to inhibiting macrophage M1 polarization and inflammatory responses.

Effect of high soil C/N ratio and nitrogen limitation caused by the long-term combined organic-inorganic fertilization on the soil microbial community structure and its dominated SOC decomposition.

The application of organic fertilizers, such as straw and manure, is an efficient approach to maintain soil productivity. However, the effect of these organic fertilizers on soil microbial nutrient balance has not yet been established. In this study, the effects of the long-term combined organic-inorganic fertilization on microbial community were investigated by conducting a 30-year-long field test. Overall, the following five fertilizer groups were employed: inorganic NP fertilizer (NP), inorganic NK fertilizer (NK), inorganic NPK fertilizer (NPK), NPK + manure (MNPK), and NPK + straw (SNPK). The results indicated that the mean natural logarithm of the soil C:N:P acquisition enzyme ratio was 1.04:1.11:1.00 under organic-inorganic treatments, which showed a deviation from its overall mean ratio of 1:1:1. This indicates that microbial resources do not have a balance. Vector analysis (vector angle <45°) and threshold elemental ratio analysis (RC:N-TERC:N > 0) further demonstrated that the microbial metabolism was limited by Nitrogen (N) under SNPK and MNPK treatments. N limitation further influenced soil microbial community structure and its dominated SOC decomposition. Specifically, Microbial communities transformed into a more oligotrophic-dominant condition (fungal, Acidobacteria, Chloroflexi) from copiotrophic-dominant (Proteobacteria, Actinobacteria) condition with increasing N limitation. Lysobacter genus and Blastocatellaceae family, in the bacterial communities along with the Mortierella elongata species in fungal communities, were markedly associated with the N limitation, which could be the critical biomarker that represented N limitation. Both correlation analysis and partial least squares path modeling showed significant positive effects of N limitation on the ratio of bacterial functional genes (Cellulase/Amylase), involved in recalcitrant SOC degradation.

Challenges Facing Percutaneous Ablation in the Treatment of Hepatocellular Carcinoma: Extension of Ablation Criteria.

As an emerging minimally invasive treatment method, percutaneous ablation is more and more widely used in the treatment of liver tumors. It has been recommended by guidelines for diagnosis and treatment of hepatocellular carcinoma (HCC) as a curative treatment alongside surgical resection and liver transplantation. In recent years, with the continuous advancement and innovation of percutaneous ablation technologies, their clinical efficacy and safety have been significantly improved, which has led to the expanded application of percutaneous ablation in the treatment of HCC-more and more patients who were previously considered unsuitable for ablation therapies are now being treated with percutaneous ablation. Obviously, percutaneous ablation can reduce the risk of treatment changes from curative strategies to palliative strategies. Based on clinical practice experience, this review enumerates the advantages and disadvantages of different ablative modalities and summarizes the existing combinations of ablation techniques, thus will help clinicians choose the most appropriate ablative modality for each patient and will provide scientific guidance for improving prognosis and making evidence-based treatment decisions. In addition, we point out the challenges and future prospects of the ablation therapies, thereby providing direction for future research.

A novel internal cold circulation radiofrequency-assisted device for liver transection.

