Ethanol extract of Herpetospermum caudigerum Wall ameliorates psoriasis-like skin inflammation and promotes degradation of keratinocyte-derived ICAM-1 and CXCL9.

OBJECTIVE: To explore whether the ethanol extract of Herpetospermum caudigerum Wall (EHC), a Xizang medicinal plant traditionally used for treating liver diseases, can improve imiquimod-induced psoriasis-like skin inflammation. METHODS: Immunohistochemistry and immunofluorescence staining were used to determine the effects of topical EHC use in vivo on the skin pathology of imiquimod-induced psoriasis in mice. The protein levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-17A (IL-17A) in mouse skin samples were examined using immunohistochemical staining. In vitro, IFN-γ-induced HaCaT cells with or without EHC treatment were used to evaluate the expression of keratinocyte-derived intercellular cell adhesion molecule-1 (ICAM-1) and chemokine CXC ligand 9 (CXCL9) using Western blotting and reverse transcription-quantitative polymerase chain reaction. The protein synthesis inhibitor cycloheximide and proteasome inhibitor MG132 were utilized to validate the EHC-mediated mechanism underlying degradation of ICAM-1 and CXCL9. RESULTS: EHC improved inflammation in the imiquimod-induced psoriasis mouse model and reduced the levels of IFN-γ, TNF-α, and IL-17A in psoriatic lesions. Treatment with EHC also suppressed ICAM-1 and CXCL9 in epidermal keratinocytes. Further mechanistic studies revealed that EHC suppressed keratinocyte-derived ICAM-1 and CXCL9 by promoting ubiquitin-proteasome-mediated protein degradation rather than transcriptional repression. Seven primary compounds including ehletianol C, dehydrodiconiferyl alcohol, herpetrione, herpetin, herpetotriol, herpetetrone and herpetetrol were identified from the EHC using ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry. CONCLUSION: Topical application of EHC ameliorates psoriasis-like skin symptoms and improves the inflammation at the lesion sites. Please cite this article as: Zhong Y, Zhang BW, Li JT, Zeng X, Pei JX, Zhang YM, Yang YX, Li FL, Deng Y, Zhao Q. Ethanol extract of Herpetospermum caudigerum Wall ameliorates psoriasis-like skin inflammation and promotes degradation of keratinocyte-derived ICAM-1 and CXCL9. J Integr Med. 2023; 21(6): 584-592.

Enhancement of PPARα-Inhibited Leucine Metabolism-Stimulated ß-Casein Synthesis and Fatty Acid Synthesis in Primary Bovine Mammary Epithelial Cells.

Leucine, a kind of branched-chain amino acid, plays a regulatory role in the milk production of mammalian mammary glands, but its regulatory functions and underlying molecular mechanisms remain unknown. This work showed that a leucine-enriched mixture (LEUem) supplementation increased the levels of milk protein and milk fat synthesis in primary bovine mammary epithelial cells (BMECs). RNA-seq of leucine-treated BMECs indicated alterations in lipid metabolism, translation, ribosomal structure and biogenesis, and inflammatory response signaling pathways. Meanwhile, the supplementation of leucine resulted in mTOR activation and increased the expression of BCKDHA, FASN, ACC, and SCD1. Interestingly, the expression of PPARα was independently correlated with the leucine-supplemented dose. PPARα activated by WY-14643 caused significant suppression of lipogenic genes expression. Furthermore, WY-14643 attenuated leucine-induced ß-casein synthesis and enhanced the level of BCKDHA expression. Moreover, promoter analysis revealed a peroxisome-proliferator-response element (PPRE) site in the bovine BCKDHA promoter, and WY-14643 promoted the recruitment of PPARα onto the BCKDHA promoter. Together, the present data indicate that leucine promotes the synthesis of ß-casein and fatty acid and that PPARα-involved leucine catabolism is the key target.

Total alkaloids of Fritillaria unibracteata var. wabuensis bulbus ameliorate chronic asthma via the TRPV1/Ca2+/NFAT pathway.

