Jiawei Dachaihu decoction protects against mitochondrial dysfunction in atherosclerosis (AS) mice with chronic unpredictable mild stress (CUMS) via SIRT1/PGC-1α/TFAM/LON signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: JiaWei DaChaiHu is composed of Bupleurum chinense, Scutellaria baicalensis, Pinellia ternata, Paeonia lactiflora, Zingiber officinaleRoscoe, Poncirus tuifoliata, Rheum palmatum L., Curcumae Radix, Herba Lysimachiae, Ziziphus. JiaWei DaChaiHu is one of the most common traditional Chinese medicines for the treatment of depression. AIM OF THE STUDY: The chronic unpredictable mild stress (CUMS) has been shown to promote atherosclerosis (AS). Dachaihu has been widely used in traditional Chinese medicine and has been known to exert distinct pharmacological effects. This investigation aims to examine the therapeutic effect of Jiawei Dachaihu extract on AS animal models with CUMS. METHODS: AS-CUMS mice model was established by Apoe-/- mice. Mice were treated with Jiawei Dachaihu. Serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C) levels were measured using ELISA kits. Aortic tissue pathologic changes detected by oil red O staining. Mice behavioral changes detected by sucrose preference test and sucrose preference test. The relative mRNA expression levels of CRH, ND1, and TFAM were determined by qRT-PCR. 5-HT1A, BDNF, LON, TFAM, PGC-1α, and SIRT1 protein expression determined by western blotting. ATP content detected by ATP kits. RESULTS: The treatment with Jiawei Dachaihu extract alleviated the veins plaque and reduced stress signs in vitro and in vivo. It increased the ATP and HDL-C levels while decreased the TC, TG, LDL-C levels. Jiawei Dachaihu extract treatment upregulated Lon, SIRT1, TFAM, PGC-1α, BDNF, and 5-HT1A protein expression and regained mitochondrial function. CONCLUSION: Jiawei Dachaihu extract could alleviate AS and reduce CUMS by upregulating the SIRT1/PGC-1α signaling and promoted its crosstalk with Lon protein to maintain mitochondrial stability.

Electroacupuncture improves peripheral neuropathy by up-regulating Sirt1/PGC-1α/TFAM pathway in type 2 diabetes rats with peripheral neuropathy. / 基于Sirt1/PGC-1α/TFAM通路探讨电针治疗2åž‹ç³–å°¿ç— å‘¨å›´ç¥žç»ç— å˜çš„æœºåˆ¶.

OBJECTIVES: To observe the effect of electroacupuncture (EA) on activation of silent information regulator 1 (Sirt1)/peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α)/mitochondrial transcription factor A (TFAM) pathway in type 2 diabetes (T2DM) rats with peripheral neuropathy (DPN) , so as to explore its possible mechanisms underlying improvement of DPN. METHODS: Thirty male SD rats were randomly divided into blank control group (n=8) and DPN model group (n=22) which were further divided into model group (n=8) and EA group (n=8) after successful modeling. The model of T2DM was established by high-fat diet and low-dose intraperitoneal injection of streptozocin (35 mg/kg). For rats of the EA group (anesthetized with isoflurane), EA stimulation (2 Hz/15 Hz, 2 mA) was applied to "Tianshu"(ST25) for 20 min, once daily, 6 times a week for 6 weeks. The blood glucose level, body weight, area under curve (AUC) of glucose tolerance test, and hind-paw mechanical pain threshold and thermal pain threshold were observed. The intra-epidermal nerve fiber density (IENFD) of the hind-foot pad was observed by immunofluorescence staining. The motor nerve conduction velocity (MNCV) of the sciatic nerve was measured by using electrophysiological method. H.E. staining was used to observe the histopathological changes of the sciatic nerve after modeling. Transmission electron microscopy (TEM) was used to observe the ultrastructural changes of the sciatic nerve. The protein expressions of energy-related Sirt1, PGC-1α and TFAM in the sciatic nerve was detected by Western blot. RESULTS: Compared with the blank control group, the model group had a higher blood glucose contents and AUC (P<0.001), a slower MNCV (P<0.01), and a decrease in the body weight and in the mechanical and thermal pain thresholds (P<0.001) and IENFD (P<0.001), and in the expression levels of Sirt1, PGC-1α and TFAM (P<0.05, P<0.01). In contrast to the model group, the EA group had a decrease in the blood glucose contents and AUC (P<0.05, P<0.01), and an increase in mechanical and thermal pain thresholds, MNCV, IENFD, and expression levels of Sirt1, PGC-1α and TFAM proteins (P<0.01, P<0.05). In addition, results of histopathological and ultrastructural changes of the sciatic nerve showed more fragmented and disordered distribution of axons on the transverse section, and extensive separation of myelin and axons, uneven myelin thickness, axonal degeneration and irregular shape in the model group, whereas in the EA group, the axons on the transverse section were relatively more dense and more complete, the myelin sheath of the sciatic nerve was relatively uniform, and the axonal shape was relatively regular with relatively milder lesions. CONCLUSIONS: EA up-regulates the expressions of Sirt1, PGC-1α, TFAM in T2DM rats with DPN, which may be associated with its functions in improving and repairing the injured peripheral nerves in rats with DPN.