PURPOSE: To evaluate the safety and efficacy of a new internal cold circulation bipolar radiofrequency compared with Habib-4X bipolar radiofrequency device in the resection of liver tumors. METHODS: A total of 85 patients with hepatocellular carcinoma who received radiofrequency-assisted liver resection from February 2017 to January 2020 were retrospectively enrolled in our study, in which 45 patients received the new internal cold circulation bipolar radiofrequency (New-RF) and 40 patients received Habib-4X bipolar radiofrequency (Habib-4X). Primary outcome measures were the speed of liver transection, the width of coagulation tissue, hemorrhage volume, blood transfusion rate, and operation time. RESULTS: The baseline characteristics of patients in the New-RF and Habib-4X groups had no significant difference (p > 0.05). Compared to Habib-4X, the New-RF had a faster average speed of liver transection (4.81 ± 1.20 cm2/min vs 3.64 ± 1.08 cm2/min, p < 0.001), a narrower width of coagulation tissue (1.42 ± 0.23 cm2 vs 1.81 ± 0.20 cm2, p < 0.001), a less operation time (55.04 ± 16.12 min vs 64.02 ± 15.09 min, p = 0.010), a lower rate of needle path bleeding (13.3% vs 35.0%, p = 0.019), and a lower carbonization rate of electrode needle (22.2% vs 77.8%, p < 0.001). Hemorrhage during the transection (85.0 ml vs 105.0 ml, p = 0.438) and hemorrhage per square centimeter (3.28 ± 0.86 ml/cm2 vs 3.60 ± 1.12 ml/cm2, p = 0.141) in the New-RF group were smaller than those in Habib-4X group with no significant difference. CONCLUSION: The new internal cold circulation bipolar radiofrequency was a safe and efficacious auxiliary device for liver resection with a faster speed of resection, lower carbonization rate of electrode needle, and more precise range of coagulation.

Shuangshen granules attenuate lung metastasis by modulating bone marrow differentiation through mTOR signalling inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine Shuangshen granules (SSG) have been used to treat lung cancer patients with Qi deficiency and blood stasis for decades. According to clinical experience, SSG indeed improve the quality of life and prolong the survival time of patients with lung cancer after surgery. Each of the components herbs was proved to be effective in anti-cancer therapy. Both the American ginseng and notoginseng belong to genus Panax of the family Araliaceae. Preclinical and clinical studies demonstrated that ginsenosides of them have anti- or preventive activities to various tumors, including cancers of gastric, breast, liver, lung, ovarian, colon, melanoma and leukemia. PDS, such as ginsenoside Rb1, and PTS, such as ginsenoside Rg1 are the main anticancer compositions. Cordyceps sinensis had also been found effective in inhibiting tumour growth and metastasis, especially on tumour associated immune cells, such as macrophages. However, limited information is available regarding potential mechanisms of SSG. Myeloid-derived suppressor cell (MDSC)-mediated immunosuppression, which is closely associated with poor clinical outcomes in cancer patients, may be the target of SSG, which regulate immune function. AIM OF THE STUDY: The present study aimed to explore whether SSG attenuate the differentiation of bone marrow cells (BMCs) into MDSCs by blocking the mTOR signalling, leading to the suppression of lung metastasis. MATERIALS AND METHODS: First, we observed the differentiation of BMCs into MDSCs in vitro and in vivo. BMCs were cultured alone or co-cultured with Lewis lung carcinoma (LLC) cell supernatant in vitro. The effects of different concentrations of SSG, or LLC cell supernatant as a control, on BMC differentiation were detected by flow cytometry and western blotting. Male C57BL/6J mice were subcutaneously implanted with LLC cells, and SSG were administered by gavage twice daily before and after implantation for 7 or 14 days, respectively. The tumour weight, proportion of MDSCs, presence of CD11b+Ly6C+Ly6G- and CD11b+Ly6C+Ly6G+ cells in the bone marrow, blood, and lungs, as well as the expression levels of differentiation-related proteins in the bone marrow and lungs were evaluated. RESULTS: SSG attenuated the differentiation of BMCs into MDSCs, and reduced the fraction of CD11b+Ly6C+Ly6G+ cells by inhibiting the mTOR/S6K1/Myc signalling pathway. In vivo, SSG attenuated differentiation-associated protein markers and reduced the fractions of MDSCs and CD11b+Ly6C+Ly6G+ cells in the bone marrow, blood, and lungs. In addition, SSG administration reduced the tumour weight and inhibited lung metastasis. CONCLUSIONS: SSG may reduce lung metastasis by attenuating BMC differentiation into CD11b+Ly6C+Ly6G+ cells by inhibiting mTOR signalling in vitro and in vivo.

