Oral post-exercise garlic extract supplementation enhances glycogen replenishment but does not up-regulate mitochondria biogenesis mRNA expression in human-exercised skeletal muscle.

PURPOSE: Garlic extract (GA) is purported to enhance antioxidant and anti-inflammatory activity and glucose regulation in humans. The present study investigated the effects of post-exercise GA supplementation on GLUT4 expression, glycogen replenishment, and the transcript factors involved with mitochondrial biosynthesis in exercised human skeletal muscle. METHODS: The single-blinded crossover counterbalanced study was completed by 12 participants. Participants were randomly divided into either GA (2000 mg of GA) or placebo trials immediately after completing a single bout of cycling exercise at 75% Maximal oxygen uptake (VO2max) for 60 minutes. Participants consumed either GA (2000 mg) or placebo capsules with a high glycemic index carbohydrate meal (2 g carb/body weight) immediately after exercise. Muscle samples were collected at 0-h and 3-h post-exercise. Muscle samples were used to measure glycogen levels, GLUT4 protein expression, as well as transcription factors for glucose uptake, and mitochondria biogenesis. Plasma glucose, insulin, glycerol, non-esterified fatty acid (NEFA) concentrations, and respiratory exchange ratio (RER) were also analyzed during the post-exercise recovery periods. RESULTS: Skeletal muscle glycogen replenishment was significantly elevated during the 3-h recovery period for GA concurrent with no difference in GLUT4 protein expression between the garlic and placebo trials. PGC1-α gene expression was up-regulated for both GA and placebo after exercise (p < 0.05). Transcript factors corresponding to muscle mitochondrial biosynthesis were significantly enhanced under acute garlic supplementation as demonstrated by TFAM and FIS1. However, the gene expression of SIRT1, ERRα, NFR1, NFR2, MFN1, MFN2, OPA1, Beclin-1, DRP1 were not enhanced, nor were there any improvements in GLUT4 expression, following post-exercise garlic supplementation. CONCLUSION: Acute post-exercise garlic supplementation may improve the replenishment of muscle glycogen, but this appears to be unrelated to the gene expression for glucose uptake and mitochondrial biosynthesis in exercised human skeletal muscle.

Study on analgesic effect of Shentong Zhuyu Decoction in neuropathic pain rats by network pharmacology and RNA-Seq.

ETHNOPHARMACOLOGICAL RELEVANCE: Shentong Zhuyu Decoction (STZYD) is a traditional prescription for promoting the flow of Qi and Blood which is often used in the treatment of low back and leg pain clinicall with unclear mechanism. Neuropathic pain (NP) is caused by disease or injury affecting the somatosensory system. LncRNAs may play a key role in NP by regulating the expression of pain-related genes through binding mRNAs or miRNAs sponge mechanisms. AIM OF THE STUDY: To investigate the effect and potential mechanism of STZYD on neuropathic pain. METHODS: Chronic constriction injury (CCI) rats, a commonly used animal model, were used in this study. The target of STZYD in NP was analyzed by network pharmacology, and the analgesic effect of STZYD in different doses (H-STZYD, M-STZYD, L-STZYD) on CCI rats was evaluated by Mechanical withdrawal thresholds (MWT) and thermal withdrawal latency (TWL). Meanwhile, RNA-seq assay was used to detect the changed mRNAs and lncRNAs in CCI rats after STZYD intervention. GO analysis, KEGG pathway analysis, and IPA analysis were used to find key target genes and pathways, verified by qPCR and Western Blot. The regulatory effect of lncRNAs on target genes was predicted by co-expression analysis and ceRNA network construction. RESULTS: We found that STZYD can improve hyperalgesia in CCI rats, and H-STZYD has the best analgesic effect. The results of network pharmacological analysis showed that STZYD could play an analgesic role in CCI rats through the MAPK/ERK/c-FOS pathway. By mRNA-seq and lncRNA-seq, we found that STZYD could regulate the expression of Cnr1, Cacng5, Gucy1a3, Kitlg, Npy2r, and Grm8, and inhibited the phosphorylation level of ERK in the spinal cord of CCI rats. A total of 27 lncRNAs were associated with the target genes and 30 lncRNAs, 83 miRNAs and 5 mRNAs participated in the ceRNA network. CONCLUSION: STZYD has the effect of improving hyperalgesia in CCI rats through the MAPK/ERK/c-FOS pathway, which is related to the regulation of lncRNAs to Cnr1 and other key targets.

Mijiao formula regulates NAT10-mediated Runx2 mRNA ac4C modification to promote bone marrow mesenchymal stem cell osteogenic differentiation and improve osteoporosis in ovariectomized rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The Mijiao (MJ) formula, a traditional herbal remedy, incorporates antlers as its primary constituent. It can effectively treat osteoporosis (OP), anti-aging, enhance immune activity, and change depression-like behavior. In this study, we investigated that MJ formula is a comprehensive treatment strategy, and may provide a potential approach for the clinical treatment of postmenopausal osteoporosis. AIM OF THE STUDY: The purpose of this study was to determine whether MJ formula promoted osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and improved osteoporosis in ovariectomized rats by regulating the NAT10-mediated Runx2 mRNA ac4C modification. MATERIALS AND METHODS: Female Sprague-Dawley (SD) rats were used to investigate the potential therapeutic effect of MJ formula on OP by creating an ovariectomized (OVX) rat model. The expression of osteogenic differentiation related proteins in BMSCs was detected in vivo, indicating their role in promoting bone formation. In addition, the potential mechanism of its bone protective effect was explored via in vitro experiments. RESULTS: Our study showed that MJ formula significantly mitigated bone mass loss in the OVX rat model, highlighting its potential as an OP therapeutic agent. We found that the possible mechanism of action was the ability of this formulation to stabilize Runx2 mRNA through NAT10-mediated ac4C acetylation, which promoted osteogenic differentiation of BMSCs and contributed to the enhancement of bone formation. CONCLUSIONS: MJ formula can treat estrogen deficiency OP by stabilizing Runx2 mRNA, promoting osteogenic differentiation and protecting bone mass. Conceivably, MJ formulation could be a safe and promising strategy for the treatment of osteoporosis.

