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

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

Preclinical Investigations on Anti-fibrotic Potential of Long-Term Oral Therapy of Sodium Astragalosidate in Animal Models of Cardiac and Renal Fibrosis.

In traditional Chinese medicine, Radix Astragali has played a vital role in treating progressive fibrotic diseases. One of its main active components, astragaloside IV, is a promising anti-fibrotic treatment despite its extremely low bioavailability. Our study aimed to optimize sodium astragalosidate (SA) by salt formation to improve solubility and oral absorption for anti-fibrotic therapy in vivo. Isoproterenol-induced myocardial fibrosis rat models and obese BKS-db mice presenting diabetic kidney fibrosis were used in this study. Daily oral administration of SA (20 mg/kg) for 14 days ameliorated cardiac fibrosis by reducing collagen accumulation and fibrosis-related inflammatory signals, including TNF-α, IL-1ß, and IL-6. In db/db mice, SA (5,10, and 20 mg/kg per day for 8 weeks) dose-dependently alleviated lipid metabolism impairment and renal dysfunction when administered orally. Furthermore, Western blot and immunohistochemistry analyses demonstrated that SA treatment inhibited renal fibrosis by suppressing TGF-ß1/Smads signaling. Taken together, our findings provide the oral-route medication availability of SA, which thus might offer a novel lead compound in preclinical trial-enabling studies for developing a long-term therapy to treat and prevent fibrosis.

Role of simulated in vitro gastrointestinal digestion on biotransformation and bioactivity of astragalosides from Radix Astragali.

Radix Astragali (RA) is commonly used in Asian herbal therapy or food supply, and astragalosides and flavonoids are its major components with diverse pharmaceutical effects. To provide new information on the potential cardiovascular benefits of RA administered orally, the bioaccessibility of these compounds with relevant in vitro digestion parameters was determined for four digestion phases (oral, gastric, small and large intestines) by ultrahigh-performance liquid chromatography quadrupole time-of-flight-mass spectrometry (UPLC-Q-TOF/MS). Meanwhile, we compared the effects of digestion products on advanced glycation end products (AGEs)-induced intracellular reactive oxygen species (ROS) levels in a human arterial endothelial cells (HAECs) model, and studied the potential of RA against oxidative stress-related cardiovascular disease. The changes of saponins and flavonoids composition and antioxidant activity after digestion in intestines were mainly due to the astragaloside IV (AS-IV) biosynthesis involving saponins acetyl isomerization and deacetylation, and the flavonoid glycosides converted to aglycone by deglycosylation processes. All these results suggest that acetyl biotransformation of RA in small intestine directly influenced the response to oxidative stress, and might provide a reference for elucidation of the multi-component action after oral RA in cardiovascular health care.

Observation on the short-term efficacy of inverse moxibustion at the Baihui point and Dazhui point in preventing post-stroke depression.

OBJECTIVE: In this study, inverse moxibustion was performed at the Baihui and Dazhui points in patients with ischemic stroke, and the Hamilton Depression Rating Scale 17 (HAMD) score, National Institute of Health Stroke Scale (NIHSS) score, modified Barthel index (MBI) score, and incidence of post-stroke depression (PSD) were observed. METHODS: Eighty patients with acute ischemic stroke were enrolled and randomly divided into two groups. All enrolled patients were given routine treatment for ischemic stroke, and those in the treatment group were also given moxibustion at the Baihui and Dazhui points. The course of treatment was four weeks. The HAMD, NIHSS, and MBI scores of the two groups were evaluated before and four weeks after the treatment. The differences between the groups and the incidence of PSD were evaluated to determine the effect of inverse moxibustion at the Baihui and Dazhui points on the HAMD, NIHSS, and MBI scores and prevention of PSD in patients with ischemic stroke. RESULTS: After the four weeks of treatment, the HAMD and NIHSS scores of the treatment group were lower than those of the control group, their MBI was higher than that of the control group, and their incidence of PSD was statistically significantly lower than that of the control group. CONCLUSION: Inverse moxibustion at the Baihui acupoint in patients with ischemic stroke can effectively promote the recovery of neurological function, improve depression, and reduce the incidence of PSD and should be considered for application in clinical practice.