Diterpenoid honatisine overcomes temozolomide resistance in glioblastoma by inducing mitonuclear protein imbalance through disruption of TFAM-mediated mtDNA transcription.

BACKGROUND: Glioblastoma (GBM) represents as the most formidable intracranial malignancy. The systematic exploration of natural compounds for their potential applications in GBM therapy has emerged as a pivotal and fruitful avenue of research. PURPOSE: In the present study, a panel of 96 diterpenoids was systematically evaluated as a repository of potential antitumour agents. The primary objective was to discern their potency in overcoming resistance to temozolomide (TMZ). Through an extensive screening process, honatisine, a heptacyclic diterpenoid alkaloid, emerged as the most robust candidate. Notably, honatisine exhibited remarkable efficacy in patient-derived primary and recurrent GBM strains. Subsequently, we subjected this compound to comprehensive scrutiny, encompassing GBM cultured spheres, GBM organoids (GBOs), TMZ-resistant GBM cell lines, and orthotopic xenograft mouse models of GBM cells. RESULTS: Our investigative efforts delved into the mechanistic underpinnings of honatisine's impact. It was discerned that honatisine prompted mitonuclear protein imbalance and elicited the mitochondrial unfolded protein response (UPRmt). This effect was mediated through the selective depletion of mitochondrial DNA (mtDNA)-encoded subunits, with a particular emphasis on the diminution of mitochondrial transcription factor A (TFAM). The ultimate outcome was the instigation of deleterious mitochondrial dysfunction, culminating in apoptosis. Molecular docking and surface plasmon resonance (SPR) experiments validated honatisine's binding affinity to TFAM within its HMG-box B domain. This binding may promote phosphorylation of TFAM and obstruct the interaction of TFAM bound to heavy strand promoter 1 (HSP1), thereby enhancing Lon-mediated TFAM degradation. Finally, in vivo experiments confirmed honatisine's antiglioma properties. Our comprehensive toxicological assessments underscored its mild toxicity profile, emphasizing the necessity for a thorough evaluation of honatisine as a novel antiglioma agent. CONCLUSION: In summary, our data provide new insights into the therapeutic mechanisms underlying honatisine's selective inducetion of apoptosis and its ability to overcome chemotherapy resistance in GBM. These actions are mediated through the disruption of mitochondrial proteostasis and function, achieved by the inhibition of TFAM-mediated mtDNA transcription. This study highlights honatisine's potential as a promising agent for glioblastoma therapy, underscoring the need for further exploration and investigation.

LncRNA GAS6-AS1 contributes to 5-fluorouracil resistance in colorectal cancer by facilitating the binding of PCBP1 with MCM3.

5-Fluorouracil (5-FU) resistance has always been a formidable obstacle in the adjuvant treatment of advanced colorectal cancer (CRC). In recent years, long non-coding RNAs have emerged as key regulators in various pathophysiological processes including 5-FU resistance. TRG is a postoperative pathological score of the chemotherapy effectiveness for CRC, of which TRG 0-1 is classified as chemotherapy sensitivity and TRG 3 as chemotherapy resistance. Here, RNA-seq combined with weighted gene correlation network analysis confirmed the close association of GAS6-AS1 with TRG. GAS6-AS1 expression was positively correlated with advanced clinicopathological features and poor prognosis in CRC. GAS6-AS1 increased the 50% inhibiting concentration of 5-FU, enhanced cell proliferation and accelerated G1/S transition, both with and without 5-FU, both in vitro and in vivo. Mechanistically, GAS6-AS1 enhanced the stability of MCM3 mRNA by recruiting PCBP1, consequently increasing MCM3 expression. Furthermore, PCBP1 and MCM3 counteracted the effects of GAS6-AS1 on 5-FU resistance. Notably, the PDX model indicated that combining chemotherapeutic drugs with GAS6-AS1 knockdown yielded superior outcomes in vivo. Together, our findings elucidate that GAS6-AS1 directly binds to PCBP1, enhancing MCM3 expression and thereby promoting 5-FU resistance. GAS6-AS1 may serve as a robust biomarker and potential therapeutic target for combination therapy in CRC.

Gastrodin alleviates mitochondrial dysfunction by regulating SIRT3-mediated TFAM acetylation in vascular dementia.