BACKGROUND: Asthma is a chronic inflammatory disease that is challenging to treat. Fritillaria unibracteata var. wabuensis (FUW) is the plant origin for the famous Chinese antitussive medicine Fritillaria Cirrhosae Bulbus. The total alkaloids of Fritillaria unibracteata var. wabuensis bulbus (TAs-FUW) have anti-inflammatory properties and may be used to treat asthma. PURPOSE: To explore whether TAs-FUW have bioactivity against airway inflammation and a therapeutic effect on chronic asthma. METHODS: The alkaloids were extracted via ultrasonication in a cryogenic chloroform-methanol solution after ammonium-hydroxide percolation of the bulbus. UPLC-Q-TOF/MS was used to characterize the composition of TAs-FUW. An ovalbumin (OVA)-induced asthmatic mouse model was established. We used whole-body plethysmography, ELISA, western blotting, RT-qPCR, and histological analyses to assess the pulmonary pathological changes in these mice after TAs-FUW treatment. Additionally, TNF-α/IL-4-induced inflammation in BEAS-2B cells was used as an in vitro model, whereby the effects of various doses of TAs-FUW on the TRPV1/Ca2+-dependent NFAT-induced expression of TSLP were assessed. Stimulation and inhibition of TRPV1 receptors by capsaicin (CAP) and capsazepine (CPZ), respectively, were used to validate the effect of TAs-FUW. RESULTS: The UPLC-Q-TOF/MS analysis revealed that TAs-FUW mainly contain six compounds (peiminine, peimine, edpetiline, khasianine, peimisine, and sipeimine). TAs-FUW improved airway inflammation and obstruction, mucus secretion, collagen deposition, and leukocyte and macrophage infiltration, and downregulated TSLP by inhibiting the TRPV1/NFAT pathway in asthmatic mice. In vitro, the application of CPZ demonstrated that the TRPV1 channel is involved in TNF-α/IL-4-mediated regulation of TSLP. TAs-FUW suppressed TNF-α/IL-4-induced TSLP generation expression by regulating the TRPV1/Ca2+/NFAT pathway. Furthermore, TAs-FUW reduced CAP-induced TSLP release by inhibiting TRPV1 activation. Notably, sipeimine and edpetiline each were sufficient to block the TRPV1-mediated Ca2+ influx. CONCLUSION: Our study is the first to demonstrate that TNF-α/IL-4 can activate the TRPV1 channel. TAs-FUW can alleviate asthmatic inflammation by suppressing the TRPV1 pathway and thereby preventing the increase in cellular Ca2+ influx and the subsequent NFAT activation. The alkaloids in FUW may be used for complementary or alternative therapies in asthma.

Role of hyperbaric oxygen therapy in PDGF-BB-mediated astrogliosis in traumatic brain injury rats associated with ERK1/2 signaling pathway inhibition.

BACKGROUND: Hyperbaric oxygen (HBO) plays positive roles in the therapy of traumatic brain injury (TBI); however, the mechanism underlying its effects on TBI is largely unknown. The study aims to elucidate the molecular mechanism implicated with the interaction between platelet-derived growth factor-BB (PDGF-BB) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway, which may play critical roles during HBO treatment both in the astrocyte scratching model in vitro and rat TBI model in vivo. METHODS: Changes in neurological function and wound healing were evaluated using the neurological severity scores (NSS) scale, immunohistochemistry, western blotting, and qRT-PCR, respectively. RESULTS: The results showed that PDGF-BBi (PDGB interfered with small RNA) dramatically improves neuronal viability in vitro when transfected into the scratched astrocytes derived from the cerebral cortex of neonatal rats. Moreover, in vivo experiments revealed that HBO therapy substantially elevated the NSS scores and simultaneously reduced the mortality in TBI rats, as indicated by the NSS scales. Notably, HBO therapy was found to possess the ability to inhibit glial cell proliferation, promote the regeneration of neurons and synapses, and ultimately facilitate the wound healing, as revealed by immunohistochemistry and glial scar formation found in TBI rats. Importantly, HBO markedly decreased the expression levels of PDGF-BB and ERK1/2. It can clearly be seen that downregulated PDGF-BB and ERK1/2 levels were corresponding with the status of significant amelioration of the therapeutic effect of HBO. Conversely, the upregulation of PDGF-BB and ERK1/2 levels was in line with the opposite effect. CONCLUSION: It has been concluded that HBO therapy may play its active role in TBI treatment dependent on astrogliosis inhibition, which may be achieved by downregulating the ERK1/2 signaling pathway mediated by PDGF-BB.