Identification and transcriptional activity analysis of core regulatory region of human guanylate binding protein 5 gene promoter / 中国生物制品学杂志

@#Objective To construct luciferase reporter plasmids of truncated fragments of different lengths of human guanylate binding protein 5(GBP5)gene promoter and analyze the transcriptional activity of each fragment to determine the core regulatory region.Methods GBP5promoter sequence was amplified by PCR,truncated into five fragments of different lengths and connected to pGL3-basic plasmid.The constructed recombinant plasmids pGL3-GBP5-11/21/31/41/51were transfected into 293FT cells and detected for luciferase activity.The binding sites of transcription factors in GBP5promoter region were predicted by JASPAR software,and Yin-Yang transcription factor 1(YY1)targeting the core regulatory region was selected and verified for the transcriptional regulatory activity.The CDS sequence of YY1 was amplified by PCR to construct the overexpression plasmid pIRES2-EGFP-YY1,which was then co-transfected to 293FT cells with plasmids pGL3-GBP5-21(-1 623 ～ +47 bp)and internal reference plasmid pRL-CMV,and detected for luciferase activity to analyze the regulation of transcription factor YY1 on GBP5 promoter activity.Results Colony PCR and double enzyme digestion identification proved that the plasmid of human GBP5 promoter reporter gene was correctly constructed;JASPAR software predicted that there were multiple transcription factor binding sites such as STAT1,YY1 and Foxp3 in GBP5promoter region.Double luciferase activity assay showed that pGL3-GBP5-21(-1 623 ～ +47 bp)showed the highest promoter activity,while the promoter activity of pGL3-GBP5-41(-520 ～ +47 bp)decreased significantly,suggesting that the core region of GBP5 promoter was located at upstream-1 623 ～-520 bp of 5 'UTR;Overexpression of YY1 significantly activated the GBP5 promoter activity and regulated the expression of GBP5.Conclusion The core regulatory region of human GBP5 promoter was located in upstream-1 623 ～-520 bp of the 5 'UTR,with a binding site of transcription factor YY1 existing in this region.Meanwhile,overexpression of YY1 significantly effected the activity of GBP5 promoter.

Docosahexaenoic acid monoglyceride induces apoptosis and autophagy in breast cancer cells via lipid peroxidation-mediated endoplasmic reticulum stress.

Epidemiologic and preclinical studieshave shown that marine n-3 polyunsaturated fatty acids (n-3 PUFAs) elicit promising chemoprevention against breast cancer. Docosahexaenoic acid monoglyceride (MAG-DHA), a docosahexaenoic acid sn-1-monoacylglycerol does not required pancreatic lipase to be absorbed, eliciting a better bioavailability when compared with other formulations such as DHA-free fatty acid, DHA-triglycerol, or DHA-ethyl ester. However, the anticancer actions and underlying mechanisms of MAG-DHA on breast cancer remain to be assessed. In this study, MAG-DHA induced significant growth inhibition in MCF-7 and MDA-MB-231 breast cancer cells in a dose-dependent manner. MAG-DHA treatment (80 µM) led to 83.8 and 94.3% growth inhibition between MCF-7 and MDA-MB-231 cells, respectively. MAG-DHA-induced growth inhibition was tightly associated with apoptosis, as evidenced by increased active forms of caspase-3, poly (ADP-ribose) polymerase (PARP) and caspase-12. In particular, MAG-DHA-induced apoptosis was triggered by oxidative stress-mediated endoplasmic reticulum (ER) stress, as evidenced by activation of the PERK-eIF2α pathway in ER. MAG-DHA treatment also strongly suppressed the growth of E0771 murine breast cancer xenografts, significant differences of tumor volume were found between MAG-DHA group (0.271 cm3 ) and control group (0.875 cm3 ) after 15 daily MAG-DHA treatments. The in vitro antibreast cancer mechanism of MAG-DHA was supported by the in vivo xenograft model. In addition, MAG-DHA-induced ER stress concomitantly triggered autophagy in these cancer cells, and the induction of autophagy suppressed its ability to induce apoptotic cell death. Our data suggested that MAG-DHA as dietary supplement, in combination with autophagy inhibitors may be a useful therapeutic strategy in treating breast cancer.

Synergistic effects of α-Mangostin and sorafenib in hepatocellular carcinoma: New insights into α-mangostin cytotoxicity.

Sorafenib remains the standard first-line treatment for advanced hepatocellular carcinoma (HCC), although other clinical trials are currently underway for treatments that show better curative effects. However, some patients are not sensitive to sorafenib. α-Mangostin, extracted from the pericarp of the mangosteen, which is widely used as a traditional medicine, has anticancer and anti-proliferative properties in various types of cancers, including HCC. In the present study, we found that combining sorafenib and α-Mangostin could be synergistically toxic to HCC both in vitro and in vivo. We then demonstrated that the combination of sorafenib and α-Mangostin enhances the inhibition of cell proliferation in HCC cell lines. Combination therapy leads directly to apoptosis. In xenograft mouse models, the in vivo safety and effectivity was confirmed by a reduction in tumor size after combination treatment. RNA sequencing and protein testing showed that the expression of LRRC8A and RNF181 genes and mTOR and MAPK pathways may be associated with the synergistic effect of the two drugs. In conclusion, our results highlight the synergistic effect of the combination of sorafenib and α-Mangostin, which indicates a potential treatment for advanced HCC for patients that are not sensitive to sorafenib therapy.