HBXIP drives metabolic reprogramming in hepatocellular carcinoma cells via METTL3-mediated m6A modification of HIF-1α.

Cancer cells sustain high levels of glycolysis and glutaminolysis via reprogramming of intracellular metabolism, which represents a driver of hepatocellular carcinoma (HCC) progression. Understanding the mechanisms of cell metabolic reprogramming may present a new basis for liver cancer treatment. Herein, we collected HCC tissues and noncancerous liver tissues and found hepatitis B virus X-interacting protein (HBXIP) was found to be upregulated in HCC tissues and associated with poor prognosis. The N6-methyladenosine (m6A) level of hypoxia-inducible factor-1α (HIF-1α) in HCC cells was evaluated after the intervention of METTL3. The possible m6A site of HIF-1α was queried and the binding relationship between METTL3 and HIF-1α was verified. The interference of HBXIP suppressed HCC malignant behaviors and inhibited the Warburg effect in HCC cells. METTL3 was upregulated in HCC tissues and positively regulated by HBXIP. Overexpression of METTL3 restored cell metabolic reprogramming in HCC cells with partial loss of HBXIP. HBXIP mediated METTL3 to promote the metabolic reprogramming and malignant biological behaviors of HCC cells. The levels of total m6A in HCC cells and m6A in HIF-1α were increased. METTL3 had a binding relationship with HIF-1α and mediated the m6A modification of HIF-1α. In conclusion, HBXIP drives metabolic reprogramming in HCC cells via METTL3-mediated m6A modification of HIF-1α.

Sinomenine regulates CD14/TLR4, JAK2/STAT3 pathway and calcium signal via α7nAChR to inhibit inflammation in LPS-stimulated macrophages.

Objective: To investigate the cellular mechanism that sinomenine (SIN) inhibits inflammation in macrophages induced by LPS through α7 nicotinic acetylcholine receptor (α7nAChR). Materials and methods: RAW264.7 cells were stimulated with LPS and treated by SIN or nicotine (Nic). A selective antagonist of α7nAChR, α-bungarotoxin (BTX) was used to block α7nAChR. AG490 was used to inhibit JAK2 activation. ELISA was performed to detect the levels of TNF-α and MCP-1. Western blotting was used to analyze the expression of MIF, MMP-9, CD14, TLR4, STAT3 and p-STAT3. Intracellular-free calcium level was measured by Fluorescent probe fluo-3/AM Results: SIN inhibited the production of TNF-α, MCP-1, MIF, and MMP-9, decreased the expression of CD14 and TLR4, and inhibited the release of intracellular-free calcium from intracellular stores in RAW 264.7 cells stimulated by LPS. JAK-specific inhibitor AG490 attenuated the inhibitory effect of SIN on TNF-α. SIN increased the phosphorylation of STAT3. And the above effects of SIN were attenuated by antagonist of α7nAChR. Conclusions: SIN can decrease the expression of CD14/TLR4 and intracellular free calcium level, activate JAK2/STAT3 pathway to inhibit inflammatory response through α7nAChR in macrophages.

Sinomenine inhibits fibroblast-like synoviocyte proliferation by regulating α7nAChR expression via ERK/Egr-1 pathway.

