Gegen Qinlian decoction ameliorates TNBS-induced ulcerative colitis by regulating Th2/Th1 and Tregs/Th17 cells balance, inhibiting NLRP3 inflammasome activation and reshaping gut microbiota.

ETHNOPHARMACOLOGICAL RELEVANCE: Chinese herbal medicine Gegen Qinlian Decoction (GQD) has been clinically shown to be an effective treatment of ulcerative colitis (UC) in China. However, the underlying mechanism of GQD's anti-ulcerative colitis properties and its effect on gut microbiota still deserve further exploration. AIM OF THE STUDY: This study observed the regulatory effects of GQD on Th2/Th1 and Tregs/Th17 cells balance, the NOD-like receptor family pyrin domain containing 3 (NLRP3) infammasome and gut microbiota in TNBS-induced UC in BALB/c mice. MATERIALS AND METHODS: 61 main chemical compounds in the GQD were determined by UPLC-Q-TOF/MS. The UC BALB/c model was established by intrarectal administration of trinitrobenzene sulfonic acid (TNBS), and GQD was orally administered at low and high dosages of 2.96 and 11.83 g/kg/day, respectively. The anti-inflammatory effects of GQD for ulcerative colitis were evaluated by survival rate, body weight, disease activity index (DAI) score, colonic weight and index, spleen index, hematoxylin-eosin (HE) staining and histopathological scores. Flow cytometry was used to detect the percentage of CD4, Th1, Th2, Th17 and Tregs cells. The levels of Th1-/Th2-/Th17-/Tregs-related inflammatory cytokines and additional proinflammatory cytokines (IL-1ß, IL-18) were detected by CBA, ELISA, and RT-PCR. The expressions of GATA3, T-bet, NLRP3, Caspase-1, IL-Iß, Occludin and Zonula occludens-1 (ZO-1) on colon tissues were detected by Western blot and RT-PCR. Transcriptome sequencing was performed using colon tissue and 16S rRNA gene sequencing was performed on intestinal contents. Fecal microbiota transplantation (FMT) was employed to assess the contribution of intestinal microbiota and its correlation with CD4 T cells and the NLRP3 inflammasome. RESULTS: GQD increased the survival rate of TNBS-induced UC in BALB/c mice, and significantly improved their body weight, DAI score, colonic weight and index, spleen index, and histological characteristics. The intestinal barrier dysfunction was repaired after GQD administration through promoting the expression of tight junction proteins (Occludin and ZO-1). GQD restored the balance of Th2/Th1 and Tregs/Th17 cells immune response of colitis mice, primarily inhibiting the increase in Th2/Th1 ratio and their transcription factor production (GATA3 and T-bet). Morever, GQD changed the secretion of Th1-/Th2-/Th17-/Tregs-related cytokines (IL-2, IL-12, IL-5, IL-13, IL-6, IL-10, and IL-17A) and reduced the expressions of IL-1ß, IL-18. Transcriptome results suggested that GQD could also remodel the immune inflammatory response of colitis by inhibiting NOD-like receptor signaling pathway, and Western blot, immunohistochemistry and RT-PCR further revealed that GQD exerted anti-inflammatory effects by inhibiting the NLRP3 inflammasome, such as down-regulating the expression of NLRP3, Caspase-1 and IL-1ß. More interestingly, GQD regulated gut microbiota dysbiosis, suppressed the overgrowth of conditional pathogenic gut bacteria like Helicobacter, Proteobacteria, and Mucispirillum, while the probiotic gut microbiota, such as Lactobacillus, Muribaculaceae, Ruminiclostridium_6, Akkermansia, and Ruminococcaceae_unclassified were increased. We further confirmed that GQD-treated gut microbiota was sufficient to relieve TNBS-induced colitis by FMT, involving the modulation of Th2/Th1 and Tregs/Th17 balance, inhibition of NLRP3 inflammasome activation, and enhancement of colonic barrier function. CONCLUSIONS: GQD might alleviate TNBS-induced UC via regulating Th2/Th1 and Tregs/Th17 cells Balance, inhibiting NLRP3 inflammasome and reshaping gut microbiota, which may provide a novel strategy for patients with colitis.

Maclurin inhibits caspase-11 non-canonical inflammasome in macrophages and ameliorates acute lethal sepsis in mice.

Maclurin is a natural phenolic compound isolated from Morus alba(white mulberry) andGarcinia mangostana (purple mangosteen) and has been reported to regulate cancer progression, oxidative stress, and melanogenesis. The regulatory role of maclurin, however, has never been demonstrated. This study investigated in vitro and in vivo anti-inflammatory roles of maclurin and the underlying mechanism in caspase-11 non-canonical inflammasome-stimulated inflammatory responses in macrophages and an animal model of acute lethal sepsis. Maclurin protected J774A.1 macrophages from LPS-induced cytotoxicity and suppressed caspase-11 non-canonical inflammasome-stimulated pyroptosis. Maclurin decreased the secretion and mRNA expression of pro-inflammatory cytokines and inflammatory mediators, such as IL-1ß, IL-18, TNF-α, IL-6, nitric oxide (NO), and inducible NO synthase (iNOS) in caspase-11 non-canonical inflammasome-stimulated J774A.1 macrophages. Mechanistic studies revealed that maclurin markedly suppressed the proteolytic activation of caspase-11 and gasdermin D (GSDMD) in caspase-11 non-canonical inflammasome-stimulated J774A.1 macrophages, while it did not inhibit caspase-11-mediated direct sensing of LPS. In vivo study revealed that maclurin ameliorated acute lethal sepsis in mice by increasing the survival rate and decreasing the serum levels of IL-1ß and IL-18 without significant toxicity. In conclusion, this study suggests that maclurin is a novel anti-inflammatory agent in inflammatory responses and against acute lethal sepsis via the inhibition of the caspase-11 non-canonical inflammasome in macrophages, which justifies its potential as an anti-inflammatory therapeutic agent in traditional medicine.