BACKGROUND: Mitochondrial dysfunction is key to the pathogenesis of vascular dementia (VaD). Sirtuin-3 (SIRT3), an essential member of the sirtuins family, has been proven to be a critical sirtuin in regulating mitochondrial function. The phenolic glucoside gastrodin (GAS), a bioactive ingredient from Gastrodiae Rhizome (known in Chinese as Tian ma) demonstrates significant neuroprotective properties against central nervous system disorders; however, the precise mechanisms through which GAS modulates VaD remain elusive. PURPOSE: This study aims to investigate whether GAS confers a protective role against VaD, and to figure out the underlying molecular mechanisms. METHODS: A bilateral common carotid artery occlusion (BCCAO)-mediated chronic cerebral hypoperfusion (CCH) VaD rat model and a hypoxia model using HT22 cells were employed to investigate pharmacological properties of GAS in mitigating mitochondrial dysfunction. A SIRT3 agonist resveratrol (RES), a SIRT3 inhibitor 3-TYP and SIRT3-knockdown in vitro were used to explore the mechanism of GAS in association with SIRT3. The ability of SIRT3 to bind and deacetylate mitochondrial transcription factor A (TFAM) was detected by immunoprecipitation assay, and TFAM acetylation sites were further validated using mass spectrometry. RESULTS: GAS increased SIRT3 expression and ameliorated mitochondrial structure, mitochondrial respiration, mitochondrial dynamics along with upregulated TFAM, mitigating oxidative stress and senescence. Comparable results were noted with the SIRT3 agonist RES, indicating an impactful neuroprotection played by SIRT3. Specifically, the attenuation of SIRT3 expression through knockdown techniques or exposure to the SIRT3 inhibitor 3-TYP in HT22 cells markedly abrogated GAS-mediated mitochondrial rescuing function. Furthermore, our findings elucidate a novel facet: SIRT3 interacted with and deacetylated TFAM at the K5, K7, and K8 sites. Decreased SIRT3 is accompanied by hyper-acetylated TFAM. CONCLUSION: The present results were the first to demonstrate that the SIRT3/TFAM pathway is a protective target for reversing mitochondrial dysfunction in VaD. The findings suggest that GAS-mediated modulation of the SIRT3/TFAM pathway, a novel mechanism, could ameliorate CCH-induced VaD, offering a potentially beneficial therapeutic strategy for VaD.

Molecular cloning, subcellular localization, and rapid recruitment to DNA damage sites of chicken Ku70.

Ku70 is a multifunctional protein with pivotal roles in DNA repair via non-homologous end-joining, V(D)J recombination, telomere maintenance, and neuronal apoptosis control. Nonetheless, its regulatory mechanisms remain elusive. Chicken Ku70 (GdKu70) cDNA has been previously cloned, and DT40 cells expressing it have significantly contributed to critical biological discoveries. GdKu70 features an additional 18 amino acids at its N-terminus compared to mammalian Ku70, the biological significance of which remains uncertain. Here, we show that the 5' flanking sequence of GdKu70 cDNA is not nearly encoded in the chicken genome. Notably, these 18 amino acids result from fusion events involving the NFE2L1 gene on chromosome 27 and the Ku70 gene on chromosome 1. Through experiments using newly cloned chicken Ku70 cDNA and specific antibodies, we demonstrated that Ku70 localizes within the cell nucleus as a heterodimer with Ku80 and promptly accumulates at DNA damage sites following injury. This suggests that the functions and spatiotemporal regulatory mechanisms of Ku70 in chickens closely resemble those in mammals. The insights and resources acquired will contribute to elucidate the various mechanisms by which Ku functions. Meanwhile, caution is advised when interpreting the previous numerous key studies that relied on GdKu70 cDNA and its expressing cells.

Increased iron uptake by splenic hematopoietic stem cells promotes TET2-dependent erythroid regeneration.

Hematopoietic stem cells (HSCs) are capable of regenerating the blood system, but the instructive cues that direct HSCs to regenerate particular lineages lost to the injury remain elusive. Here, we show that iron is increasingly taken up by HSCs during anemia and induces erythroid gene expression and regeneration in a Tet2-dependent manner. Lineage tracing of HSCs reveals that HSCs respond to hemolytic anemia by increasing erythroid output. The number of HSCs in the spleen, but not bone marrow, increases upon anemia and these HSCs exhibit enhanced proliferation, erythroid differentiation, iron uptake, and TET2 protein expression. Increased iron in HSCs promotes DNA demethylation and expression of erythroid genes. Suppressing iron uptake or TET2 expression impairs erythroid genes expression and erythroid differentiation of HSCs; iron supplementation, however, augments these processes. These results establish that the physiological level of iron taken up by HSCs has an instructive role in promoting erythroid-biased differentiation of HSCs.

Natural small-molecules reverse Xeroderma Pigmentosum Complementation Group C (XPC) deficient-mediated drug-resistance in renal cell carcinoma.