Water extract from Herpetospermum pedunculosum attenuates oxidative stress and ferroptosis induced by acetaminophen via regulating Nrf2 and NF-κB pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: The seeds of Herpetospermum pedunculosum seeds is a traditional Tibetan medicine possessing hepatoprotective effect, but their protective effect on APAP-induced liver injury has not yet been explored. AIM OF THE STUDY: This study aimed at exploring the protective effect and mechanism of the water extract from the seeds of Herpetospermum pedunculosum (HPWE) on APAP-induced liver injury in vitro and in vivo. MATERIALS AND METHODS: In vitro and in vivo models of liver injury were established by APAP treatment of BRL-3A cells or mice. The effect and mechanism of action of HPWE were explored by using cell viability assay, ELISA, immunofluorescence assay, RT-qPCR, histological observation and immunohistochemistry staining, western blotting and high-content imaging system. RESULTS: In vitro experiments showed that HPWE treatment significantly promoted the cell viability, decreased ALT/AST level, and inhibited the ROS accumulation induced by APAP. Furthermore, HPWE and Fer-1 alleviated erastin-induced cell ferroptosis, upregulated GPX4 and SLC7A11 expression, and reduced lipid peroxides production. Further study showed that APAP could also downregulate the expression of GPX4 and SLC7A11, causing cell ferroptosis, and HPWE and Fer-1 counteracted this process. Our in vivo experiments showed that pretreatment with HPWE in APAP-treated mice significantly alleviated the serum ALT/AST level, decreased necrotic cells and inflammatory cell infiltration, upregulated the expression of GPX4 and SLC7A11. Further, it was demonstrated that HPWE treatment downregulated Nrf2 and its downstream target genes, i.e. HO-1 and NQO1 expression at the mRNA and protein levels. HPWE treatment also inhibited the activation of NF-κB p65 and downregulated its target genes, i.e. TNF-α and IL-1ß, expression. CONCLUSION: The present study showed that HPWE could relieve oxidative stress and ferroptosis via activating Nrf2 signaling pathway and inhibiting NF-κB mediated pathway.

Dietary folate drives methionine metabolism to promote cancer development by stabilizing MAT IIA.

Folic acid, served as dietary supplement, is closely linked to one-carbon metabolism and methionine metabolism. Previous clinical evidence indicated that folic acid supplementation displays dual effect on cancer development, promoting or suppressing tumor formation and progression. However, the underlying mechanism remains to be uncovered. Here, we report that high-folate diet significantly promotes cancer development in mice with hepatocellular carcinoma (HCC) induced by DEN/high-fat diet (HFD), simultaneously with increased expression of methionine adenosyltransferase 2A (gene name, MAT2A; protein name, MATIIα), the key enzyme in methionine metabolism, and acceleration of methionine cycle in cancer tissues. In contrast, folate-free diet reduces MATIIα expression and impedes HFD-induced HCC development. Notably, methionine metabolism is dynamically reprogrammed with valosin-containing protein p97/p47 complex-interacting protein (VCIP135) which functions as a deubiquitylating enzyme to bind and stabilize MATIIα in response to folic acid signal. Consistently, upregulation of MATIIα expression is positively correlated with increased VCIP135 protein level in human HCC tissues compared to adjacent tissues. Furthermore, liver-specific knockout of Mat2a remarkably abolishes the advocating effect of folic acid on HFD-induced HCC, demonstrating that the effect of high or free folate-diet on HFD-induced HCC relies on Mat2a. Moreover, folate and multiple intermediate metabolites in one-carbon metabolism are significantly decreased in vivo and in vitro upon Mat2a deletion. Together, folate promotes the integration of methionine and one-carbon metabolism, contributing to HCC development via hijacking MATIIα metabolic pathway. This study provides insight into folate-promoted cancer development, strongly recommending the tailor-made folate supplement guideline for both sub-healthy populations and patients with cancer expressing high level of MATIIα expression.