Fibroblast like synoviocyte (FLS) is a crucial in the pathogenesis of rheumatoid arthritis (RA), and involved in inflammation and joint destruction. Sinomenine (SIN), an alkaloid derived from the plant Sinomenium acutum, has anti-inflammatory and analgesic effect and been used for RA treatment in China. Alpha 7 nicotinic acetylcholine receptors (α7nAChR), as the key receptor in cholinergic anti-inflammatory pathway (CAP) to inhibit inflammation, has been detected in RA patients synovium, but its role is still unclear. Here we investigated the association between the aggressive proliferation of FLS and α7nAChR expression and the effect of sinomenine. FLS was isolated from synovial tissues of adjuvant-induced-arthritis (AIA) rat. Tumor necrosis factor(TNF)-α was used to induce the aggressive proliferation of FLS. MTT assay was applied to evaluate the proliferation of FLS. The messenger RNA (mRNA) and protein levels of α7nAChR and early growth response gene-1 (Egr-1) were measured. The results showed that TNF-α induced FLS proliferation in vitro (P < .01) and increased the phosphorylation of ERK1/2 and the expression of Egr-1 and α7nAChR (P < .05 or P < .01). U0126, the inhibitor of ERK1/2 inhibited α7nAChR expression and FLS proliferation significantly (P < .05 or P < .01). Specific short interference RNA(siRNA) of α7nAChR decreased α7nAChR expression and inhibited FLS proliferation as well. SIN inhibited the proliferation of FLS and decreased the phosphorylation of ERK1/2, and the expression of Egr-1 and α7nAChR induced by TNF-α (P < .05). In conclusion, the expression of α7nAChR involved in the aggressive proliferation of FLS induced by TNF-α and was regulated by ERK/Egr-1 signal pathway. SIN inhibited FLS proliferation and α7nAChR expression through inhibiting ERK/Egr-1 signal pathway, this may contribute to the anti-inflammatory and anti-arthritic effect of SIN.

Biology of the Tongue Coating and Its Value in Disease Diagnosis.

Tongue diagnosis is one of the most important diagnostic tools in traditional Chinese medicine and has been verified for thousands of years. However, its subjectivity and repeatability has been disputed continuously. The tongue coating as the primary coverage of tongue diagnosis provides more objectivity and reproducibility due to its relatively clear molecular basis; it also has a close relationship with many system diseases and may be used as a potentially valuable disease diagnostic tool. This article describes the material basis of the tongue coating, including its biology (epithelial cells, blood cells, vascular endothelial cells, and bacteria) and its metabolites; moreover, we summarize the diseases that are most correlated with the tongue coating. This will be valuable not only for fundamental research of tongue diagnosis but also for the diagnosis and differential diagnosis of disease. We suppose that the tongue coating could serve as a valuable auxiliary diagnosis tool in many diseases, and more research should focus on how to colligate the various information about the tongue and provide useful information for disease diagnosis.

Estrogen receptor ß promoter methylation: a potential indicator of malignant changes in breast cancer.

INTRODUCTION: Estrogen receptor ß (ERß) always lacks expression in estrogen-dependent tumors, which may result from gene inactivation by methylation. In this study, we aimed to determine whether aberrant methylation of the ERß promoter is associated with decreased ERß gene expression in breast cancer. MATERIAL AND METHODS: ERß methylation status was determined for 132 pairs of breast cancer and adjacent normal tissues via the MethyLight method. Additionally, mRNA relative expression was quantified by real-time polymerase chain reaction (RT-PCR) to determine whether aberrant methylation had a negative correlation with expression. The correlation of ERß promoter methylation and clinical parameters is also discussed. RESULTS: Methylation was observed in 96 (72.7%) breast cancer samples, and the median percentage of fully methylated reference (PMR) among methylated tissues was 0.83. Meanwhile, 94 (71.2%) adjacent normal tissues were methylated and the median PMR was 0.48. Compared to adjacent normal tissues, the methylation level of breast cancer was significantly higher (p < 0.001) and mRNA expression was much lower (p < 0.001). There was a significant correlation between ERß methylation and mRNA expression in adjacent normal breast tissues (p = 0.004). In addition, the methylation rate of cancer tissues whose maximum diameter < 3 cm was significantly higher than those > 3 cm (p = 0.025). CONCLUSIONS: ERß promoter methylation level varies between cancerous and adjacent normal breast tissues. There was significant downregulation of ERß methylation expression in pre-cancerous stages of breast cancer. Therefore, demethylation drugs may offer a potential strategy for preventing the development of pre-cancerous cells.

Natural attenuation model and biodegradation for 1,1,1-trichloroethane contaminant in shallow groundwater.