BACKGROUND: Renal cancer is insensitive to radiotherapy or most chemotherapies. While the loss of the XPC gene was correlated with drug resistance in colon cancer, the expression of XPC and its role in the drug resistance of renal cancer have not yet been elucidated. With the fact that natural small-molecules have been adopted in combinational therapy with classical chemotherapeutic agents to increase the drug sensitivity and reduce adverse effects, the use of herbal compounds to tackle drug-resistance in renal cancer is advocated. PURPOSE: To correlate the role of XPC gene deficiency to drug-resistance in renal cancer, and to identify natural small-molecules that can reverse drug-resistance in renal cancer via up-regulation of XPC. METHODS: IHC was adopted to analyze the XPC expression in human tumor and adjacent tissues. Clinical data extracted from The Cancer Genome Atlas (TCGA) database were further analysed to determine the relationship between XPC gene expression and tumor staging of renal cancer. Two types of XPC-KD renal cancer cell models were established to investigate the drug-resistant phenotype and screen XPC gene enhancers from 134 natural small-molecules derived from herbal plants. Furthermore, the identified XPC enhancers were verified in single or in combination with FDA-approved chemotherapy drugs for reversing drug-resistance in renal cancer using MTT cytotoxicity assay. Drug resistance gene profiling, ROS detection assay, immunocytochemistry and cell live-dead imaging assay were adopted to characterize the XPC-related drug resistant mechanism. RESULTS: XPC gene expression was significantly reduced in renal cancer tissue compared with its adjacent tissue. Clinical analysis of TCGA database also identified the downregulated level of XPC gene in renal tumor tissue of stage IV patients with cancer metastasis, which was also correlated with their lower survival rate. 6 natural small-molecules derived from herbal plants including tectorigenin, pinostilbene, d-pinitol, polygalasaponin F, atractylenolide III and astragaloside II significantly enhanced XPC expression in two renal cancer cell types. Combinational treatment of the identified natural compound with the treatment of FDA-approved drug, further confirmed the up-regulation of XPC gene expression can sensitize the two types of XPC-KD drug-resistant renal cancer cells towards the FDA-approved drugs. Mechanistic study confirmed that GSTP1/ROS axis was activated in drug resistant XPC-KD renal cancer cells. CONCLUSION: XPC gene deficiency was identified in patient renal tumor samples, and knockdown of the XPC gene was correlated with a drug-resistant phenotype in renal cancer cells via activation of the GSTP1/ROS axis. The 6 identified natural small molecules were confirmed to have drug sensitizing effects via upregulation of the XPC gene. Therefore, the identified active natural small molecules may work as an adjuvant therapy for circumventing the drug-resistant phenotype in renal cancer via enhancement of XPC expression.

StHsfB5 Promotes Heat Resistance by Directly Regulating the Expression of Hsp Genes in Potato.

With global warming, high temperatures have become a major environmental stress that inhibits plant growth and development. Plants evolve several mechanisms to cope with heat stress accordingly. One of the important mechanisms is the Hsf (heat shock factor)-Hsp (heat shock protein) signaling pathway. Therefore, the plant transcription factor Hsf family plays important roles in response to heat stress. All Hsfs can be divided into three classes (A, B, and C). Usually, class-A Hsfs are transcriptional activators, while class-B Hsfs are transcriptional repressors. In potato, our previous work identified 27 Hsfs in the genome and analyzed HsfA3 and HsfA4C functions that promote potato heat resistance. However, the function of HsfB is still elusive. In this study, the unique B5 member StHsfB5 in potato was obtained, and its characterizations and functions were comprehensively analyzed. A quantitative real-time PCR (qRT-PCR) assay showed that StHsfB5 was highly expressed in root, and its expression was induced by heat treatment and different kinds of phytohormones. The subcellular localization of StHsfB5 was in the nucleus, which is consistent with the characterization of transcription factors. The transgenic lines overexpressing StHsfB5 showed higher heat resistance compared with that of the control nontransgenic lines and inhibitory lines. Experiments on the interaction between protein and DNA indicated that the StHsfB5 protein can directly bind to the promoters of target genes small Hsps (sHsp17.6, sHsp21, and sHsp22.7) and Hsp80, and then induce the expressions of these target genes. All these results showed that StHsfB5 may be a coactivator that promotes potato heat resistance ability by directly inducing the expression of its target genes sHsp17.6, sHsp21, sHsp22.7, and Hsp80.

Effect of electroacupuncture intervention on the spinal cord PPIA/NF-κB signaling pathway in mice with amyotrophic lateral sclerosis. / ç”µé’ˆå¯¹è‚ŒèŽç¼©ä¾§ç´¢ç¡¬åŒ–ç—‡å°é¼ è„Šé«“PPIA/NF-κB信号通路的影响.