Effects of moxibustion on intestinal barrier function and TLR4/NF-κB p65 signaling pathway in obese rats. / è‰¾ç¸å¯¹è‚¥èƒ–å¤§é¼ è‚ é“å±éšœåŠŸèƒ½åŠTLR4/NF-κB p65信号通路的影响.

OBJECTIVES: To observe the effects of moxibustion on intestinal barrier function and Toll-like receptor 4 (TLR4)/nuclear factor-κB p65 (NF-κB p65) signaling pathway in obese rats and explore the mechanism of moxibustion in the intervention of obesity. METHODS: Fifty-five Wistar rats of SPF grade were randomly divided into a normal group (10 rats) and a modeling group (45 rats). In the modeling group, the obesity model was established by feeding high-fat diet. Thirty successfully-modeled rats were randomized into a model group, a moxibustion group, and a placebo-control group, with 10 rats in each one. In the moxibustion group, moxibustion was applied at the site 3 cm to 5 cm far from the surface of "Zhongwan" (CV 12), with the temperature maintained at (46±1 ) ℃. In the placebo-control group, moxibustion was applied at the site 8 cm to 10 cm far from "Zhongwan" (CV 12), with the temperature maintained at (38±1) ℃. The intervention was delivered once daily for 8 weeks in the above two groups. The body mass and food intake of the rats were observed before and after intervention in each group. Using ELISA methool, the levels of serum triacylglycerol (TG), total cholesterol (TC) and lipopolysaccharide (LPS) were detected and the insulin resistance index (HOMA-IR) was calculated. HE staining was used to observe the morphology of colon tissue. The mRNA expression of zonula occludens-1 (ZO-1), Occludin, Claudin-1, TLR4 and NF-κB p65 in the colon tissue was detected by quantitative real-time PCR; and the protein expression of ZO-1, Occludin, Claudin-1, TLR4 and NF-κB p65 was detected by Western blot in the rats of each group. RESULTS: Compared with the normal group, the body mass, food intake, the level of HOMA-IR, and the serum levels of TC, TG and LPS were increased in the rats of the model group (P<0.01); those indexes in the moxibustion group were all reduced when compared with the model group and the placebo-control group respectively (P<0.01, P<0.05). Compared with the normal group, a large number of epithelial cells in the mucosa of colon tissue was damaged, shed, and the inflammatory cells were infiltrated obviously in the interstitium in the rats of the model group. When compared with the model group, in the moxibustion group, the damage of the colon tissue was recovered to various degrees and there were few infiltrated inflammatory cells in the interstitium, while, the epithelial injury of the colon tissue was slightly recovered and the infiltrated inflammatory cells in the interstitium were still seen in the placebo-control group. The mRNA and protein expressions of ZO-1, Occludin and Caudin-1 were decreased in the model group compared with those in the normal group (P<0.01). When compared with the model group and the placebo-control group, the mRNA and protein expressions of these indexes were increased in the moxibustion group (P<0.01, P<0.05). In the model group, the mRNA and protein expressions of TLR4 and NF-κB p65 were increased when compared with those in the normal group (P<0.01), and the mRNA and protein expressions of these indexes were reduced in the moxibustion group when compared with those in the model group and the placebo-control group (P<0.01). CONCLUSIONS: Moxibustion can reduce the body mass and food intake, regulate the blood lipid and improve insulin resistance in the rats of obesity. It may be related to alleviating inflammatory response through improving intestinal barrier function and modulating the intestinal TLR4/NF-κB p65 signaling pathway.

[Ligusticum cycloprolactam inhibits IL-1ß-induced apoptosis and inflammation of rat chondrocytes via HMGB1/TLR4/NF-κB signaling pathway].