Inhibitory effect of Incarvillea diffusa Royle extract in the formation of calcium oxalate nephrolithiasis by regulating ROS-induced Nrf2/HO-1 pathway in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Calcium oxalate (CaOx) kidney stones are widely acknowledged as the most prevalent type of urinary stones, with high incidence and recurrence rates. Incarvillea diffusa Royle (ID) is a traditionally used medicinal herb in the Miao Minzu of Guizhou province, China, for treating urolithiasis. However, the active components and the underlying mechanism of its pharmacodynamic effects remain unclear. AIM OF THE STUDY: This study aimed to investigate the potential inhibitory effect of the active component of ID on the formation of CaOx nephrolithiasis and elucidate the underlying mechanism. MATERIALS AND METHODS: In vivo, a CaOx kidney stone model was induced in Sprague-Dawley (SD) rats using an ethylene glycol and ammonium chloride protocol for four weeks. Forty-eight male SD rats were randomly assigned to 6 groups (n = 8): blank group, model group, apocynin group, and low, medium, and high dose of ID's active component (IDW) groups. After three weeks of administration, rat urine, serum, and kidney tissues were collected. Renal tissue damage and crystallization, Ox, BUN, Ca2+, CRE, GSH, MDA, SOD contents, and levels of IL-1ß, IL-18, MCP-1, caspase-1, IL-6, and TNF-α in urine, serum, and kidney tissue were assessed using HE staining and relevant assay kits, respectively. Protein expression of Nrf2, HO-1, p38, p65, and Toll-4 in kidney tissues was quantified via Western blot. The antioxidant capacities of major compounds were evaluated through DPPH, O2·-, and ·OH radical scavenging assays, along with their effects on intracellular ROS production in CaOx-induced HK-2 cells. RESULTS: We found that IDW could significantly reduce the levels of CRE, GSH, MDA, Ox, and BUN, and enhancing SOD activity. Moreover, it could inhibit the secretion of TNF-α, IL-1ß, IL-18, MCP-1, caspase-1, and decreased protein expression of Nrf2, HO-1, p38, p65, and Toll-4 in renal tissue. Three major compounds isolated from IDW exhibited promising antioxidant activities and inhibited intracellular ROS production in CaOx-induced HK-2 cells. CONCLUSIONS: IDW facilitated the excretion of supersaturated Ca2+ and decreased the production of Ox, BUN in SD rat urine, and mitigated renal tissue damage by regulating Nrf2/HO-1 signaling pathway. Importantly, the three major compounds identified as active components of IDW contributed to the inhibition of CaOx nephrolithiasis formation. Overall, IDW holds significant potential for treating CaOx nephrolithiasis.

A novel mechanism for regulating lung immune homeostasis: Zukamu granules alleviated acute lung injury in mice by inhibiting NLRP3 inflammasome activation and regulating Th17/Treg cytokine balance.

ETHNOPHARMACOLOGICAL RELEVANCE: Acute lung injury (ALI) is a severely acute lung inflammation with high morbidity and mortality. Zukamu granules (ZKMG) is one of the Uygur patent drugs commonly used in clinic, which is included in the National Essential Drugs List (2018 edition). Clinical studies have shown that ZKMG has a significant effect on acute upper respiratory tract infection, and has better anti-inflammatory and antipyretic effects. However, the immunomodulatory mechanism of ZKMG on ALI is still not clear. AIM OF THE STUDY: The aim of this study is to investigate the lung protective effect and immunomodulatory mechanism of ZKMG on lipopolysaccharide (LPS) -induced ALI mice, and to provide an important basis for the treatment strategy and theoretical basis of ALI. MATERIALS AND METHODS: First, network pharmacology was used to predict the potential signaling pathways and biological processes of ZKMG related to immunology. Molecular docking technique was used to predict the possibility between the core components of ZKMG acting on NLRP3 protein. In addition, protein levels of F4/80 in lung tissues were assessed by Immunohistochemistry (IHC). The contents of IL-1ß, IL-18, IL-17A and IL-10 in the lung tissue and serum, MPO in the lung tissue were detected by enzyme-linked immunosorbent assay (ELISA). Real-time quantitative PCR analysis (RT-qPCR) was used to detect NLRP3 mRNA in lung tissue. Protein levels of NLRP3, Caspase-1, Cleaved caspase-1 p20, ASC, and GSDMD were detected by Western blot (WB). RESULTS: The results of network pharmacology showed that the immune pathways of ZKMG were mainly Th17 signaling pathway, IL-17 signaling pathway, NOD-like receptor signaling pathway, etc. Molecular docking results showed that the core components of ZKMG had good binding ability to NLRP3 protein. The verification experiments showed that ZKMG can reduce the degree of lung injury, and reduce the level of inflammatory infiltration of neutrophils and macrophages by reducing the content of MPO and F4/80. In addition, ZKMG can reduce NLRP3 mRNA, inhibit the expression of NLRP3/Caspase-1/GSDMD and other related pathway proteins, and reduce inflammatory factors such as IL-1ß and IL-18. It can also reduce the content of pro-inflammatory cytokine IL-17A, increase the content of anti-inflammatory cytokine IL-10 in lung tissue. CONCLUSION: ZKMG can reduce the degree of lung tissue injury in ALI by inhibiting NLRP3/Caspase-1/GSDMD signaling pathway and restoring the IL-17A/IL-10 cytokine balance, and its protective mechanism may be related to the regulation of lung immune homeostasis. It will provide a new strategy for studying the regulation of lung immune homeostasis.

Tryptophan alleviates chronic heat stress-induced impairment of antioxidant capacities, inflammatory response, and mitochondrial function in broilers.