OBJECTIVES: To observe the effects of electroacupuncture (EA) on motor function, expression of extracellular cyclophile A(PPIA) and PPIA/nuclear factor-κB (NF-κB) signaling pathway in spinal cord of amyotrophic la-teral sclerosis (ALS) mice, so as to explore the mechanism of EA intervention in regulating extracellular PPIA on neuroinflammation in ALS mice. METHODS: Thirty ALS-SOD1G93A mice with hSOD1-G93A gene were randomly divided into model, EA and Riluzole groups , with 10 mice in each group, and other 10 ALS-SOD1G93A negative mice were used as the blank group. EA was applied to bilateral "Yanglingquan"(GB34) and "Zusanli"(ST36) for 20 min once daily, 5 days a week for 2 weeks. In the Riluzole group, riluzole solution (30 mg·kg-1·d-1) was administrated intragastrically, and the treatment time was the same as that in the EA group.Rotating rod experiment and open field experiment were used to evaluate the changes in motor function of mice .The morphology of motor neurons in the anterior horn of spinal cord was observed by HE staining.The relative protein expression levels of PPIA, TDP-43 and NF-κB in the spinal cord were detected by Western blot.The positive expression level of TDP-43 in the spinal cord was detected by immunohistochemistry. The positive expression level of PPIA in spinal cord was marked by immunofluorescence. Serum PPIA content was determined by ELISA. RESULTS: Compared with the blank group, the time of rod dropping and the total distance of open field movement in the model group were shortened (P<0.01), the number of motor neurons in the anterior horn of the spinal cord was reduced, the cell morphology was incomplete, the cell body was atrophied, the protein expression and positive expression of TDP-43 were increased (P<0.01), the protein expressions of PPIA and NF-κB in the spinal cord were increased(P<0.01), the serum content of PPIA and immunofluorescence expression of PPIA in spinal cord were increased (P<0.01). Compared with the model group, the time of rod dropping and the total distance of open field movement of mice in the EA group and the Riluzole group were prolonged (P<0.05, P<0.01), and the injury of motor neuron in the anterior horn of the spinal cord was decreased, the protein expression and positive expression of TDP-43 in the spinal cord were decreased (P<0.05, P<0.01)；the relative expression levels of PPIA and NF-κB proteins were decreased (P<0.05, P<0.01), and the content of PPIA in serum and the immunofluorescence expression of PPIA in the spinal cord were decreased (P<0.05, P<0.01) in the EA group；the relative protein expression of NF-κB and fluorescence expression of PPIA in spinal cord of mice in the Riluzole group were decreased (P<0.05). CONCLUSIONS: EA intervention can improve motor function in ALS mice, and its mechanism may be related to the inhibition of PPIA/NF-κB signaling pathway by EA to alleviating neuroinflammatory response.

ATAD2 is a driver and a therapeutic target in ovarian cancer that functions by upregulating CENPE.

Ovarian cancer is a complex disease associated with multiple genetic and epigenetic alterations. The emergence of treatment resistance in most patients causes ovarian cancer to become incurable, and novel therapies remain necessary. We identified epigenetic regulator ATPase family AAA domain-containing 2 (ATAD2) is overexpressed in ovarian cancer and is associated with increased incidences of metastasis and recurrence. Genetic knockdown of ATAD2 or its pharmacological inhibition via ATAD2 inhibitor BAY-850 suppressed ovarian cancer growth and metastasis in both in vitro and in vivo models. Transcriptome-wide mRNA expression profiling of ovarian cancer cells treated with BAY-850 revealed that ATAD2 inhibition predominantly alters the expression of centromere regulatory genes, particularly centromere protein E (CENPE). In ovarian cancer cells, changes in CENPE expression following ATAD2 inhibition resulted in cell-cycle arrest and apoptosis induction, which led to the suppression of ovarian cancer growth. Pharmacological CENPE inhibition phenotypically recapitulated the cellular changes induced by ATAD2 inhibition, and combined pharmacological inhibition of both ATAD2 and CENPE inhibited ovarian cancer cell growth more potently than inhibition of either alone. Thus, our study identified ATAD2 as regulators of ovarian cancer growth and metastasis that can be targeted either alone or in combination with CENPE inhibitors for effective ovarian cancer therapy.

Cangfu Daotan Wan alleviates polycystic ovary syndrome with phlegm-dampness syndrome via disruption of the PKP3/ERCC1/MAPK axis.

BACKGROUND/AIM: Cangfu Daotan Wan (CFDTW) has been widely used for polycystic ovary syndrome (PCOS) patients in the type of stagnation of phlegm and dampness. In this study, we aimed to evaluate the mechanism underlying the therapeutic effect of CFDTW on PCOS with phlegm-dampness syndrome (PDS). METHODS: In silico analysis was adopted to identify CFDTW potential targets and the downstream pathways in the treatment of PCOS. Expression of PKP3 was examined in the ovarian granulosa cells from PCOS patients with PDS and rat PCOS models induced by dehydroepiandrosterone (DHEA). PKP3/ERCC1 was overexpressed or underexpressed or combined with CFDTW treatment in ovarian granulosa cells to assay the effect of CFDTW on ovarian granulosa cell functions via the PKP3/MAPK/ERCC1 axis. RESULTS: Clinical samples and ovarian granulosa cells of rat models were characterized by hypomethylated PKP3 promoter and upregulated PKP3 expression. CFDTW reduced PKP3 expression by enhancing the methylation of PKP3 promoter, leading to proliferation of ovarian granulosa cells, increasing S and G2/M phase-arrested cells, and arresting their apoptosis. PKP3 augmented ERCC1 expression by activating the MAPK pathway. In addition, CFDTW facilitated the proliferation of ovarian granulosa cells and repressed their apoptosis by regulating PKP3/MAPK/ERCC1 axis. CONCLUSION: Taken together, this study illuminates how CFDTW confers therapeutic effects on PCOS patients with PDS, which may offer a novel theranostic marker in PCOS.