Chondrocytes are unique resident cells in the articular cartilage, and the pathological changes of them can lead to the occurrence of osteoarthritis(OA). Ligusticum cycloprolactam(LIGc) are derivatives of Z-ligustilide(LIG), a pharmacodynamic marker of Angelica sinensis, which has various biological functions such as anti-inflammation and inhibition of cell apoptosis. However, its protective effect on chondrocytes in the case of OA and the underlying mechanism remain unclear. This study conducted in vitro experiments to explore the molecular mechanism of LIGc in protecting chondrocytes from OA. The inflammation model of rat OA chondrocyte model was established by using interleukin-1ß(IL-1ß) to induce. LIGc alone and combined with glycyrrhizic acid(GA), a blocker of the high mobility group box-1 protein(HMGB1)/Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) signaling pathway, were used to intervene in the model, and the therapeutic effects were systematically evaluated. The viability of chondrocytes treated with different concentrations of LIGc was measured by the cell counting kit-8(CCK-8), and the optimal LIGc concentration was screened out. Annexin V-FITC/PI apoptosis detection kit was employed to examine the apoptosis of chondrocytes in each group. The enzyme-linked immunosorbent assay(ELISA) was employed to measure the expression of cyclooxygenase-2(COX-2), prostaglandin-2(PGE2), and tumor necrosis factor-alpha(TNF-α) in the supernatant of chondrocytes in each group. Western blot was employed to determine the protein levels of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), caspase-3, HMGB1, TLR4, and NF-κB p65. The mRNA levels of HMGB1, TLR4, NF-κB p65, and myeloid differentiation factor 88(MyD88) in chondrocytes were determined by real-time fluorescent quantitative PCR(RT-qPCR). The safe concentration range of LIGc on chondrocytes was determined by CCK-8, and then the optimal concentration of LIGc for exerting the effect was clarified. Under the intervention of IL-1ß, the rat chondrocyte model of OA was successfully established. The modeled chondrocytes showed increased apoptosis rate, promoted expression of COX-2, PGE2, and TNF-α, up-regulated protein levels of Bax, caspase-3, HMGB1, TLR4, and NF-κB p65 and mRNA levels of HMGB1, TLR4, NF-κB p65, and MyD88, and down-regulated protein level of Bcl-2. However, LIGc reversed the IL-1ß-induced changes of the above factors. Moreover, LIGc combined with GA showed more significant reversal effect than LIGc alone. These fin-dings indicate that LIGc extracted and derived from the traditional Chinese medicine A. sinensis can inhibit the inflammatory response of chondrocytes and reduce the apoptosis of chondrocytes, and this effect may be related to the HMGB1/TLR4/NF-κB signaling pathway. The pharmacological effect of LIGc on protecting chondrocytes has potential value in delaying the progression of OA and improving the clinical symptoms of patients, and deserves further study.

[Mechanism of ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix regulates HIF-1α signaling pathway mediated by renal hypoxia to ameliorate renal fibrosis in DKD rats].

This study explored the mechanism of the ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix in ameliorating renal fibrosis in the rat model of diabetic kidney disease(DKD) based on the expression of hypoxia-inducible factor-1α(HIF-1α)/vascular endothelial growth factor(VEGF) and HIF-1α/platelet-derived growth factor(PDGF)/platelet-derived growth factor receptor(PDGFR) signaling pathways in the DKD rats. After 1 week of adaptive feeding, 50 male SPF-grade Wistar rats were randomized into a blank group(n=7) and a modeling group. After 24 h of fasting, the rats in the modeling group were subjected to intraperitoneal injection of streptozocin and fed with a high-sugar and high-fat diet to establish a DKD model. After modeling, the rats were randomly assigned into model(n=7), low-dose ultrafiltration extract(n=7), medium-dose ultrafiltration extract(n=7), irbesartan(n=8), and high-dose ultrafiltration extract(n=8) groups. After intervention by corresponding drugs for 12 weeks, the general conditions of the rats were observed. The body weights and blood glucose levels of the rats were measured weekly, and the 24 h urinary protein(24hUP) was measured at the 6th and 12th weeks of drug administration. After the last drug administration, the renal function indicators were determined. Masson staining was employed to observe the pathological changes of the renal tissue. The expression of prolyl hydroxylase domain 2(PHD2) and HIF-1α in the renal tissue was detected by immunohistochemistry(IHC). Real-time qPCR was employed to determine the mRNA levels of PHD2, VEGF, PDGF, and PDGFR in the renal tissue. Western blot was employed to determine the protein levels of HIF-1α, VEGF, PDGF, and PDGFR in the renal tissue. The results showed that compared with the model group, drug administration lowered the levels of glycosylated serum protein(GSP), aerum creatinine(Scr), and blood urea nitrogen(BUN) in a dose-dependent manner(P<0.05 or P<0.01) and mitigated the pathological changes in the renal tissue. Furthermore, drug administration up-regulated mRNA level of PHD2(P<0.05 or P<0.01), down-regulated the mRNA levels of VEGF, PDGF, and PDGFR(P<0.05 or P<0.01) and the protein levels of HIF-1α, VEGF, PDGF, and PDGFR(P<0.01) in the renal tissue, and increased the rate of PHD2-positive cells(P<0.01). In conclusion, the ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix effectively alleviated the renal fibrosis in DKD rats by inhibiting the expression of key proteins in the HIF-1α signaling pathway mediated by renal hypoxia and reducing extracellular matrix(ECM) deposition.

[Intervention effect of Erchen Decoction on methionine and choline deficient diet-induced non-alcoholic steatohepatitis in mice].