Rhubarb extract relieves constipation by stimulating mucus production in the colon and altering the intestinal flora.

BACKGROUND: Constipation, mainly characterized by the difficulty in defecation, is a clinical symptom caused by a variety of factors. It can be manifested as normal or slow colonic transport abnormalities, which can occur alone or concurrently with defecation disorders. As there is not uniform definition and assessment standard, no clear plan could be used for the treatment of constipation. Although rhubarb, a traditional Chinese medicine, plays a therapeutic role in diseases involving constipation symptoms, the detailed mechanism of it in treating constipation remains unclear. METHODS: A model of constipation-induced by diphenoxylate was prepared. Immunofluorescent staining was used to detect the expression of mucin 2 (MUC2), calnexin and chymase in colon. Western blotting was used to detect changes of tryptase and calnexin in the colon. And real-time polymerase chain reaction (PCR) was utilized to detect the changes of immunoglobulin-binding protein (Bip), X-box binding protein 1 (Xbp1) and C/EBP homologous protein (CHOP) of colonic goblet cells in mRNA levels. ELISA and biochemical kits were utilized to detect the changes of MUC2, Trefoil factor 3 (TFF3), acetylcholine, histamine and C-C motif chemokine ligand 5 (CCL5) in the colon. And the changes of colonic mucosa and intestinal flora of constipation model mice caused by rhubarb extract (RE) were analyzed to identify the mechanism of RE on the treatment of constipation. RESULTS: RE promotes the secretion of colonic mucus by recruiting mast cells and enhancing the content of histamine and Ach in the mice colon. In the process, RE causes up-regulation of Bip and CHOP mRNA expression and down-regulation of Xbp1 and Xbp1s mRNA expression that induces ER stress of colonic epithelium associated with changes in the intestinal flora diversity and short-chain fatty acids content. CONCLUSION: RE could relieve constipation by promoting the secretion of colonic mucus via mast cells activation and improving the intestinal microenvironment.

Discovery and development of novel rhodanine derivatives targeting enoyl-acyl carrier protein reductase.

A series of rhodanine derivatives RB1-RB23 were synthesized through a two-round screening. Their Mycobacterial tuberculosis (Mtb) InhA inhibitory activity and Mtb growth blocking capability were evaluated. The most potent hit compound RB23 indicated comparable InhA inhibiton (IC50 = 2.55 µM) with the positive control Triclosan (IC50 = 6.14 µM) and Isoniazid (IC50 = 8.29 µM). Its improved growth-blocking effect on Mtb and low toxicity were attractive for further development. The docking simulation revealed the possible binding pattern of this series and picked the key interacted residues as Ser20, Phe149, Lys165 and Thr196. The 3D-QSAR model visualized the SAR discussion and hinted new information. Modifying the surroundings near rhodanine moiety might be promising attempts in later investigations.

[Re-operation treatment in uremic patients complicated with persistent secondary hyperparathyroidism after parathyroidectomy with autotransplantation].

Objective:To analyze the clinical profile and therapeutic effect of re-operation treatment in uremic patients complicated with persistent secondary hyperparathyroidism(SHPT)after parathyroidectomy with autotransplantation.Method:Twelve persistent SHPT patients who were treated with reoperation of paramyroidectomy(PTX)were enrolled in this study during the period from Jan 2014 to Jul 2017 in our hospital.We evaluated the location of the remaining parathyroid glands by ultrasonography,dual-phase 99 Tcm-sestamibi scintigraphy,CT and MR imaging of the neck before the operation.We resected the parathyroid gland tissue in situ,and the ectopic parathyroid glands hiding in thymus,mediastinal,tracheal esophageal groove,thyroid gland and other locations in the neck.During the surgery,nanocarbon imaging was used to help identify the parathyroid gland and parathyroid hormone assay(IOPTH)was measured at the end of the surgery.We observed the changes of clinical symptoms after the surgery and collected blood parameters including serum intact aramyroidhomone(i-PTH),calcium(Ca),phosphoms(P),calcium and phosphorus product before and after surgery.Complications and failure were also analyzed.Result:All the 12 patients underwented successful operation.The postoperative pathological results were hyperplastic parathyroid glands tissue.22 parathyroid glands were resected,among which 14 were located at the neck in situ,8 were ectopic,i.e.,located at thymus in 4 cases,superior mediastinum in 2 cases and thyroid parenchyma in 2 cases.The clinical symptoms were significantly improved including osteoarthritis,skin itching and limb weakness.The levels of serum iPTH,calcium,phosphorus and calcium and phosphorus product were significantly lower than those before operation(P<0.05).Ten patients presented hypocalcemia after surgery and the level of calcium returned to normal after supplement of calcium.Temporary injury of laryngeal nerve was found in4 cases,but there was no patient with transient bucking,dyspnea or death.No recurrence was found during 1 year follow-up.Conclusion:It was very important to locate the residual parathyroid gland accurately with a variety of imaging methods in uremic patients complicated with persistent or recurrent SHPT when they needed re-operation.Surgeons should explorate ectopic parathyroid gland according to the concept of the superior mediastinum dissection and the central compartment neck dissection.Meanwhile,the use of nanocarbon assisted parathyroid gland negative imaging and rapid IOPTH can significantly improve the success rate of surgery and reduce surgical complications.