Physiological mechanisms by which gypsum increases the growth and yield of Lentinula edodes.

Lentinula edodes is one of the most important commercially cultivated edible mushrooms. It is well known that gypsum (CaSO4·2H2O) supplementation in sawdust medium increases the yield of L. edodes, while the physiological mechanisms remain unclear. Our previous study showed that the acidification of the medium to pH 3.5-4.0 was essential for the growth of L. edodes. In this study, it was found that the oxalic acid excreted by L. edodes was responsible for the acidification of the medium. The biosynthesis of oxalic acid was regulated by the ambient pH and buffer capacity of the medium. To acidify the sawdust medium, the concentrations of total and soluble oxalate were 51.1 mmol/kg and 10.8 mmol/kg, respectively. However, when the concentration of soluble oxalate was 8.0 mmol/kg, the mycelial growth rate decreased by 29% compared with the control. Soluble oxalate was toxic to L. edodes, while soluble sulfate was nontoxic. CaSO4 reacted with soluble oxalate to form nontoxic insoluble CaC2O4 and the strong acid H2SO4. When the CaSO4 supplemented in sawdust medium was more than 25 mmol/kg, the soluble oxalate decreased to less than 1 mmol/kg, and the mycelial growth rate increased by 32% compared with the control. In conclusion, gypsum improved the growth and yield by relieving the toxicity of oxalate and facilitating the acidification of sawdust medium. KEY POINTS: • L. edodes excretes oxalic acid to acidify the ambient environment for growth. • Soluble oxalate is toxic to L. edodes. • Gypsum increases growth by reacting with oxalate to relieve its toxicity.

Ginsenoside Rg1 improves Alzheimer's disease by regulating oxidative stress, apoptosis, and neuroinflammation through Wnt/GSK-3ß/ß-catenin signaling pathway.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder that can cause cognitive impairment. Ginsenoside Rg1 (Rg1) has a significant neuroprotective effect on animals with memory impairment. However, the mechanism of how Rg1 mediates the Wnt signaling pathway and improves cognitive function by regulating oxidative stress, apoptosis, and neuroinflammation is still unclear. In this study, the spatial memory ability of tree shrews was tested by Morris water maze, the expression levels of amyloid protein (Aß1-42), ionized calcium-binding adapter molecule 1 (iba-1), nitrotyrosine (NT), and 8-hydroxyguanine (8-OHG) were detected by immunohistochemistry. Subsequently, the activity of catalase (CAT) and the glutathione peroxidase (GSH-Px) was, respectively, measured by the ammonium molybdate method and the 5,5'-dithiobis (2-nitrobenzoic acid). Furthermore, the malondialdehyde (MDA) concentration was determined by the thiobarbituric acid test. Finally, the expression levels of Beta-secretase (BACE1), superoxide dismutase (SOD), BCL2-Associated X (Bax), B-cell lymphoma-2 (Bcl-2), caspase-anti-apoptotic factor Cleaved-caspase-3 (Caspase-3), microtubule-associated proteins 2 (MAP2), Neuronal nuclear antigen (NeuN), as well as the phosphorylation of GSK-3ß and ß-catenin were detected by Western blot. This study implied that Rg1 reduced the phosphorylation of Tau protein, the deposition of Aß1-42, and the expression of BACE1. It also showed that Rg1 increased the antioxidant activity of SOD, CAT, GPx, and instead reduced the oxidation products of NT, 8-OHG, and MDA, as wells as the inflammatory factor interleukin-1 and iba-1. It further showed that Rg1 increased the ratio of Bcl-2 to Bax and expression of neuronal markers MAP2 and NeuN, but instead reduced the expression of Caspase-3, GSK-3ß, and ß-catenin. In conclusion, by regulating the Wnt/GSK-3ß/ß-catenin signaling pathway, Rg1 of moderate and high dose could alleviate oxidative stress damage, improve neuroinflammation, protect neurons, finally improve the cognitive impairment of the AD tree shrew. This study provides theoretical basis for the Rg1 clinical application in AD.