The aim of this study was to investigate the effect of dietary tryptophan (Trp) supplementation on serum biochemical indices, antioxidant indices, cytokine levels, mitochondrial biosynthesis, and mitochondrial morphology of heat-stressed broilers. A total of 180 female Arbor Acres broilers (18-day-old) were randomly allocated into three groups with six replicates of 10 broilers each. Broilers in thermoneutral (TN) (23 ± 1 °C) group were fed a basal diet; the other two groups were fed the basal diet supplemented with 0 or 0.18% Trp under heat stress (HS) (34 ± 1 °C for 8 h/day (h/day) and 23 ± 1°C for the remaining time) condition. The heat stress lasted for 21 days (days 21 to 42). The results indicated that heat stress reduced serum total protein content (TP) and decreased the activities of serum superoxide dismutase (SOD) and total antioxidant capacity (T-AOC), but increased the levels of serum uric acid (UA), interleukin (IL)-1ß, IL-6, and IL-18 (P < 0.05) compared to the TN group. However, dietary supplementation with 0.18% Trp enhanced serum TP content, glutathione peroxidase (GSH-Px), SOD, catalase (CAT) activities, and T-AOC; decreased aspartate aminotransferase (AST) activities (P < 0.05); and lowered serum IL-1ß, IL-6, IL-18 contents (P < 0.05). Meanwhile, heat stress exposure downregulated the mRNA expression of mitochondrial transcription factor A (TFAM), cytochrome c oxidase subunit 1 (COX1), and cytochrome c oxidase subunit 5A (COX5A) in ileum (P < 0.05) as compared to the TN group. Dietary Trp supplementation enhanced the mitochondrial membrane potential (MMP) and the mRNA expression of TFAM, COX1 in ileum mucosa (P < 0.05) and ameliorated the damage of mitochondrial structure. Collectively, dietary supplementation with Trp could improve antioxidant capacity and mitochondrial structure and regulate mitochondrial function-related genes and decrease inflammatory response in heat-stressed broilers. Dietary Trp supplementation might be an effective nutritional strategy to protect against heat stress impairment.

Effect of Shao's five-needle therapy pretreatment on airway inflammatory response in asthmatic rats based on ROS/TXNIP/NLRP3 pathway. / 基于ROS/TXNIP/NLRP3通路探讨"邵氏五针法"é¢„å¤„ç†å¯¹å“®å–˜å¤§é¼ æ°”é“ç‚Žæ€§ååº”çš„å½±å“.

OBJECTIVES: To explore the possible mechanism of Shao's five-needle therapy pretreatment on relieving airway inflammatory response in asthmatic rats. METHODS: Forty SPF-grade SD rats were randomly divided into a blank group, a model group, an acupuncture group, and a medication group, with 10 rats in each group. Except the blank group, asthma model was established by aerosol inhalation of ovalbumin in the other 3 groups. The rats in the acupuncture group were treated with acupuncture at "Dazhui" (GV 14) and bilateral "Feishu" (BL 13) and "Fengmen" (BL 12), with each session lasting for 20 min. Acupuncture was given before each motivating, once daily for 7 consecutive days. The rats in the medication group were treated with intraperitoneal injection of dexamethasone sodium phosphate solution before each motivating, once daily for 7 days. General situation of the rats was observed in each group; ELISA method was used to detect the levels of inflammatory cytokines interleukin (IL)-1ß and IL-18 in serum; immunofluorescence staining method was performed to assess the expression of reactive oxygen species (ROS) in lung tissues; Western blot method was used to measure the protein expression of thioredoxin interacting protein (TXNIP), NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), and Caspase-1 in lung tissues. RESULTS: The rats in the blank group exhibited normal behavior, while those in the model group showed signs of respiratory distress, ear scratching, cheek rubbing, and dysphoria. Compared with the model group, the rats in the acupuncture group and the medication group showed stable respiration and relatively agile responses. Compared with those in the blank group, the serum levels of IL-18 and IL-1ß were elevated (P<0.01), the expression intensity of ROS was increased, and the protein expressions of TXNIP, NLRP3, ASC and Caspase-1 in lung tissues were increased (P<0.01) in the model group. Compared with those in the model group, the serum levels of IL-18 and IL-1ß were reduced (P<0.01), the expression intensity of ROS was lowered, and the protein expressions of TXNIP, NLRP3, ASC and Caspase-1 in lung tissues were reduced (P<0.01) in the acupuncture group and the medication group. Compared with the medication group, the protein expression of ASC in lung tissue was reduced in the acupuncture group (P<0.05). CONCLUSIONS: Pretreatment of Shao's five-needle therapy could alleviate airway inflammatory response in asthmatic rats by reducing ROS levels and decreasing the aggregation and activation of pathway-related proteins in the ROS/TXNIP/NLRP3 pathway, ultimately leading to decreased secretion of IL-1ß and IL-18. This mechanism may contribute to the effectiveness of Shao's five-needle therapy in preventing and treating asthma.

[Terpinen-4-ol inhibits proliferation of VSMCs exposed to high glucose via regulating KLF4/NF-κB signaling pathway].

This study aimed to observe the effect of terpinen-4-ol(T4O) on the proliferation of vascular smooth muscle cells(VSMCs) exposed to high glucose(HG) and reveal the mechanism via the Krüppel-like factor 4(KLF4)/nuclear factor kappaB(NF-κB) signaling pathway. The VSMCs were first incubated with T4O for 2 h and then cultured with HG for 48 h to establish the model of inflammatory injury. The proliferation, cell cycle, and migration rate of VSMCs were examined by MTT method, flow cytometry, and wound healing assay, respectively. The content of inflammatory cytokines including interleukin(IL)-6 and tumor necrosis factor-alpha(TNF-α) in the supernatant of VSMCs was measured by enzyme-linked immunosorbent assay(ELISA). Western blot was employed to determine the protein levels of proliferating cell nuclear antigen(PCNA), Cyclin D1, KLF4, NF-κB p-p65/NF-κB p65, IL-1ß, and IL-18. The KLF4 expression in VSMCs was silenced by the siRNA technology, and then the effects of T4O on the cell cycle and protein expression of the HG-induced VSMCs were observed. The results showed that different doses of T4O inhibited the HG-induced proliferation and migration of VSMCs, increased the percentage of cells in G_1 phase, and decreased the percentage of cells in S phase, and down-regulated the protein levels of PCNA and Cyclin D1. In addition, T4O reduced the HG-induced secretion and release of the inflammatory cytokines IL-6 and TNF-α and down-regulated the expression of KLF4, NF-κB p-p65/NF-κB p65, IL-1ß, and IL-18. Compared with si-NC+HG, siKLF4+HG increased the percentage of cells in G_1 phase, decreased the percentage of cells in S phase, down-regulated the expression of PCNA, Cyclin D1, and KLF4, and inhibited the activation of NF-κB signaling pathway. Notably, the combination of silencing KLF4 with T4O treatment further promoted the changes in the above indicators. The results indicate that T4O may inhibit the HG-induced proliferation and migration of VSMCs by down-regulating the level of KLF4 and inhibiting the activation of NF-κB signaling pathway.

[Effect of multi-glycosides of Tripterygium wilfordii on renal injury in diabetic kidney disease rats through NLRP3/caspase-1/GSDMD pyroptosis pathway].

