Comprehensive Study in the Inhibitory Effect of Berberine on Gene Transcription, Including TATA Box

Berberine (BBR) is an established natural DNA intercalator with numerous pharmacological functions. However, currently there are neither detailed reports concerning the distribution of this alkaloid in living cells nor reports concerning the relationship between BBR's association with DNA and the function of DNA. Here we report that the distribution of BBR within the nucleus can be observed 30 minutes after drug administration, and that the content of berberine in the nucleus peaks at around 4 µmol, which is twelve hours after drug administration. The spatial conformation of DNA and chromatin was altered immediately after their association with BBR. Moreover, this association can effectively suppress the transcription of DNA in living cell systems and cell-free systems. Electrophoretic mobility shift assays (EMSA) demonstrated further that BBR can inhibit the association between the TATA binding protein (TBP) and the TATA box in the promoter, and this finding was also attained in living cells by chromatin immunoprecipitation (ChIP). Based on results from this study, we hypothesize that berberine can suppress the transcription of DNA in living cell systems, especially suppressing the association between TBP and the TATA box by binding with DNA and, thus, inhibiting TATA box-dependent gene expression in a non-specific way. This novel study has significantly expanded the sphere of knowledge concerning berberine's pharmacological effects, beginning at its paramount initial interaction with the TATA box

UPLC/Q-TOFMS-Based Metabolomics Approach to Reveal the Protective Role of Other Herbs in An-Gong-Niu-Huang Wan Against the Hepatorenal Toxicity of Cinnabar and Realgar

An-Gong-Niu-Huang Wan (AGNH) is a well-known traditional Chinese medicine (TCM) recipe containing cinnabar (HgS) and realgar (As2S2). However, the application of AGNH is limited by the hepato- and nephrotoxicity of cinnabar and realgar. It should be noted that cinnabar and realgar in AGNH are not used alone, but rather combined with other herbs as formula to use. In this study, the protective effects and mechanisms of the other herbs in AGNH against the hepatorenal toxicity induced by cinnabar and realgar were investigated. The combination use of the other herbs in AGNH alleviated inflammatory cell infiltration and damage in the liver and kidney and restored the disturbed serum metabolic profile induced by cinnabar and realgar insults. By UPLC/Q-TOFMS combined with pattern recognition approaches, we identified 41 endogenous metabolites in the sera of mice that were related to the hepatorenal toxicity of cinnabar and realgar, 36 of which were restored to normal levels when various kinds of herbs were combined as compound recipe. These metabolites function as modulators in inflammation-associated glycerophospholipid, arachidonic acid, linoleic acid, sphingolipid, and ether lipid metabolic pathways. Notably, lysophosphatidylcholines (LysoPCs) were the most elevated among all of the metabolites detected after cinnabar and realgar treatment, while these LysoPCs did not show overt differences between the AGNH and saline control groups, which was associated with relatively unaffected or even up-regulated expression of lysophosphatidylcholine acyltransferase 1 (LPCAT1) and autotaxin (ATX). These findings indicated that other herbs in AGNH could have a protective effect against cinnabar- and realgar-induced hepatic and renal damage via modulating the disordered homeostasis of the glycerophospholipid, arachidonic acid, linoleic acid, ether lipid, and sphingolipid metabolism

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Anti-atherosclerosis effect of Angong Niuhuang pill via regulating Th17/Treg immune balance and inhibiting chronic inflammatory on apoE^-/- Mice model of early and mid-term atherosclerosis