This study investigated the effect of Erchen Decoction(ECD) on the prevention of non-alcoholic steatohepatitis(NASH) in mice and explored its possible mechanism, so as to provide scientific data for the clinical application of ECD in the prevention of NASH. C57BL/6 male mice were randomly divided into normal group(methionine and choline supplement, MCS), model group(methionine and choline deficient, MCD), low-dose ECD group(ECD＿L, 6 g·kg~(-1)), medium-dose ECD group(ECD＿M, 12 g·kg~(-1)), and high-dose ECD group(ECD＿H, 24 g·kg~(-1)), with eight mice in each group. The MCS group was fed with an MCS diet, and the other groups were fed with an MCD diet. The mice in each group were given corresponding diets, but the drug intervention group was given low-, medium-, and high-dose ECD(10 mL·kg~(-1)·d~(-1)) by intragastric administration for six weeks on the basis of MCD diet feeding, and the mice could eat and drink freely during the whole experiment. At the end of the experiment, mice were fasted overnight(12 h) and were anesthetized with 20% urethane. Thereafter, the blood and liver tissue were collected. The serum was used to detect the levels of alanine aminotransferase(ALT), aspartate aminotransaminase(AST), interleukin-1ß(IL-1ß), interleukin-6(IL-6), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Liver tissue was processed by hematoxylin-eosin(HE) staining and used for hepatic histological analysis and detection of the expression levels of genes and proteins related to nuclear factor erythroid 2-related factor 2/glutathione peroxidase 4(Nrf2/GPX4) pathway by real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR) and Western blot analysis, respectively. The results showed that compared with the MCS group, the MCD group showed higher serum ALT and AST levels; the HE staining exhibited fat vacuoles and obvious inflammatory cell infiltration in liver tissue; serum IL-1ß, IL-6, and TNF-α levels were significantly increased, and the serum IL-10 level was significantly decreased. The mRNA expressions of fatty acid synthase(FASN), monocyte chemoattractant protein-1(MCP-1), and IL-1ß in liver tissue were significantly up-regulated, while those of GPX4, Nrf2, and NAD(P)H:quinine oxidoreductase(NQO1) were significantly down-regulated. Compared with the MCD group, the serum ALT and AST levels of ECD＿M and ECD＿H groups were significantly decreased, and the AST level in the ECD＿L group was significantly decreased. The number of fat vacuoles and the degree of inflammatory cell infiltration in liver tissue were improved; serum IL-1ß, IL-6, and TNF-α levels were significantly decreased, but the serum IL-10 level was significantly increased only in the ECD＿H group. The mRNA expressions of FASN, MCP-1, and IL-1ß in liver tissue were significantly down-regulated, and those of GPX4 and NQO1 were significantly up-regulated. The mRNA expressions of Nrf2 in ECD＿M and ECD＿H groups were significantly up-regulated. Western blot results showed that compared with the MCD group, the protein expression levels of Nrf2 and GPX4 in each group were significantly increased after ECD administration, and the protein expression level of FASN was significantly decreased; the protein expression of NQO1 was increased in ECD＿M and ECD＿H groups. In summary, ECD can reduce hepatic lipid accumulation, oxidative stress, liver inflammation, and liver injury in NASH mice, which may be related to the activation of the Nrf2/GPX4 pathway.

[Improvement effect of cinnamaldehyde on reserpine-induced Parkinson's disease rat model].

In order to study the neuroprotective mechanism of cinnamaldehyde on reserpine-induced Parkinson's disease(PD) rat models, 72 male Wistar rats were randomly divided into blank group, model group, Madopar group, and cinnamaldehyde high-, medium-, and low-dose groups. Except for the blank group, the other groups were intraperitoneally injected with reserpine of 0.1 mg·kg~(-1) once every other morning, and cinnamaldehyde and Madopar solutions were gavaged every afternoon. Open field test, rotarod test, and oral chewing movement evaluation were carried out in the experiment. The brain was taken and fixed. The positive expression of dopamine receptor D1(DRD1) was detected by TSA, and the changes in neurotransmitters such as dopamine(DA) and 3,4-dihydroxyphenylacetic acid(DOPAC) in the brain were detected by enzyme-linked immunosorbent assay(ELISA). The protein and mRNA expression levels of tyrosine hydroxylase(TH) and α-synuclein(α-Syn) in substantia nigra(SN) were detected by RT-PCR and Western blot. The results showed that after the injection of reserpine, the hair color of the model group became yellow and dirty; the arrest behavior was weakened, and the body weight was reduced. The spontaneous movement and exploration behavior were reduced, and the coordination exercise ability was decreased. The number of oral chewing was increased, but the cognitive ability was decreased, and the proportion of DRD1 positive expression area in SN was decreased. The expression of TH protein and mRNA was down-regulated, and that of α-Syn protein and mRNA was up-regulated. After cinnamaldehyde intervention, it had an obvious curative effect on PD model animals. The spontaneous movement behavior, the time of staying in the rod, the time of movement, the distance of movement, and the number of standing times increased, and the number of oral chewing decreased. The proportion of DRD1 positive expression area in SN increased, and the protein and mRNA expression levels of α-Syn were down-regulated. The protein and mRNA expression levels of TH were up-regulated. In addition, the levels of DA, DOPAC, and homovanillic acid(HVA) neurotransmitters in the brain were up-regulated. This study can provide a new experimental basis for clinical treatment and prevention of PD.

Melatonin improves the viability and ultrastructure of bovine oocyte-granulosa complexes of in vitro cultured early antral follicles.