This study investigated the effect of multi-glycosides of Tripterygium wilfordii(GTW) on renal injury in diabetic kidney disease(DKD) rats through Nod-like receptor protein 3(NLRP3)/cysteine-aspartic acid protease-1(caspase-1)/gsdermin D(GSDMD) pyroptosis pathway and the mechanism. To be specific, a total of 40 male SD rats were randomized into the normal group(n=8) and modeling group(n=34). In the modeling group, a high-sugar and high-fat diet and one-time intraperitoneal injection of streptozotocin(STZ) were used to induce DKD in rats. After successful modeling, they were randomly classified into model group, valsartan(Diovan) group, and GTW group. Normal group and model group were given normal saline, and the valsartan group and GTW group received(ig) valsartan and GTW, respectively, for 6 weeks. Blood urea nitrogen(BUN), serum creatinine(Scr), alanine ami-notransferase(ALT), albumin(ALB), and 24 hours urinary total protein(24 h-UTP) were determined by biochemical tests. The pathological changes of renal tissue were observed based on hematoxylin and eosin(HE) staining. Serum levels of interleukin-1ß(IL-1ß) and interleukin-18(IL-18) were detected by enzyme-linked immunosorbent assay(ELISA). Western blot was used to detect the expression of pyroptosis pathway-related proteins in renal tissue, and RT-PCR to determine the expression of pyroptosis pathway-related genes in renal tissue. Compared with the normal group, the model group showed high levels of BUN, Scr, ALT, and 24 h-UTP and serum levels of IL-1ß and IL-18(P<0.01), low level of ALB(P<0.01), severe pathological damage to kidney, and high protein and mRNA levels of NLRP3, caspase-1, and GSDMD in renal tissue(P<0.01). Compared with the model group, valsartan group and GTW group had low levels of BUN, Scr, ALT, and 24 h-UTP and serum levels of IL-1ß and IL-18(P<0.01), high level of ALB(P<0.01), alleviation of the pathological damage to the kidney, and low protein and mRNA levels of NLRP3, caspase-1, and GSDMD in renal tissue(P<0.01 or P<0.05). GTW may inhibit pyroptosis by decreasing the expression of NLRP3/caspase-1/GSDMD in renal tissue, thereby relieving the inflammatory response of DKD rats and the pathological injury of kidney.

["Trichosanthis Fructus-Allii Macrostemonis Bulbus" combination inhibits NLRP3 inflammasome activation and reduces inflammatory response in RAW264.7 macrophage by inducing autophagy].

This study aims to explore the effect of "Trichosanthis Fructus-Allii Macrostemonis" combination(GX) on the activation of NOD-, LRR-, and pyrin domain-containing protein 3(NLRP3) inflammasome, the release of inflammatory cytokines, and the level of autophagy in RAW264.7 macrophage damaged by lipopolysaccharide(LPS), and the mechanism of GX against inflammatory response in macrophages. To be specific, LPS was used to induce the injury of RAW264.7 cells. Cell Counting Kit-8(CCK-8) assay was employed to measure the survival rate of cells, and Western blot to detect the protein expression of NLRP3, apoptosis-associated speck-like protein(ASC), cysteine-aspartic acid protease(caspase)-1, interleukin(IL)-18, IL-1ß, microtubule-associated protein light chain 3(LC3)-Ⅱ, and selective autophagy junction protein p62/sequestosome 1 in RAW264.7 macrophages. ELISA was used to measure the levels of IL-18 and IL-1ß in RAW264.7 cells. Transmission electron microscopy was applied to observe the number of autophagosomes in RAW264.7 cells. Immunofulourescence staining was used to detect the expression of LC3-Ⅱ and p62 in RAW264.7 cells. The result showed that GX significantly reduced the protein expression of NLRP3, ASC, and caspase-1 in RAW264.7 cells, significantly increased the protein expression of LC3Ⅱ, decreased the expression of p62, significantly inhibited the secretion of IL-18 and IL-1ß, significantly increased the number of autophagosomes, significantly enhanced the immunofluorescence of LC3Ⅱ, and reduced the immunofluorescence of p62. Furthermore, 3-methyladenine(3-MA) could reverse the inhibitory effect of GX on NLRP3, ASC, and caspase-1 and reduce the release of IL-18 and IL-1ß. In summary, GX can increase of the autophagy activity of RAW264.7 and inhibit the activation of NLRP3 inflammasome, thereby reducing the release of inflammatory cytokines and suppressing inflammatory response in macrophages.

Tibetan medicine Qi-Sai-Er-Sang-Dang-Song Decoction inhibits TNF-α-induced rheumatoid arthritis in human fibroblast-like synoviocytes via regulating NOTCH1/NF-κB/NLRP3 pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Qi-Sai-Er-Sang-Dang-Song Decoction (QSD, ཆུ་སེར་སེང་ལྡེང་སུམ་ཐང་།), a Tibetan classical herbal formula, is commonly used in Tibetan hospital preparation for the treatment of rheumatoid arthritis (RA). Its efficacy is to relieve inflammation, dispel cold, remove dampness, and alleviate pain. However, its anti-RA mechanism is still unclear. AIM OF THE STUDY: This study aimed to investigate the effect of QSD on rheumatoid arthritis and explore its anti-inflammatory mechanism against human fibroblast-like synoviocytes (HFLSs) by regulating the notch family of receptors (NOTCH1)/Nuclear factor-κB (NF-κB)/nucleotide-binding (NLRP3) pathway. MATERIALS AND METHODS: We used ultra-performance liquid chromatography coupled with Q-TOF mass spectrometry (UPLC-Q-TOF-MS) to identify the chemical composition of QSD. Then, HFLSs were exposed to drug-containing serum. The effect of QSD drug-containing serum on HFLS viability was detected using the cell counting kit-8 (CCK-8) assay. Next, we explored the anti-inflammatory effect of QSD using enzyme-linked immunosorbent assay (ELISA) for inflammatory factors, such as interleukin-18 (IL-18), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6). The expression of NOTCH-related proteins, a member of the NOTCH1, Cleaved NOTCH1, hairy and enhancer of split-1 (HES-1), NF-κB p65, NF-κB pp65, NLRP3, and delta-like 1 (DLL-1), was examined using western blotting. Furthermore, the relative mRNA expression levels of NOTCH1, NF-κB p65, NLRP3, DLL-1, and HES-1 were detected using real-time quantitative (RT-qPCR). To explore the mechanism underlying the anti-RA effect of QSD, we the used the NOTCH signaling pathway inhibitor LY411575 and transfection with a NOTCH1 siRNA. In addition, we employed immunofluorescence to determine the expression of HES-1 and NF-κB p65 in vitro. RESULT: Our results revealed that QSD ameliorated inflammation in HFLSs. Compared with the model group, the QSD drug-containing serum group had obviously down-regulated levels of IL-18, IL-1ß, and IL-6. Consistently, the CCK-8 results showed that the QSD drug-containing serum had no obvious toxicity towards HFLSs. Moreover, both LY411575 and siNOTCH1, QSD could reduce NOTCH1, NLRP3, and HES-1 protein expression levels, and LY411575 could significantly inhibit the expression levels of NF-κB p65, NF-κB pp65, and Cleaved NOTCH1 (p < 0.05). siNOTCH1 could also suppress the expression of DLL-1. The RT-qPCR results indicated that QSD could downregulate the relative mRNA expression levels of NOTCH1, NF-κB p65, NLRP3, DLL-1, and HES-1 in HFLSs (p < 0.05). In the immunofluorescence experiment, the fluorescence intensities of HES-1 and NF-κB p65 in HFLSs were found to decrease after exposure to QSD drug-containing serum (p < 0.05). Ultimately, 44 chemical components were detected in QSD using UPLC-Q-TOF-MS. CONCLUSION: This study reveals that the QSD can markedly ameliorate inflammation induced by TNF-α on HFLS. The effect of QSD on HFLS may be exerted by inhibition of the NOTCH1/NF-κB/NLRP3 signaling pathway.