Angong Niuhuang Pill (ANP) is a well-known patented Chinese medicine which is used for hundreds of years for treating the central nervous system diseases. Atherosclerosis is a polyaetiological chronic inflammatory vascular disease. Preventing inflammation is fundamental for treating atherosclerosis in early stages. In this study, we investigated the protective effects and possible mechanisms of ANP action on a high-fat diet induced early and mid-term atherosclerosis ApoE-/- mice. The effects of ANP were compared with accepted drug simvastatin. Twelve male C57BL/6J mice were used as the control group, and 60 male ApoE-/- mice were randomly divided into five groups: Model group, Simvastatin group, Low-, Medium-, and High-dose ANP group these groups received, respectively, saline, simvastatin (3.0mg/kg), low-dose ANP (0.25 g/kg), medium-dose ANP (0.50 g/kg), and high-dose ANP (1.0 g/kg), once every other day for 10 weeks. After administration, serum biochemical indices were detected by the automatic biochemical analyzer, the concentrations of IL-6 and IL-10 in the serum were assayed by ELISA, expression levels of IL-1b, TNF-a, MMP-2, MMP-9, CCL2, and its receptor CCR2 in the full-length aorta, and expression levels of transcription factors Foxp3, RORgt in the spleen were assayed via western blotting and RT-qPCR. Flow cytometry was used to analyze Th17 cells and Treg cells. Pathological and histological analysis was completed on aortic root. ANP decreased LDL/HDL ratio, concentrations of IL-6 while increased IL-10 in serum. Moreover, ANP down-regulated the expression levels of IL-1b, TNF-a, MMP-2, MMP-9, CCL2, and CCR2 receptor in the full-length aorta. In addition, ANP decreased Th17 cells and expression levels of transcription factor RORgt, increased Treg cells and expression levels of transcription factor Foxp3. ANP decreased content of collagen fibers and infiltration of inflammatory cells in the aortic root. In conclusion, we demonstrated that ANP has anti-atherosclerosis effects on a high-fat diet induced ApoE-/mice early and mid-term AS model via regulating Th17/Treg balance, inhibiting chronic inflammation, reducing plaque collagen fibers, and reducing inflammatory cells infiltration, to exert its multi-channel multi-target anti-early and mid-term AS effects.</p

Image_2_UPLC/Q-TOFMS-Based Metabolomics Approach to Reveal the Protective Role of Other Herbs in An-Gong-Niu-Huang Wan Against the Hepatorenal Toxicity of Cinnabar and Realgar.PDF

<p>An-Gong-Niu-Huang Wan (AGNH) is a well-known traditional Chinese medicine (TCM) recipe containing cinnabar (HgS) and realgar (As₂S₂). However, the application of AGNH is limited by the hepato- and nephrotoxicity of cinnabar and realgar. It should be noted that cinnabar and realgar in AGNH are not used alone, but rather combined with other herbs as formula to use. In this study, the protective effects and mechanisms of the other herbs in AGNH against the hepatorenal toxicity induced by cinnabar and realgar were investigated. The combination use of the other herbs in AGNH alleviated inflammatory cell infiltration and damage in the liver and kidney and restored the disturbed serum metabolic profile induced by cinnabar and realgar insults. By UPLC/Q-TOFMS combined with pattern recognition approaches, we identified 41 endogenous metabolites in the sera of mice that were related to the hepatorenal toxicity of cinnabar and realgar, 36 of which were restored to normal levels when various kinds of herbs were combined as compound recipe. These metabolites function as modulators in inflammation-associated glycerophospholipid, arachidonic acid, linoleic acid, sphingolipid, and ether lipid metabolic pathways. Notably, lysophosphatidylcholines (LysoPCs) were the most elevated among all of the metabolites detected after cinnabar and realgar treatment, while these LysoPCs did not show overt differences between the AGNH and saline control groups, which was associated with relatively unaffected or even up-regulated expression of lysophosphatidylcholine acyltransferase 1 (LPCAT1) and autotaxin (ATX). These findings indicated that other herbs in AGNH could have a protective effect against cinnabar- and realgar-induced hepatic and renal damage via modulating the disordered homeostasis of the glycerophospholipid, arachidonic acid, linoleic acid, ether lipid, and sphingolipid metabolism.</p

Cytotoxicity assay of berberine.

<p>(A) represents the cytotoxicity of BBR from the MTT assay: A(I) and A(II) represent the cytotoxicity of BBR to PC12 cells 12 hours and 24 hours after drug administration, respectively; (B) represents the LDH release of PC12 cells after BBR addition: B(I) and B(II) represent the amount of LDH released from PC12 cells 12 hours and 24 hours since BBR incubation. In the MTT assay, the group with 0 µmol of BBR was considered the control group; in LDH release assay, the spontaneous release of LDH from the BBR-free group (0 µmol) was considered the control group. The percentage of LDH release was calculated by the equation: LDH release (%) = (Experimental LDH release-Spontaneous LDH release)/Maximum LDH release. * p<0.05, ** p<0.01 vs control. Data were presented as mean ± S.D. from twelve independent experiments. (n = 12).</p