This study aims to investigate the effects of melatonin on follicular growth, viability and ultrastructure, as well as on the levels of mRNA for antioxidant enzymes, reactive oxygen species (ROS) and meiotic progression in oocytes from in vitro cultured bovine early antral follicles. To this end, isolated early antral follicles (500-600 µm) were cultured in TCM-199+ alone or supplemented with 10-6 , 10-7 or 10-8 M melatonin at 38.5°C with 5% CO2 for 8 days. Follicle diameters were evaluated at days 0, 4 and 8 of culture. At the end of culture, ultrastructure, chromatin configuration, viability (calcein-AM and ethidium homodimer-1 staining), and the levels of ROS and mRNA for catalase (CAT), superoxide dismutase (SOD) and peroxiredoxin 6 (PRDX6) and glutathione peroxidase (GPx) were investigated in oocyte-granulosa cell complexes (OGCs). The results showed that early antral follicles cultured with 10-6 and 10-8 M melatonin had a progressive and significant increase in their diameters throughout the culture period (p < .05). Additionally, oocytes from follicles cultured with 10-7 or 10-8 M melatonin had increased fluorescence for calcein-AM, while those cultured with 10-6 or 10-7 M had reduced fluorescence for ethidium homodimer-1. Different from follicles cultured in other treatments, those cultured with 10-8 M melatonin had well-preserved ultrastructure of oocyte and granulosa cells. Melatonin, however, did not influence the levels of ROS, the mitochondrial activity, oocyte meiotic resumption and expression mRNA for SOD, CAT, GPX1 and PRDX6. In conclusion, the presence of 10-8 M melatonin in culture medium improves viability and preserves the ultrastructure of oocyte and granulosa cells of early antral follicles cultured in vitro.

Effect and mechanism of acupuncture on airway smooth muscle relaxation during acute asthma attack in rats. / é’ˆåˆºæ¾å¼›å“®å–˜æ€¥æ€§å‘ä½œå¤§é¼ æ°”é“å¹³æ»‘è‚Œçš„ä½œç”¨æœºåˆ¶.

OBJECTIVES: To explore the effect and mechanism of acupuncture at "Feishu" (BL 13) and "Dingchuan" (EX-B 1), and "Kongzui" (LU 6) and "Yuji" (LU 10) for relaxing the airway smooth muscle in the rats during acute asthma attack and compare the effect among the two pairs of acupoints and the acupoints combination. METHODS: Forty SD male rats with SPF grade were randomly divided into a blank group, a model group, a pair-point A group (acupuncture at "Feishu" [BL 13] and "Dingchuan" [EX-B 1]), a pair-point B group (acupuncture at "Kongzui" [LU 6] and "Yuji" [LU 10]) and a point combination group (acupuncture at "Feishu" [BL 13] , "Dingchuan" [EX-B 1], "Kongzui" [LU 6] and "Yuji" [LU 10]), with 8 rats in each group. Except the rats in the blank group, the model of acute asthma attack was induced by ovalbumin (OVA) combined with aluminum hydroxide gel in the rest groups. Started on the 15th day of modeling, except in the blank group and the model group, acupuncture was delivered in the other groups, 30 min in each intervention, once daily, for 14 days. In each group, the latent period of asthma inducing was measured; the lung resistance (LR) and dynamic lung compliance (Cdyn) were determined using lung function detector; the levels of endothelin-1 (ET-1), tumor necrosis factor-α (TNF-α), cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in serum and bronchoalveolar lavage fluid (BALF) were measured by ELISA; with Masson staining and electron microscopy adopted, the morphology and ultrastructure of airway smooth muscle of the rats were observed; the mRNA and protein expressions of ET-1 and beta-2 adrenergic receptor (ß2-AR) were detected by quantitative real-time fluorescence and Western blot, respectively. RESULTS: Compared with the blank group, the latent period of asthma inducing was shortened (P<0.05), RL increased and Cdyn decreased (P<0.05) with the different concentrations of methacholine (0.025 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.2 mg/kg) in the model group. In the pair-point A group, the pair-point B group and the point combination group, the latent period of asthma inducing was prolonged (P<0.05), RL decreased and Cdyn increased (P<0.05) with different concentrations of methacholine when compared with those in the model group; and the latent period of asthma inducing in the point combination group was longer than that in the pair-point A group (P<0.05). Compared with the blank group, the levels of ET-1, TNF-α and cGMP in the serum and BALF were elevated (P<0.05), and those of cAMP reduced (P<0.05) in the model group. The levels of ET-1, TNF-α and cGMP in the serum and BALF were reduced (P<0.05), and those of cAMP elevated (P<0.05) in the pair-point A group, the pair-point B group and the point combination group when compared with those in the model group. In the blank group, the lung tissue was normal structurally. In the model group, the collagen fibers were proliferated increasingly, the smooth muscle was thickened, the mitochondria were swollen, and their cristae disrupted and reduced massively. In the pair-point B group, the collagen fibers were proliferated, the smooth muscle was thicker compared with that in the blank group, the mitochondria were mildly swollen and their cristae disrupted partially. In the pair-point A group and the point combination group, the lung tissue changes were obviously alleviated in comparison with the model group, the mitochondria were slightly swollen and their cristae disrupted occasionally. Compared with the blank group, the mRNA and protein expression of ET-1 increased and that of ß2-AR decreased in the lung tissue of the model group (P<0.05). In the pair-point A group, the pair-point B group and the point combination group, the mRNA and protein expression of ET-1 was reduced and that of ß2-AR elevated in the lung tissue when compared with those in the model group (P<0.05). In comparison with the pair-point A group, the mRNA expression of ß2-AR was elevated in the point combination group (P<0.05). When compared with the pair-point B group, the mRNA expression of ß2-AR increased, the protein expression of ET-1 decreased (P<0.05) in the point combination group. CONCLUSIONS: Acupuncture at "Feishu" (BL 13) and "Dingchuan" (EX-B 1), "Kongzui" (LU 6) and "Yuji" (LU 10), two pairs of acupoints relieves the airway smooth muscle spasm in the rats during acute asthma attack, which may be related to inhibiting the mRNA and protein expression of ET-1 to reduce the excretion of ET-1 and TNF-α; while enhancing the mRNA and protein expression of ß2-AR to balance the levels of cAMP and cGMP. The effect is optimal when acupuncture is delivered at two pairs of acupoints simultaneously.