GPX4 utilization by selenium is required to alleviate cadmium-induced ferroptosis and pyroptosis in sheep kidney.

Cadmium (Cd), a persistent and harmful heavy metal in the environment, can accumulate in the kidneys and cause nephrotoxicity. Selenium (Se) is a beneficial natural element that alleviates the toxicity of Cd. To ascertain the relationship between the protective mechanism of Se against Cd nephrotoxicity and ferroptosis and pyroptosis, we randomly divided 48 sheep into four groups and treated them with Cd chloride and/or sodium selenite for 50 days. The data confirmed that Cd apparently resulted in impaired kidney histology and function, depletion of GSH and nicotinamide adenine dinucleotide phosphate contents and CAT and SOD activities, elevation of MDA level, as well as the reduction in selenoprotein mRNA (GPX1, GPX4, TXNRD1, SELP) levels and GPX4 protein level and immunofluorescence intensity. Meanwhile, Cd induced ferroptosis by causing iron overload, up-regulating PTGS2, NCOA4, TFR1, and LC3B mRNA levels and PTGS2 and LC3B-II/LC3B-I protein levels, reducing SLC7A11 and FTH1 mRNA and protein levels, and enhancing the immunofluorescence co-localization of FTH1/LC3B. Moreover, it was also found that Cd triggered pyroptosis, which was evidenced by the increase of NLRP3 immunohistochemical positive signal, GSDMD-N immunofluorescence intensity, IL-1ß and IL-18 release and the levels of pyroptosis-related mRNA (NLRP3, ASC, Caspase-1, GSDMD, IL-1ß and IL-18) and proteins (NLRP3, Caspase-1p20, GSDMD-N, IL-1ß and IL-18). Notably, Se increased the expression level of GPX4 and the transcription factors TFAP2c and SP1, and ameliorated Cd-induced changes in aforementioned factors. In conclusion, GPX4 utilization by Se might be required to alleviate Cd-induced ferroptosis and pyroptosis in sheep kidney.

Exploring the effect of Er miao San-containing serum on macrophage polarization through miR-33/NLRP3 pathway.

HEADINGS ETHNOPHARMACOLOGICAL RELEVANCE: Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease. Er miao San (EMS) has been shown to have good anti-inflammatory effects and is widely used in the clinical treatment of RA. However, the exact mechanism is not completely understood. AIM OF THE STUDY: The aim of this study was to explore that EMS-containing serum affects M1/M2 polarization of macrophages and may be mediated through the microRNA (miRNA)-33/NLRP3 pathway, thereby elucidating the molecular mechanism of EMS treatment of RA. MATERIALS AND METHODS: We screened for safe concentrations of EMS-containing serum by using CCK-8 measurement. RAW264.7 cells were cultured with lipopolysaccharide (LPS) (100 ng/mL) and interferon-γ (20 ng/mL) for 24 h to induce M1-type macrophages. Adenosine triphosphate (ATP) (5 mM) was added in the last 30 min to activate NLRP3. The content of miR-33 was detected by RT‒qPCR after transfection of the miRNA-33 mimic. The protein expression levels of NLRP3, ASC, caspase-1, Inducible Nitric Oxide Synthase (iNOS) and Arginase-1 (Arg-1) were detected by Western blot. The contents of IL-1ß, IL-10, TNF-α, TGF-ß and IL-18 in serum and cell supernatant were determined by ELISA. The fluorescence intensity of CD86 and CD206 was detected by immunofluorescence. RESULTS: The results showed that EMS-containing serum promoted the protein expression level of Arg-1 and the secretion levels of TGF-ß and IL-10, inhibited the levels of iNOS, IL-1ß and TNF-α, and regulated the balance of pro-inflammatory factors and anti-inflammatory factors. RT‒qPCR results showed that EMS-containing serum could reduce the level of miRNA-33. EMS-containing serum could reduce the expression of NLRP3 inflammasome-related proteins and downregulate the expression levels of IL-1ß and IL-18. These results suggest that EMS exerts its effect on macrophage polarization through the miRNA-33/NLRP3 pathway. CONCLUSION: EMS-containing serum inhibits the activation of the NLRP3 inflammasome by downregulating miRNA-33, thus preventing the polarization of M1-type macrophages.

Effects of n-butanol extract of Pulsatilla decoction on the NLRP3 inflammasome in macrophages infected with Candida albicans.