[Exploration on an overview of history (from Chunqiu to Ming and Qing Dynasties) and species of Chinese exotic traditional medicine].

This paper collected and analyzed literatures about starting of traditional medicine and trading of different medicine from Chunqiu Zhanguo to Ming and Qing Dynasties, in order to preliminarily explore on an overview of specie of chinese exotic traditional medicine and trade of different country in different dynasty（from Chunqiu to Ming and Qing Dynasties）, as well as the amount of exotic medicine over two thousand years. (remove repeating and doubtful species), find peculiarity of exotic medicine, sreen species that used to develop the Silk Route and establish quality standards.Finally, We get conclusions are as follows. First, primary,.prosperous and declining stages of development of exotic traditional medicine are Qin and Han, Song-Jin-Yuan, Ming and Qing Dynasties, respectively. Second, according to literature, the stage that has the most species of exotic medicine is song dynasty,approximately have 300. Removing repeating and doubtful species, the believable species are approximate 230 to 250. Meanwhile, the unknown species are approximate 30, which may be different name of one medicine or processed goods, now these medicines are named as "doubtful species". Third, the medicinal parts of exotic medicine are different from Chinese medicine of Han nation. The number of Resin, fruits and seeds kind are more than root and rhizomes kind,mineral medicines are more than animal. Fourth, the major producing area of exotic medicine is some countries and territories related to the Silk Route in the history. Ultimately, this paper preliminarily figure out basic information of exotic medicine of different dynasty in China,that provide reference for learning study and decision of industrial development.

Direct and indirect induction by 1,25-dihydroxyvitamin D3 of the NOD2/CARD15-defensin beta2 innate immune pathway defective in Crohn disease.

Vitamin D signaling through its nuclear vitamin D receptor has emerged as a key regulator of innate immunity in humans. Here we show that hormonal vitamin D, 1,25-dihydroxyvitamin D(3), robustly stimulates expression of pattern recognition receptor NOD2/CARD15/IBD1 gene and protein in primary human monocytic and epithelial cells. The vitamin D receptor signals through distal enhancers in the NOD2 gene, whose function was validated by chromatin immunoprecipitation and chromatin conformation capture assays. A key downstream signaling consequence of NOD2 activation by agonist muramyl dipeptide is stimulation of NF-kappaB transcription factor function, which induces expression of the gene encoding antimicrobial peptide defensin beta2 (DEFB2/HBD2). Pretreatment with 1,25-dihydroxyvitamin D(3) synergistically induced NF-kappaB function and expression of genes encoding DEFB2/HBD2 and antimicrobial peptide cathelicidin in the presence of muramyl dipeptide. Importantly, this synergistic response was also seen in macrophages from a donor wild type for NOD2 but was absent in macrophages from patients with Crohn disease homozygous for non-functional NOD2 variants. These studies provide strong molecular links between vitamin D deficiency and the genetics of Crohn disease, a chronic incurable inflammatory bowel condition, as Crohn's pathogenesis is associated with attenuated NOD2 or DEFB2/HBD2 function.

Cutting edge: 1,25-dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression.

The hormonal form of vitamin D(3), 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), is an immune system modulator and induces expression of the TLR coreceptor CD14. 1,25(OH)(2)D(3) signals through the vitamin D receptor, a ligand-stimulated transcription factor that recognizes specific DNA sequences called vitamin D response elements. In this study, we show that 1,25(OH)(2)D(3) is a direct regulator of antimicrobial innate immune responses. The promoters of the human cathelicidin antimicrobial peptide (camp) and defensin beta2 (defB2) genes contain consensus vitamin D response elements that mediate 1,25(OH)(2)D(3)-dependent gene expression. 1,25(OH)(2)D(3) induces antimicrobial peptide gene expression in isolated human keratinocytes, monocytes and neutrophils, and human cell lines, and 1,25(OH)(2)D(3) along with LPS synergistically induce camp expression in neutrophils. Moreover, 1,25(OH)(2)D(3) induces corresponding increases in antimicrobial proteins and secretion of antimicrobial activity against pathogens including Pseudomonas aeruginosa. 1,25(OH)(2)D(3) thus directly regulates antimicrobial peptide gene expression, revealing the potential of its analogues in treatment of opportunistic infections.