Hypolipidemic Effect of Rice Bran Oil Extract Tocotrienol in High-Fat Diet-Induced Hyperlipidemia Zebrafish (Danio Rerio) Induced by High-Fat Diet.

In recent years, the potent influence of tocotrienol (T3) on diminishing blood glucose and lipid concentrations in both Mus musculus (rats) and Homo sapiens (humans) has been established. However, the comprehensive exploration of tocotrienol's hypolipidemic impact and the corresponding mechanisms in aquatic species remains inadequate. In this study, we established a zebrafish model of a type 2 diabetes mellitus (T2DM) model through high-fat diet administration to zebrafish. In the T2DM zebrafish, the thickness of ocular vascular walls significantly increased compared to the control group, which was mitigated after treatment with T3. Additionally, our findings demonstrate the regulatory effect of T3 on lipid metabolism, leading to the reduced synthesis and storage of adipose tissue in zebrafish. We validated the expression patterns of genes relevant to these processes using RT-qPCR. In the T2DM model, there was an almost two-fold upregulation in pparγ and cyp7a1 mRNA levels, coupled with a significant downregulation in cpt1a mRNA (p < 0.01) compared to the control group. The ELISA revealed that the protein expression levels of Pparγ and Rxrα exhibited a two-fold elevation in the T2DM group relative to the control. In the T3-treated group, Pparγ and Rxrα protein expression levels consistently exhibited a two-fold decrease compared to the model group. Lipid metabolomics showed that T3 could affect the metabolic pathways of zebrafish lipid regulation, including lipid synthesis and decomposition. We provided experimental evidence that T3 could mitigate lipid accumulation in our zebrafish T2DM model. Elucidating the lipid-lowering effects of T3 could help to minimize the detrimental impacts of overfeeding in aquaculture.

Reyanning mixture inhibits M1 macrophage polarization through the glycogen synthesis pathway to improve lipopolysaccharide-induced acute lung injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Reyanning (RYN) mixture is a traditional Chinese medicine composed of Taraxacum, Polygonum cuspidatum, Scutellariae Barbatae and Patrinia villosa and is used for the treatment of acute respiratory system diseases with significant clinical efficacy. AIM OF THE STUDY: Acute lung injury (ALI) is a common clinical disease characterized by acute respiratory failure. This study was conducted to evaluate the therapeutic effects of RYN on ALI and to explore its mechanism of action. MATERIALS AND METHODS: Ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to analyze the chemical components of RYN. 7.5 mg/kg LPS was administered to induce ALI in rats. RYN was administered by gavage at doses of 2 ml/kg, 4 ml/kg or 8 ml/kg every 8 h for a total of 6 doses. Observations included lung histomorphology, lung wet/dry (W/D) weight ratio, lung permeability index (LPI), HE staining, Wright-Giemsa staining. ELISA was performed to detect the levels of TNF-α, IL-6, IL-10, Arg-1,UDPG. Immunohistochemical staining detected IL-6, F4/80 expression. ROS, MDA, SOD, GSH/GSSG were detected in liver tissues. Multiple omics techniques were used to predict the potential mechanism of action of RYN, which was verified by in vivo closure experiments. Immunofluorescence staining detected the co-expression of CD86 and CD206, CD86 and P2Y14, CD86 and UGP2 in liver tissues. qRT-PCR detected the mRNA levels of UGP2, P2Y14 and STAT1, and immunoblotting detected the protein expression of UGP2, P2Y14, STAT1, p-STAT1. RESULTS: RYN was detected to contain 1366 metabolites, some of the metabolites with high levels have anti-inflammatory, antibacterial, antiviral and antioxidant properties. RYN (2, 4, and 8 ml/kg) exerted dose-dependent therapeutic effects on the ALI rats, by reducing inflammatory cell infiltration and oxidative stress damage, inhibiting CD86 expression, decreasing TNF-α and IL-6 levels, and increasing IL-10 and Arg-1 levels. Transcriptomics and proteomics showed that glucose metabolism provided the pathway for the anti-ALI properties of RYN and that RYN inhibited lung glycogen production and distribution. Immunofluorescence co-staining showed that RYN inhibited CD86 and UGP2 expressions. In vivo blocking experiments revealed that blocking glycogen synthesis reduced UDPG content, inhibited P2Y14 and CD86 expressions, decreased P2Y14 and STAT1 mRNA and protein expressions, reduced STAT1 protein phosphorylation expression, and had the same therapeutic effect as RYN. CONCLUSION: RYN inhibits M1 macrophage polarization to alleviate ALI. Blocking glycogen synthesis and inhibiting the UDPG/P2Y14/STAT1 signaling pathway may be its molecular mechanism.

Therapeutic potential of salidroside in preserving rat cochlea organ of corti from gentamicin-induced injury through modulation of NRF2 signaling and GSK3ß/NF-κB pathway.