ETHNOPHARMACOLOGICAL RELEVANCE: Pulsatilla decoction is a traditional Chinese medicine from Shang Han Lun that has been reported to have therapeutic efficacy in vulvovaginal candidiasis (VVC), and is a growth inhibitor of Candida albicans (C. albicans) in vitro, the causative agent of VVC. AIM OF THE STUDY: In previous studies, Pulsatilla decoction has shown therapeutic benefits for VVC. With further chemical extraction of the decoction, the n-butanol extract (of Pulsatilla decoction; BEPD) was most effective against C. albicans and therapeutic for VVC. The mechanism, however, has not been elucidated. The regulation of NOD-like receptor protein 3 (NLRP3) inflammasome has recently been demonstrated as a critical component of the inflammasome complex that initiates the vaginal inflammatory response. Therefore, the effect of BEPD on NLRP3 associated with VVC was investigated. MATERIALS AND METHODS: Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for detecting the principal compounds of BEPD (Anemoside B4, Esculin, esculetin, Epiberberine, Berberine, Jatrorrhizine and Phellodendrine). BEPD-containing serum is prepared by intragastric administration of BEPD (4.6875 g/kg for seven days) in rats. PMA-induced THP-1 cells were challenged with C. albicans. The expression of CD68 to identify macrophages was examined by flow cytometry, the viability of THP-1 cells were assessed by CCK8 assay, the release of lactate dehydrogenase (LDH) was detected by LDH kit, and the secretion levels of IL-1ß and IL-18 were evaluated through enzyme-linked immunosorbent assay (ELISA), the levels of NLRP3 were quantified by immunofluorescence, the levels of reactive oxygen species (ROS) were measured by ROS kit, and the expression of Dectin-1, Syk, TLR2, TLR4, MyD88, NF-κB, NLRP3, Caspase-1, and ASC proteins were detected by Western blot. RESULTS: After administration of BEPD-containing serum, the levels of IL-1ß, IL-18 and LDH released from macrophages were reduced in the BEPD-containing serum group compared to the model group. In addition, BEPD-containing serum inhibited the expression of ROS in macrophages, suppressed the expression of NLRP3 and inhibited the expression of TLRs/MyD88 and Dectin-1/Syk signaling pathway-related proteins. CONCLUSIONS: BEPD suppressed the NLRP3 inflammasome and related signaling pathways in macrophages infected with C. albicans in vitro, thereby providing insight into the mechanism of BEPD action on VVC.

Effects of Saccharomyces cerevisiae boulardii (CNCM I-1079) on feed intake, blood parameters, and production during early lactation.

The periparturient period is a metabolically demanding time for dairy animals because of increased nutrient requirements for milk yield. The objective of this study was to investigate the effect of feeding Saccharomyces cerevisiae boulardii (CNCM I-1079), a commercial active dry yeast (ADY), in dairy cows on productive and metabolic measures during the periparturient period. Primiparous (n = 33) and multiparous (n = 35) cows were fed a close-up total mixed ration (TMR) before calving and a lactation TMR postpartum. Three weeks before expected calving time, animals were blocked by parity and body weight and then randomly assigned to either control group (control; n = 34) or treatment (ADY; n = 34). All animals were housed in a tie-stall barn with individual feed bunks; the ADY animals received supplementary Saccharomyces cerevisiae boulardii (CNCM I-1079), top dressed daily at a predicted dosage of 1.0 × 1010 cfu (12.5 g) per head. Blood samples were collected weekly along with milk yield and milk composition data; feed intake data were collected daily. Serum samples were analyzed for glucose, nonesterified fatty acid, ß-hydroxybutyrate, haptoglobin (Hp), and the cytokines tumor necrosis factor-α, IL-6, and IL-18. Colostrum samples collected within the first 6 to 10 h were analyzed for somatic cell score and IgG, IgA, and IgM concentrations. Data were analyzed using PROC GLIMMIX in SAS with time as a repeated measure; model included time, parity, treatment, and their interactions. The ADY groups had greater milk yield (39.0 ± 2.4 vs. 36.7 ± 2.3 kg/d) and tended to produce more energy-corrected milk with better feed efficiency. There was no difference in plasma glucose, serum nonesterified fatty acid, serum ß-hydroxybutyrate, Hp, IL-6, or IL-18 due to ADY treatment. The tumor necrosis factor-α increased in ADY-supplemented animals (1.17 ± 0.69 vs. 4.96 ± 7.7 ng/mL), though week, parity, and their interactions had no effect. Serum amyloid A tended to increase in ADY-supplemented animals when compared to control animals and was additionally affected by week and parity; there were no significant interactions. No difference in colostrum IgG, IgA, and IgM was observed between treatments. Supplementing transition cow TMR with ADY (CNCM I-1079) improved milk production and tended to improve efficiency in early lactation; markers of inflammation were also influenced by ADY treatment, though the immunological effect was inconsistent.

Nootkatone Improves Chronic Unpredictable Mild Stress-Induced Depressive-Like Behaviors by Repressing NF-κB/NLRP3-Mediated Neuroinflammation.

OBJECTIVE: To explore the effect of nootkatone (NKT) on chronic unpredictable mild stress (CUMS)-induced depressive-like behaviors and the mechanism underlying NKT improving the depressive-like behaviors. METHODS: The CUMS-induced depression model was established in mice. Fifty mice were randomized into 5 groups (n=10) in accordance with a random number table: control group, CUMS group, CUMS + NKT (6 mg/kg) group, CUMS + NKT (12 mg/kg) group, and CUMS + ketamine group. From the 22th day, NKT (6 or 12 mg/kg) or ketamine (0.5 mg/kg) was given with intragastric administration every day for 21 days. Behavioral tests including forced swimming test (FST), tail suspension test (TST), sucrose preference test (SPT) and open-field test (OFT) were carried out. The mRNA and protein expressions of interleukin (IL)-1ß, IL-18, IL-6, and tumor necrosis factor (TNF)-α in hippocampus were assessed using quantitative realtime polymerase chain reaction (PCR), Western blot analysis, and enzyme linked immunosorbent assay. The nuclear factor-κB (NF-κB)/NOD-like receptor 3 (NLRP3) inflammasome pathway was analyzed using Western blot and immunofluorescence analysis. RESULTS: NKT treatment improved CUMS-induced depressive-like behaviors in mice (P<0.05 or P<0.01). NKT significantly decreased the mRNA and protein levels of IL-1ß, IL-18, IL-6, and TNF-α in hippocampus of CUMS mice (P<0.05 or P<0.01). Furthermore, NKT repressed CUMS-induced activation of NF-κB signaling and NLRP3 inflammasome (P<0.01). More important, Nigericin, a NLRP3 activator, destroyed the effect of NKT on repressing neuroinflammation and improving depressive-like behaviors (P<0.05 or P<0.01). CONCLUSION: NKT ameliorates the depressive-like symptoms, in part by repressing NF-κB/NLRP3-mediated neuroinflammation.