Shenqi formula delayed Alzheimer's disease-like symptoms by skn-1 pathway in Caernorhabditis elegans.

ETHNOPHARMACOLOGICAL RELEVANCE: Shenqi formula is composed of Codonopsis pilosula (Cp) and Lycium barbarum (Lb), and it is traditionally used for promoting qi and nourishing the spleen, liver and kidney. Cp and Lb have been reported to improve cognitive performance in APP/PS1 mice, prevent the accumulation of Aß, and reduce the neurotoxicity of Aß to achieve the anti-Alzheimer's disease (AD) effect. AIM OF THE STUDY: Shenqi formula was explored the therapeutic effect on Caenorhabditis elegans AD pathological model and the underlying mechanism of action. MATERIALS AND METHODS: Paralysis assay and serotonin sensitivity assay was used to detect whether Shenqi formula can alleviate AD paralysis phenotype, and then DPPH, ABTS, NBT and Fenton methods were applied to investigate the scavenging capacity to free radical, ROS, ·O2- and ·OH of Shenqi formula in vitro. H2DCF-DA and MitoSOX™ Red were employed to measure ROS and .O2- accumulation, respectively. RNAi was used to knock down the expression of skn-1 and daf-16 related to oxidative stress resistance signalling pathway. Fluorescence microscopy was used to record the expression of SOD-3::GFP, GST-4::GFP, SOD-1::YFP, and the nuclear translocation of SKN-1 and DAF-16. Western blot assay was carried out to test Aß monomers and oligomers. RESULTS: Shenqi formula delayed the AD-like pathological characteristics in C. elegans, and the complete Shenqi formula was more effective than Cp or Lb alone. The effect of Shenqi formula on delaying worm paralysis was partially eliminated by skn-1 RNAi, but not daf-16 RNAi. Shenqi formula significantly inhibited the abnormal deposition of Aß protein, decreased Aß protein monomers and oligomers. It increased the expressions of gst-4, sod-1, and sod-3 similar to paraquat, companied by rise then fall of ROS and .O2- in AD worms. CONCLUSIONS: Shenqi formula at least partially depended on SKN-1 signalling pathway to exert its anti-AD effect, and it is potential to be used as a kind of health food to prevent the progress of AD.

MYB-bHLH-TTG1 in a Multi-tiered Pathway Regulates Arabidopsis Seed Coat Mucilage Biosynthesis Genes Including PECTIN METHYLESTERASE INHIBITOR14 Required for Homogalacturonan Demethylesterification.

MYB-bHLH-TTG1 (MBW) transcription factor (TF) complexes regulate Arabidopsis seed coat biosynthesis pathways via a multi-tiered regulatory mechanism. The MYB genes include MYB5, MYB23 and TRANSPARENT TESTA2 (TT2), which regulate GLABRA2 (GL2), HOMEODOMAIN GLABROUS2 (HDG2) and TRANSPARENT TESTA GLABRA2 (TTG2). Here, we examine the role of PECTIN METHYLESTERASE INHIBITOR14 (PMEI14) in seed coat mucilage pectin methylesterification and provide evidence in support of multi-tiered regulation of seed coat mucilage biosynthesis genes including PMEI14. The PMEI14 promoter was active in the seed coat and developing embryo. A pmei14 mutant exhibited stronger attachment of the outer layer of seed coat mucilage, increased mucilage homogalacturonan demethylesterification and reduced seed coat radial cell wall thickness, results consistent with decreased PMEI activity giving rise to increased PME activity. Reduced mucilage release from the seeds of myb5, myb23, tt2 and gl2, hdg2, ttg2 triple mutants indicated that HDG2 and MYB23 play minor roles in seed coat mucilage deposition. Chromatin immunoprecipitation analysis found that MYB5, TT8 and seven mucilage pathway structural genes are directly regulated by MYB5. Expression levels of GL2, HDG2, TTG2 and nine mucilage biosynthesis genes including PMEI14 in the combinatorial mutant seeds indicated that these genes are positively regulated by at least two of those six TFs and that TTG1 and TTG2 are major regulators of PMEI14 expression. Our results show that MYB-bHLH-TTG1 complexes regulate mucilage biosynthesis genes, including PMEI14, both directly and indirectly via a three-tiered mechanism involving GL2, HDG2 and TTG2.