Salidroside (SAL) is a phenol glycoside compound found in plants of the Rhodiola genus which has natural antioxidant and free radical scavenging properties. SAL are able to protect against manganese-induced ototoxicity. However, the molecular mechanism by which SAL reduces levels of reactive oxygen species (ROS) is unclear. Here, we established an in vitro gentamicin (GM) ototoxicity model to observe the protective effect of SAL on GM-induced hair cells (HC) damage. Cochlear explants of postnatal day 4 rats were obtained and randomly divided into six groups: two model groups (treatment with 0.2 mM or 0.4 mM GM for 24 h); two 400 µmol/L SAL-pretreated groups pretreatment with SAL for 3 h followed by GM treatment (0.2 mM or 0.4 mM) for 24 h; 400 µmol/L SAL group (treatment with SAL for 24 h); control group (normal cultured cochlear explants). The protective effects of SAL on GM-induced HC damage, and on mRNA and protein levels of antioxidant enzymes were observed. HC loss occurred after 24 h of GM treatment. Pretreatment with SAL significantly reduced GM-induced OHC loss. In cochlear tissues, mRNA and protein levels of NRF2 and HO-1 were enhanced in the GM alone group compared with the SAL pretreatment GM treatment group. SAL may protect against GM-induced ototoxicity by regulating the antioxidant defense system of cochlear tissues; SAL can activate NRF2/HO-1 signaling, inhibit NF-κB activation, activate AKT, and increase inhibitory phosphorylation of GSK3ß to decrease GSK3 activity, all of which exert antioxidant effects.

Regulation of STAT1 and STAT4 Expression by Growth Factor and Interferon Supplementation in Sjögren's Syndrome Cell Culture Models.

Our goal was to investigate the effects of epidermal growth factor (EGF) and interferons (IFNs) on signal transducer and activator of transcription STAT1 and STAT4 mRNA and active phosphorylated protein expression in Sjögren's syndrome cell culture models. iSGECs (immortalized salivary gland epithelial cells) and A253 cells were treated with EGF, IFN-alpha, -beta, -gamma, or mitogen-activated protein kinase p38 alpha (p38-MAPK) inhibitor for 0-24-48-72 h. STAT1 and STAT4 mRNA expression was quantified by qRT-PCR. Untreated and treated cells were compared using the delta-delta-CT method based on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) normalized relative fold changes. phospho-tyrosine-701-STAT1 and phospho-serine-721-STAT4 were detected by Western blot analysis. STAT4 mRNA expression decreased 48 h after EGF treatment in A253 cells, immortalized salivary gland epithelial cells iSGECs nSS2 (sicca patient origin), and iSGECs pSS1 (anti-SSA negative Sjögren's Syndrome patient origin). EGF and p38-MAPK inhibitor decreased A253 STAT4 mRNA levels. EGF combined with IFN-gamma increased phospho-STAT4 and phospho-STAT1 after 72 h in all cell lines, suggesting additive effects for phospho-STAT4 and a major effect from IFN-gamma for phospho-STAT1. pSS1 and nSS2 cells responded differently to type I and type II interferons, confirming unique functional characteristics between iSGEC cell lines. EGF/Interferon related pathways might be targeted to regulate STAT1 and STAT4 expression in salivary gland epithelial cells. Further investigation is required learn how to better target the Janus kinases/signal transducer and activator of transcription proteins (JAK/STAT) pathway-mediated inflammatory response in Sjögren's syndrome.

Biochemical properties and biological potential of Syzygium heyneanum with antiparkinson's activity in paraquat induced rodent model.

Syzygium heyneanum is a valuable source of flavonoids and phenols, known for their antioxidant and neuroprotective properties. This research aimed to explore the potential of Syzygium heyneanum ethanol extract (SHE) in countering Parkinson's disease. The presence of phenols and flavonoids results in SHE displaying an IC50 value of 42.13 when assessed in the DPPH scavenging assay. Rats' vital organs (lungs, heart, spleen, liver, and kidney) histopathology reveals little or almost no harmful effect. The study hypothesized that SHE possesses antioxidants that could mitigate Parkinson's symptoms by influencing α-synuclein, acetylcholinesterase (AChE), TNF-α, and IL-1ß. Both in silico and in vivo investigations were conducted. The Parkinson's rat model was established using paraquat (1 mg/kg, i.p.), with rats divided into control, disease control, standard, and SHE-treated groups (150, 300, and 600 mg/kg) for 21 days. According to the ELISA statistics, the SHE treated group had lowers levels of IL-6 and TNF-α than the disease control group, which is a sign of neuroprotection. Behavioral and biochemical assessments were performed, alongside mRNA expression analyses using RT-PCR to assess SHE's impact on α-synuclein, AChE, TNF-α, and interleukins in brain homogenates. Behavioral observations demonstrated dose-dependent improvements in rats treated with SHE (600 > 300 > 150 mg/kg). Antioxidant enzyme levels (catalase, superoxide dismutase, glutathione) were significantly restored, particularly at a high dose, with notable reduction in malondialdehyde. The high dose of SHE notably lowered acetylcholinesterase levels. qRT-PCR results indicated reduced mRNA expression of IL-1ß, α-synuclein, TNF-α, and AChE in SHE-treated groups compared to disease controls, suggesting neuroprotection. In conclusion, this study highlights Syzygium heyneanum potential to alleviate Parkinson's disease symptoms through its antioxidant and modulatory effects on relevant biomarkers.

XinJiaCongRongTuSiZiWan protects triptolide-induced rats from oxidative stress injury via mitophagy mediated PINK1/Parkin signaling pathway.