Hydrogen sulfide attenuates uranium-induced kidney cells pyroptosis via upregulation of PI3K/AKT/mTOR signaling.

We have identified that hydrogen sulfide (H2 S), a gaseous mediator, plays a crucial role in antioxidative, anti-inflammatory, and cytoprotective effects on uranium (U)-triggered rat nephrotoxicity. Pyroptosis is a special mode of inflammation and programmed cell death involved in the activation of inflammasome and Caspase-1 and the release of inflammatory cytokines. This study aims to confirm whether H2 S can alleviate U-induced rat NRK-52E cell pyroptosis and to investigate the H2 S underlying regulatory mechanism. Our results indicate that pretreatment with NaHS (an H2 S donor) significantly inhibited U-increased reactive oxygen species level, NLRP3, apoptosis-related speck-like protein consisting of a caspase recruitment domain (ASC), and cleaved Caspase-1 proteins expression, gasdermin D messenger RNA (GSDMD mRNA) expression, interleukin (IL)-1ß and IL-18 contents, lactate dehydrogenase leakage, and numbers of double-positive dying kidney cells. NaHS application evidently augmented phosphorylated PI3K, AKT, and mTOR expression as well as ratios of their respective phosphorylation to the corresponding total proteins which were downregulated by U treatment. But, LY294002 (a PI3K inhibitor) administration effectively abrogated the consequences of NaHS on the levels of p-PI3K, cleaved Caspase-1, ASC and NLRP3 proteins, GSDMD mRNA expression, and (IL)-1ß and IL-18 contents. Simultaneously, LY294002 significantly reversed the effects of NaHS on U-induced pyroptosis rate and cytotoxicity. Taken together, these results indicate that H2 S ameliorated U-triggered NRK-52E cells pyroptosis via upregulation of PI3K/AKT/mTOR pathway, suggesting a novel role for H2 S in the management of nephrotoxicity caused by U exposure.

Xianglian Pill attenuates ulcerative colitis through TLR4/MyD88/NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Xianglian Pill (XLP) is a classical Chinese medicine prescription applied for controlling ulcerative colitis (UC). Whereas, the underlying mechanism remains unclear. AIM OF THE STUDY: The present work was aimed to investigate the mechanism of XLP in dextran sulfate sodium (DSS)-induced UC via the Toll Like Receptor 4 (TLR4)/Myeloid Differentiation factor 88 (MyD88)/Nuclear Factor kappa-B (NF-κB) signaling in mice. MATERIALS AND METHODS: The major components of XLP were detected by high-performance liquid chromatography-diode array detection (HPLC-DAD). The ulcerative colitis model was induced by DSS in mice. 5-Amino Salicylic Acid (5-ASA) group and XLP group were intragastrically treated. Disease activity index (DAI) and colon length were monitored and hematoxylin-eosin (HE) staining was conducted. Gasdermin D (GSDMD)-N and TLR4 expressions in colon tissues were visualized by immunofluorescence. TLR4 mRNA was measured by Real Time Quantitative PCR (RT-qPCR). The expressions of NOD-like receptor thermal protein domain associated protein 3 (NLRP3), active-caspase-1, GSDMD-N, TLR4, MYD88, NF-κB, p-NF-κB, and the ubiquitination of TLR4 in colon tissues were detected by Western blot. Myeloperoxidase (MPO) enzyme activity was examined and serum inflammatory factors Interleukin (IL)-1ß, IL-6, Tumor Necrosis Factor-α (TNF-α), and IL-18 were determined by Enzyme-linked Immunosorbent Assay (ELISA). TLR4-/- mice were applied for verifying the mechanism of XLP attenuated DSS symptoms. RESULTS: The XLP treatment extended colon length, reduced DAI, and attenuated histopathological alteration in DSS-induced mice. XLP administration suppressed MPO activity and reduced the content of IL-1ß, IL-6, TNF-α and IL-18 in serum. XLP also inhibited the expression levels of GSDMD-N, TLR4, NLRP3, active-caspase-1, MyD88, p-NF-κB/NF-κB in colon tissues of DSS-induced mice. TLR4-/- mice proved that TLR4 was involved in XLP-mediated beneficial effect on DSS-induced ulcerative colitis. CONCLUSIONS: XLP might treat ulcerative colitis by regulating the TLR4/MyD88/NF-κB signaling pathway.

Effect of Berberine on Activation of TLR4-NFκB Signaling Pathway and NLRP3 Inflammasome in Patients with Gout.