Integrated Omic Analysis Delineates Pathways Modulating Toxic TDP-43 Protein Aggregates in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a multi-systemic, incurable, amyloid disease affecting the motor neurons, resulting in the death of patients. The disease is either sporadic or familial with SOD1, C9orf72, FUS, and TDP-43 constituting the majority of familial ALS. Multi-omics studies on patients and model systems like mice and yeast have helped in understanding the association of various signaling and metabolic pathways with the disease. The yeast model system has played a pivotal role in elucidating the gene amyloid interactions. We carried out an integrated transcriptomic and metabolomic analysis of the TDP-43 expressing yeast model to elucidate deregulated pathways associated with the disease. The analysis shows the deregulation of the TCA cycle, single carbon metabolism, glutathione metabolism, and fatty acid metabolism. Transcriptomic analysis of GEO datasets of TDP-43 expressing motor neurons from mice models of ALS and ALS patients shows considerable overlap with experimental results. Furthermore, a yeast model was used to validate the obtained results using metabolite addition and gene knock-out experiments. Taken together, our result shows a potential role for the TCA cycle, cellular redox pathway, NAD metabolism, and fatty acid metabolism in disease. Supplementation of reduced glutathione, nicotinate, and the keto diet might help to manage the disease.

Cornus officinalis Seed Extract Inhibits AIM2-Inflammasome Activation and Attenuates Imiquimod-Induced Psoriasis-like Skin Inflammation.

The AIM2 inflammasome is an innate immune system component that defends against cytosolic bacteria and DNA viruses, but its aberrant activation can lead to the progression of various inflammatory diseases, including psoriasis. However, there have been few reports of specific inhibitors of AIM2 inflammasome activation. In this study, we aimed to investigate the inhibitory activity of ethanolic extracts of seeds of Cornus officinalis (CO), a herb and food plant used in traditional medicine, on AIM2-inflammasome activation. We found that CO inhibited the release of IL-1ß induced by dsDNA in both BMDMs and HaCaT cells, but that it showed no effect on the release of IL-1ß induced by NLRP3 inflammasome triggers, such as nigericin and silica, or the NLRC4 inflammasome trigger flagellin. Furthermore, we demonstrated that CO inhibited the cleavage of caspase-1, an inflammasome activation marker, and an upstream event, the translocation and speck formation of ASC. In addition, further experiments and mechanistic investigations revealed that CO can inhibit AIM2 speck formation induced by dsDNA in AIM2-overexpressing HEK293T cells. To verify the correlation in vivo, we investigated the efficacy of CO in an imiquimod (IMQ)-induced psoriasis model, which has reported associations with the AIM2 inflammasome. We found that topical application of CO alleviated psoriasis-like symptoms, such as erythema, scaling, and epidermal thickening, in a dose-dependent manner. Moreover, CO also significantly decreased IMQ-induced expression of AIM2 inflammasome components, including AIM2, ASC, and caspase-1, and led to the elevation of serum IL-17A. In conclusion, our results suggest that CO may be a valuable candidate for the discovery of AIM2 inhibitors and the regulation of AIM2-related diseases.

Electroacupuncture Ameliorates Depression-Like Behaviors Comorbid to Chronic Neuropathic Pain via Tet1-Mediated Restoration of Adult Neurogenesis.

Although electroacupuncture (EA) stimulation is a widely used therapy for chronic pain and comorbid psychiatric disorders, its long-term effects on chronic neuropathic pain-induced depression and the underlying mechanisms remain elusive. In the present study, we found that EA stimulation was able to restore adult neurogenesis in the ventral dentate gyrus (DG), by both increasing neuronal differentiation and restoring the normal morphology of newborn dendrites, in mice with spared nerve injury surgery. By ablating the Nestin+ neural stem cells (NSCs) via diphtheria toxin fragment A expression, we further proved that neurogenesis in the ventral DG was crucial to the long-term, but not the immediate antidepressant effect of EA, nor was it associated with nociception. Furthermore, we found that the restoration of neurogenesis was dependent on Tet1-mediated epigenetic modification upon EA treatment. Tet1 could bind to the promoter of the Prox1 gene, thus catalyzing its demethylation and facilitating its expression, which finally contributed to the restoration of neurogenesis and amelioration of depression-like behaviors induced by chronic neuropathic pain. Thus, we conclude that EA stimulation restores inhibited Tet1 expression in hippocampal NSCs of mice with chronic neuropathic pain, and increased Tet1 expression ameliorates hypermethylation of Prox1 and restores normal adult neurogenesis in the ventral DG, which contributes to the long-term antidepressant effect of EA.

Zhen Wu decoction represses renal fibrosis by invigorating tubular NRF2 and TFAM to fuel mitochondrial bioenergetics.