Breviscapine exerts neuroprotective effects through multiple mechanisms in APP/PS1 transgenic mice.

Alzheimer's disease (AD) is one of the most serious neurodegenerative diseases and is characterized by progressive cognitive impairment and multiple neurological changes. To date, there are no effective drugs to delay or cure AD. Breviscapine (Bre) is an active ingredient of flavonoids extracted from breviscapus. Previous research suggests that Bre is an effective medicine for the prevention and treatment of AD. In the present study, we sought to explore the molecular mechanisms responsible for short-term beneficial effects of Breviscapine on Aß burden, neuronal and synaptic, cognitive function in APP/PS1 transgenic mice at 6 months age. Our results showed that 3 months of intraperitoneal treatment with Bre rescued learning deficits, relieved memory retention, improved the ability to explore the outside world, markedly decreased Aß burden, attenuated function of neocortical and hippocampal neuron, and increased the synaptic proteins levels in the brain of APP/PS1 mice by decreasing BACE1, promoting Aß-degrading enzyme IDE expression, suppressing RAGE expression, and regulating p38/p53/NT4 pathway. This finding provides more evidence of neuroprotective effects and action mechanisms of Bre antagonist AD, suggesting that Bre may have potential as anti-AD agent.

Immunogenicity of a plant-derived edible rotavirus subunit vaccine transformed over fifty generations.

Major efforts have been put forth for the development of effective rotavirus vaccines including transgenic plant vaccines. Previous studies have reported that rotavirus VP7 maintains its neutralizing immunity when it is transformed into the potato genome. The present study was aimed at investigating the hereditary stability of VP7-transformed potatoes over fifty generations. The VP7 gene was stably transcribed and expressed in potato cells as detected by RT-PCR and Western blotting. Humeral and mucosal responses were successfully induced in BALB/c mice fed with the fiftieth generation transformed potato tubers. There were no significant differences in serum IgG and fecal IgA between the mice fed with the first and fiftieth generation potatoes (P>0.05). Profiles of cytokines such as IFN-gamma, IL-2, IL-4, IL-5 and TGF-beta in immunized mice showed a naive T-cells bias to Th1 and Th3 polarization. Moreover, specific CTL responses were also detected in C57BL/6 mice fed with transformed potatoes. This research represents a significant step towards the development of rotavirus vaccines derived from a transgenic plant that can be obtained by long-term and large-scale vegetative reproduction. To our knowledge, this is the first finding regarding vaccines derived from plants that can be propagated for many generations.

Oral immunization with rotavirus VP7 expressed in transgenic potatoes induced high titers of mucosal neutralizing IgA.

Rotaviruses (RV) are a common cause of severe diarrhea in young children, resulting in nearly one million deaths worldwide annually. Rotavirus VP7 was the rotavirus neutralizing protein. Previous study reported that VP7 DNA vaccine can induce high levels of IgG in mice but cannot protect mice against challenge (Choi, A.H., Basu, M., Rae, M.N., McNeal, M.M., Ward, R.L., 1998. Virology 250, 230-240). We found that rotavirus VP7 could maintain its neutralizing immunity when it was transformed into the potato genome. Mice immunized with the transformed tubers successfully elicited serum IgG and mucosal IgA specific for VP7. The mucosal IgA titer was as high as 1000, while serum IgG titer was only 600. Neutralizing assays indicated that IgA could neutralize rotavirus. These results indicate the potential usefulness of plants for production and delivery of edible rotavirus vaccines.