OBJECTIVE: To determine the effects of berberine (BBR) on the activation of toll-like receptor 4 (TLR4), nuclear factor (NF)κB (NF-κB) signaling and NLRP3 inflammasome in patients with gout. METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from 24 acute (AP) and 41 non-acute (NAP) phases of primary gout patients, respectively, as well as 30 healthy controls (HC). TLR4, NF-κB (p65), NLRP3, apoptosis-associated specklike protein containing a CARD (PYCARD), cysteinyl aspartate specific proteinase-1 (CASP1), and interleukin-1ß (IL-1ß) mRNA expression levels in PBMCs were measured by quantitative reverse transcriptase polymerase chain reaction. The protein levels of TLR4, myeloid differentiation factor 88 (MyD88), NF-κB (p50/65), inhibitor of kappa B kinase α/ß (IKKα/ß), NF-κB inhibitor α (IKBα), phospho-IKKα/ß (p-IKKα/ß), NLRP3, PYCARD, and CASP1 were monitored by Western blotting. Serum IL-1ß protein level was measured using enzyme-linked immunosorbent assay (ELISA). In addition, PBMCs from HC and macrophages derived from a spontaneously immortalized monocyte-like cell line (THP-1) were stimulated using monosodium urate (MSU, 100 µg/mL), 0.1% dimethyl sulfoxide, 25 µmol/L BBR, and 10, 25, and 50 µmol/L BBR+100 µg/mL MSU for different time periods. The protein levels of IL-1ß and IL-18 in cell culture supernatants was measured by ELISA, and the protein expressions of TLR4, MyD88, NF-κB (p50/p65), IKKα/ß, I κBß, p-IKKα/ß, NLRP3, PYCARD, and CASP1 in macrophages were analyzed by Western blotting. RESULTS: (1) TLR4, NF-κB (p65), PYCARD, CASP1, and IL-1ß mRNA levels in PBMCs were significantly higher in the AP group than in the HC group (P<0.05). The NLRP3 mRNA expression levels in PBMCs were found to be significantly lower in the AP and NAP groups than in the HC group (P<0.05, P<0.01). (2) The protein levels of TLR4, IKKß, MyD88, NF-κB, p-IKKα/ß, PYCARD, and CASP1 in PBMCs were significantly higher, and those of IκBα, IKKα, and NLRP3 were found to be significantly lower in the AP group than in the HC group (P<0.05 or P<0.01). (3) The serum IL-1ß protein levels were significantly higher in the AP and NAP groups than in the HC group (P<0.01). (4) The IL-1ß protein level was significantly lower in the culture supernatants of the PBMCs stimulated with MSU for 3 and 6 h in the 25 and 50 µmoL/L BBR groups compared with that in the MSU group (P<0.01). (5) The protein levels of IL-1ß and IL-18 were also significantly lower in the culture supernatants of macrophages stimulated with MSU for 3 and 6 h in BBR groups compared with those in the MSU group (P<0.01). (6) The protein levels of TLR4, MyD88, NF-κB (p50, p65, p105), IKKα/ß, p-IκBα, p-IKKα/ß, PYCARD, and CASP1 were significantly differed between the macrophages stimulated with MSU for 0.5 and 6 h in BBR groups compared with those in the MSU group (P<0.05 or P<0.01). CONCLUSIONS: Activation of TLR4-NFκB signaling and NLRP3 inflammasome by MSU crystals drives the progression of gout inflammation. BBR ameliorates gouty inflammation, which is mechanistically associated with its regulation of TLR4-NF-κB signaling and NLRP3 inflammasome expression.

[Shenfu Injection improves chronic heart failure by regulating pyroptosis based on NLRP3/caspase-1 pathway].

This study investigated the mechanisms and targets of Shenfu Injection in the intervention in chronic heart failure(CHF) through the NOD-like receptor thermal protein domain associated protein 3(NLRP3)/caspase-1 signaling pathway. A CHF model was induced in rats by subcutaneous injection of isoproterenol. Model rats were randomly divided into a model group, a Shenfu Injection group, and a MCC950(NLRP3 inhibitor) group, and a blank group was also set up as a control. After 15 days of treatment, echocardiography was performed to measure cardiac function parameters [left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS)]. Enzyme-linked immunosorbent assay(ELISA) was used to measure serum levels of N-terminal pro-brain natriuretic peptide(NT-proBNP), interleukin(IL)-1ß, and IL-18. Hematoxylin-eosin(HE) and Masson staining were used to observe morphological changes in myocardial tissues, and Western blot was used to measure the expression levels of NLRP3/caspase-1 pathway-related proteins [NLRP3, caspase-1, apoptosis-associated speck-like protein containing a CARD(ASC), gasdermin D(GSDMD), IL-1ß, and IL-18]. The study found that isoproterenol-induced CHF in rats resulted in decreased cardiac function, worsened myocardial fibrosis, increased expression levels of NLRP3, ASC, caspase-1, GSDMD-N, IL-1ß, and IL-18 in myocardial tissues, elevated serum inflammatory factors, and induced myocardial cell pyroptosis. Following Shenfu Injection intervention, the Shenfu Injection group showed significantly improved LVEF and LVFS, a significant decrease in NT-proBNP, a marked downregulation of NLRP3, ASC, caspase-1, GSDMD-N, IL-1ß, and IL-18 protein expression levels, reduced serum inflammatory factors IL-1ß and IL-18 expression in CHF rats, and a decrease in the rate of TUNEL-positive cells. Shenfu Injection can significantly improve cardiac function in CHF, inhibit myocardial fibrosis, and alleviate the progression of myocardial cell pyroptosis through the inhibition of the NLRP3/caspase-1 pathway.

[Effects of electroacupuncture on the blood-brain-barrier and proinflammatory cytokine IL-1ß and IL-18 in the hippocampus of rats with vascular dementia].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on blood-brain barrier (BBB) permeability and proinflammatory cytokines interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) in the hippocampus of vascular dementia (VD) rats, so as to explore the mechanism of EA on treatment of VD. METHODS: SD male rats were randomly divided into sham operation, model, and EA groups, with 15 rats in each group. The VD rat model was established by permanently occlusion of the bilateral middle cerebral artery. Rats of the EA group received EA at "Baihui" (GV20), "Dazhui" (GV14), and bilateral "Shenshu"(BL23) for 30 min, 6 days a week for a total of 4 weeks. Morris water maze test was used to assess the cognitive function of rats. Evans blue staining was used to detect the BBB permeability, transmission electron microscopy and ELISA were used to detect the ultrastructure of BBB and the contents of hippocampal IL-1ß and IL-18, respectively. RESULTS: Following modeling, compared with the sham operation group, the mean escape latency of model group was significantly prolonged (P<0.01), the times of crossing the platform were significantly decreased (P<0.01), the content of Evans blue, and the contents of IL-1ß and IL-18 in hippocampus were increased (P<0.01). After the intervention, comparison between the model and EA groups showed that the average escape latency of rats in EA group was significantly shortened (P<0.01), the times of crossing the platform were increased (P<0.05), the content of Evans blue, and the contents of IL-1ß and IL-18 in hippocampus were significantly decreased (P<0.01). The ultrastructure of BBB was moderately damaged in the model group, which was evidenced by blurred endothelial cell membrane structure, obviously dropsical astrocyte foot process, and decreased tight junctions. The ultrastructure of BBB was slightly damaged and astrocyte foot had no obvious edema in the EA group. CONCLUSION: EA can significantly improve the learning and memory ability of VD rats and improve the BBB permeability, which may be related to its effect in inhibiting the expression of IL-1ß and IL-18 in the hippocampus.