BACKGROUND: Zhen Wu Decoction (ZWD) is a prescription from the classical text "Treatise on Exogenous Febrile Disease" and has been extensively used to control kidney diseases since the time of the Eastern Han Dynasty. HYPOTHESIS: We hypothesized that ZWD limits tubular fibrogenesis by reinvigorating tubular bio-energetic capacity. STUDY DESIGN / METHODS: A mouse model of chronic kidney disease (CKD) was established using unilateral ureteral obstruction (UUO). Three concentrations of ZWD, namely 25.2 g/kg (high dosage), 12.6 g/kg (middle dosage), and 6.3 g/kg (low dosage), were included to study the dose-effect relationship. Real-time qPCR was used to observe gene transcription in blood samples from patients with CKD. Different siRNAs were designed to study the role of mitochondrial transcription factor A (TFAM) and nuclear factor (erythroid-derived 2)-related factor 2 (NRF2) in transforming growth factor (TGF)-ß1 induced fibrogenesis and mitochondrial damage. RESULTS: We showed that ZWD efficiently attenuates renal function impairment and reduces renal interstitial fibrosis. TFAM and NRF2 were repressed, and the stimulator of interferon genes (STING) was activated in CKD patient blood sample. We further confirmed that ZWD activated TFAM depended on NRF2 as an important negative regulator of STING in mouse kidneys. Treatment with ZWD significantly reduced oxidative stress and inflammation by regulating the levels of oxidative phosphorylation (OXPHOS) and pro-inflammatory factors, such as interleukin-6, interleukin-1ß, tumor necrosis factor receptor 1, and mitochondrial respiratory chain subunits. NRF2 inhibitors can weaken the ability of ZWD to increase TFAM expression and heal injured mitochondria, playing a similar role to that of STING inhibitors. Our study showed that ZWD elevates the expression of TFAM and mitochondrial respiratory chain subunits by promoting NRF2 activation, after suppressing mitochondrial membrane damage and cristae breakdown and restricting mitochondrial DNA (mtDNA) leakage into the cytoplasm to reduce STING activation. CONCLUSION: ZWD maintains mitochondrial integrity and improves OXPHOS which represents an innovative insight into "strengthening Yang-Qi" theory. ZWD limits tubular fibrogenesis by reinvigorating tubular bioenergetic capacity.

IL-17A and TNF synergistically drive expression of proinflammatory mediators in synovial fibroblasts via IκBζ-dependent induction of ELF3.

OBJECTIVE: IL-17A and TNF act in synergy to induce proinflammatory mediators in synovial fibroblasts thus contributing to diseases associated with chronic arthritis. Many of these factors are regulated by transcription factor E74-like factor-3 (ELF3). Therefore, we sought to investigate ELF3 as a downstream target of IL-17A and TNF signalling and to characterize its role in the molecular mechanism of synergy between IL-17A and TNF. METHODS: Regulation of ELF3 expression by IL-17A and TNF was studied in synovial fibroblasts of RA and OA patients and RA synovial explants. Signalling leading to ELF3 mRNA induction and the impact of ELF3 on the response to IL-17A and TNF were studied using siRNA, transient overexpression and signalling inhibitors in synovial fibroblasts and HEK293 cells. RESULTS: ELF3 was marginally affected by IL-17A or TNF alone, but their combination resulted in high and sustained expression. ELF3 expression was regulated by the nuclear factor-κB (NF-κB) pathway and CCAAT/enhancer-binding protein ß (C/EBPß), but its induction required synthesis of the NF-κB co-factor IκB (inhibitor of NF-κB) ζ. siRNA-mediated depletion of ELF3 attenuated the induction of cytokines and matrix metalloproteinases by the combination of IL-17A and TNF. Overexpression of ELF3 or IκBζ showed synergistic effect with TNF in upregulating expression of chemokine (C-C motif) ligand 8 (CCL8), and depletion of ELF3 abrogated CCL8 mRNA induction by the combination of IκBζ overexpression and TNF. CONCLUSION: Altogether, our results establish ELF3 as an important mediator of the synergistic effect of IL-17A and TNF in synovial fibroblasts. The findings provide novel information of the pathogenic mechanisms of IL-17A in chronic arthritis and implicate ELF3 as a potential therapeutic target.

G-rich motifs within phosphorothioate-based antisense oligonucleotides (ASOs) drive activation of FXN expression through indirect effects.

Friedreich's ataxia is an incurable disease caused by frataxin (FXN) protein deficiency, which is mostly induced by GAA repeat expansion in intron 1 of the FXN gene. Here, we identified antisense oligonucleotides (ASOs), complementary to two regions within the first intron of FXN pre-mRNA, which could increase FXN mRNA by ∼2-fold in patient fibroblasts. The increase in FXN mRNA was confirmed by the identification of multiple overlapping FXN-activating ASOs at each region, two independent RNA quantification assays, and normalization by multiple housekeeping genes. Experiments on cells with the ASO-binding sites deleted indicate that the ASO-induced FXN activation was driven by indirect effects. RNA sequencing analyses showed that the two ASOs induced similar transcriptome-wide changes, which did not resemble the transcriptome of wild-type cells. This RNA-seq analysis did not identify directly base-paired off-target genes shared across ASOs. Mismatch studies identified two guanosine-rich motifs (CCGG and G4) within the ASOs that were required for FXN activation. The phosphorodiamidate morpholino oligomer analogs of our ASOs did not activate FXN, pointing to a PS-backbone-mediated effect. Our study demonstrates the importance of multiple, detailed control experiments and target validation in oligonucleotide studies employing novel mechanisms such as gene activation.