Natural attenuation is an effective and feasible technology for controlling groundwater contamination. This study investigated the potential effectiveness and mechanisms of natural attenuation of 1,1,1-trichloroethane (TCA) contaminants in shallow groundwater in Shanghai by using a column simulation experiment, reactive transport model, and 16S rRNA gene clone library. The results indicated that the majority of the contaminant mass was present at 2-6 m in depth, the contaminated area was approximately 1000 m × 1000 m, and natural attenuation processes were occurring at the site. The effluent breakthrough curves from the column experiments demonstrated that the effectiveness of TCA natural attenuation in the groundwater accorded with the advection-dispersion-reaction equation. The kinetic parameter of adsorption and biotic dehydrochlorination of TCA was 0.068 m(3)/kg and 0.0045 d(-1). The contamination plume was predicted to diminish and the maximum concentration of TCA decreased to 280 µg/L. The bacterial community during TCA degradation in groundwater belonged to Trichococcus, Geobacteraceae, Geobacter, Mucilaginibacter, and Arthrobacter.

Aerobic biodegradation of trichloroethylene and phenol co-contaminants in groundwater by a bacterial community using hydrogen peroxide as the sole oxygen source.

Trichloroethylene (TCE) and phenol were often found together as co-contaminants in the groundwater of industrial contaminated sites. An effective method to remove TCE was aerobic biodegradation by co-metabolism using phenol as growth substrates. However, the aerobic biodegradation process was easily limited by low concentration of dissolved oxygen (DO) in groundwater, and DO was improved by air blast technique with difficulty. This study enriched a bacterial community using hydrogen peroxide (H2O2) as the sole oxygen source to aerobically degrade TCE by co-metabolism with phenol in groundwater. The enriched cultures were acclimatized to 2-8 mM H2O2 which induced catalase, superoxide dismutase and peroxidase to decompose H2O2 to release O2 and reduce the toxicity. The bacterial community could degrade 120 mg/L TCE within 12 days by using 8 mM H2O2 as the optimum concentration, and the TCE degradation efficiency reached up to 80.6%. 16S rRNA gene cloning and sequencing showed that Bordetella, Stenotrophomonas sp., Sinorhizobium sp., Variovorax sp. and Sphingobium sp. were the dominant species in the enrichments, which were clustered in three phyla: Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria. Polymerase chain reaction detection proved that phenol hydroxylase (Lph) gene was involved in the co-metabolic degradation of phenol and TCE, which indicated that hydroxylase might catalyse the epoxidation of TCE to form the unstable molecule TCE-epoxide. The findings are significant for understanding the mechanism of biodegradation of TCE and phenol co-contamination and helpful for the potential applications of an aerobic bioremediation in situ the contaminated sites.

A single nucleotide polymorphism in PIK3CA gene is inversely associated with P53 protein expression in breast cancer.

Single nucleotide polymorphism (SNP) rs17849071 was recently reported to be inversely associated with PIK3CA amplification in follicular thyroid cancer, but the main function of this SNP remains unclear. In this study, by using PCR and sequencing method, we explored whether this SNP was associated with P53 expression status and other clinicopathological characteristics in 62 Chinese breast cancer (BCa) patients. In our results, P53 protein accumulation was significantly associated with HER2 overexpression (P = 0.013) and Ki-67 expression (P = 0.007), which were in accord with previous studies. Besides, there was a significantly inverse relationship between P53 protein expression and rs17849071 GT+GG genotype in Chinese BCa patients (P = 0.044). The SNP was not related to other important BCa markers such as estrogen receptor, progestin receptor, and HER2. Among different BCa intrinsic subtypes, no significant differences were found on P53 expression status (P = 0.356) or rs17849071 polymorphism (T>G) (P = 0.813). In conclusion, SNP rs17849071 GT+GG genotype was inversely associated with P53 protein accumulation in BCa samples. Studies with larger sample size focusing on exploring the relationship of rs17849071 polymorphisms, P53 accumulation, P53 mutations, and PIK3CA amplification might be needed.