Image_1_UPLC/Q-TOFMS-Based Metabolomics Approach to Reveal the Protective Role of Other Herbs in An-Gong-Niu-Huang Wan Against the Hepatorenal Toxicity of Cinnabar and Realgar.PDF

<p>An-Gong-Niu-Huang Wan (AGNH) is a well-known traditional Chinese medicine (TCM) recipe containing cinnabar (HgS) and realgar (As₂S₂). However, the application of AGNH is limited by the hepato- and nephrotoxicity of cinnabar and realgar. It should be noted that cinnabar and realgar in AGNH are not used alone, but rather combined with other herbs as formula to use. In this study, the protective effects and mechanisms of the other herbs in AGNH against the hepatorenal toxicity induced by cinnabar and realgar were investigated. The combination use of the other herbs in AGNH alleviated inflammatory cell infiltration and damage in the liver and kidney and restored the disturbed serum metabolic profile induced by cinnabar and realgar insults. By UPLC/Q-TOFMS combined with pattern recognition approaches, we identified 41 endogenous metabolites in the sera of mice that were related to the hepatorenal toxicity of cinnabar and realgar, 36 of which were restored to normal levels when various kinds of herbs were combined as compound recipe. These metabolites function as modulators in inflammation-associated glycerophospholipid, arachidonic acid, linoleic acid, sphingolipid, and ether lipid metabolic pathways. Notably, lysophosphatidylcholines (LysoPCs) were the most elevated among all of the metabolites detected after cinnabar and realgar treatment, while these LysoPCs did not show overt differences between the AGNH and saline control groups, which was associated with relatively unaffected or even up-regulated expression of lysophosphatidylcholine acyltransferase 1 (LPCAT1) and autotaxin (ATX). These findings indicated that other herbs in AGNH could have a protective effect against cinnabar- and realgar-induced hepatic and renal damage via modulating the disordered homeostasis of the glycerophospholipid, arachidonic acid, linoleic acid, ether lipid, and sphingolipid metabolism.</p

Spatial conformational change induced by berberine.

<p>(A) represents the dose-dependent alterations in circular dichroic spectrum of genome, chromatin, and plasmid, respectively. (B) represents the time-dependent alterations in circular dichroic spectrum of genome, chromatin, and plasmid, respectively. (C) represents the effect of BBR (500 µmol) on the intensity distribution (%) of chromatin (92.4 µmol), genome (423.2 µmol), and plasmid (808 µmol) in size (diameter) obtained from DLS measurements, respectively.</p

The suppressive effect of berberine on gene transcription in live cells.

<p>(A) portrays the time-dependent effect of BBR on gene transcription on a global level. (B) shows the time-dependent recovery of the global RNA level after the elimination of a 12 hour-BBR treatment. The linear equation of the control group in (B) is: <i>y</i> = 4.9836×<i>+21.516</i>, R² = 0.8162; the linear equation of the BBR group in (B)is: <i>y</i> = 6.6219×<i>+15.472</i>, R² = 0.8941. (C) displays the protective effect of BBR on the global RNA level. (D) depicts the protective effect of BBR on the mRNA level of the transfected artificial plasmid, pEGFP-N1. The dosage of BBR in all of the experiments was 2.69 µmol. Data in D was the ratio of target gene in the BBR group to the same target gene in the BBR-free group at the same time-point (control group). * p<0.05, ** p<0.01 vs the corresponding control indicated above. Data were presented as mean ± S.D. from four independent experiments (n = 4).</p

Distribution of berberine in a living cell.

<p>(A) (from I to V) depicts images of the subcellular location of BBR in PC12 cells one hour after BBR administration. The fluorescence of BBR is shown in blue in A (I); A (II) represents PC12 cells in visible light; A (III) and A (IV) represents the nucleus stained by PI and AO, respectively; figure A (V) is the merged image of A (I), A (II), A (III), and A (IV). (B) represents the process of BBR entering the nucleus. The fluorescence of BBR is shown in green in this figure, and the red arrow points to the nuclear region. All of the images in (B) were taken under the same conditions with a time interval of 1.2 min. (C) represents the fluorescence intensity of BBR in the nucleus. * p<0.05, ** p<0.01 vs control (0 minutes after BBR administration). (D) represents the time-resolution change of berberine in live cells and the nucleus, which was detected by HPLC. (E) represents the chemical structure of BBR. Data were presented as mean ± S.D. from three independent experiments (n = 3).</p