PURPOSE: XinJiaCongRongTuSiZiWan (XJCRTSZW) is a traditional Chinese medicine compound for invigorating the kidney, nourishing blood, and promoting blood circulation. This study aimed to explore the effect of XJCRTSZW on triptolide (TP)-induced oxidative stress injury. METHODS: Adult female Sprague-Dawley rats and human ovarian granulosa cell lines were treated with TP and XJCRTSZW. Hematoxylin and eosin staining, enzyme-linked immunosorbent assay, flow cytometry, CCK-8, JC-1 staining, transmission electron microscopy, reverse transcription-quantitative polymerase chain reaction, and Western blotting were performed in this study. RESULTS: XJCRTSZW treatment observably ameliorated the TP-induced pathological symptoms. Furthermore, XJCRTSZW treatment observably enhanced the TP-induced reduction of estradiol, anti-Mullerian hormone, progesterone, superoxide dismutase, ATP content, mitochondrial membrane potential, p62, and Hsp60 mRNA, and protein levels in vivo and in vitro (p < 0.05). However, TP-induced elevation of follicle stimulating hormone and luteinizing hormone concentrations, malondialdehyde levels, reactive oxygen species levels, apoptosis rate, mitophagy, and the mRNA and protein expressions of LC3-II/LC3-I, PTEN-induced kinase 1 (PINK1), and Parkin were decreased (p < 0.05). In addition, XJCRTSZW treatment markedly increased cell viability in vitro (p < 0.05). CONCLUSIONS: XJCRTSZW protects TP-induced rats from oxidative stress injury via the mitophagy-mediated PINK1/Parkin pathway.

Investigating the therapeutic effects and mechanisms of Carthamus tinctorius L.-derived nanovesicles in atherosclerosis treatment.

BACKGROUND: Carthamus tinctorius L., a traditional herbal medicine used for atherosclerosis (AS), lacks a clear understanding of its therapeutic mechanisms. This study aimed to investigate the therapeutic effects and mechanisms of Carthamus tinctorius L.-derived nanovesicles (CDNVs) in AS treatment. METHODS: CDNVs were isolated and characterized using improved isolation methods. Transmission electron microscopy, nanoparticle tracking analysis, and protein analysis confirmed their morphology, size, and protein composition. Small RNA sequencing was performed to identify the miRNA profile of CDNVs, and bioinformatics analysis was used to determine their potential biological roles. In vivo biodistribution and toxicity studies were conducted in mice to assess the stability and safety of orally administered CDNVs. The anti-atherosclerotic effects of CDNVs were evaluated in ApoE-/- mice through plaque burden analysis. The protective effects of CDNVs on ox-LDL-treated endothelial cells were assessed through proliferation, apoptosis, reactive oxygen species activation, and monocyte adhesion assays. miRNA and mRNA sequencing of CDNV-treated endothelial cells were performed to explore their regulatory effects and potential target genes. RESULTS: CDNVs were successfully isolated and purified from Carthamus tinctorius L. tissue lysates. They exhibited a saucer-shaped or cup-shaped morphology, with an average particle size of 142.6 ± 0.7 nm, and expressed EV markers CD63 and TSG101. CDNVs contained proteins, small RNAs, and metabolites, including the therapeutic compound HSYA. Small RNA sequencing identified 95 miRNAs, with 10 common miRNAs accounting for 72.63% of the total miRNAs. These miRNAs targeted genes involved in cell adhesion, apoptosis, and cell proliferation, suggesting their relevance in cardiovascular disease. Orally administered CDNVs were stable in the gastrointestinal tract, absorbed into the bloodstream, and accumulated in the liver, lungs, heart, and aorta. They significantly reduced the burden of atherosclerotic plaques in ApoE-/- mice and exhibited superior effects compared to HSYA. In vitro studies demonstrated that CDNVs were taken up by HUVECs, promoted proliferation, attenuated ox-LDL-induced apoptosis and ROS activation, and reduced monocyte adhesion. CDNV treatment resulted in significant changes in miRNA and mRNA expression profiles of HUVECs, with enrichment in inflammation-related genes. CXCL12 was identified as a potential direct target of miR166a-3p. CONCLUSION: CDNVs isolated from Carthamus tinctorius L. tissue lysates represent a promising oral therapeutic option for cardiovascular diseases. The delivery of miRNAs by CDNVs regulates inflammation-related genes, including CXCL12, in HUVECs, suggesting their potential role in modulating endothelial inflammation. These findings provide valuable insights into the therapeutic potential of CDNVs and their miRNAs in cardiovascular disease.

Ningxin-Tongyu-Zishen formula alleviates the senescence of granulosa cells on D-galactose-induced premature ovarian insufficiency mice.

Ningxin-Tongyu-Zishen formula (NTZF) is a clinical experience formula for the treatment of premature ovarian insufficiency (POI) in traditional Chinese medicine (TCM), and the potential mechanism is unknown. For in vivo experiments, POI mouse models (C57BL/6 mice), were constructed by subcutaneous injection of D-galactose (D-gal, 200 mg/kg). After treatment of NTZF (10.14, 20.27, 40.54 g/kg;) or estradiol valerate (0.15 mg/kg), ovarian function, oxidative stress (OS) and protein expression of Sirt1/p53 were evaluated. For in vitro experiments, H2O2 (200 µM) was used to treat KGN to construct ovarian granulosa cells (OGCs) cell senescence model. Pretreatment with NTZF (1.06 mg/mL) or p53 inhibitor (Pifithrin-α, 1 µM) was performed before induction of senescence, and further evaluated the cell senescence, OS, mRNA and protein expression of Sirt1/p53. In vivo, NTZF improved ovarian function, alleviated OS and Sirt1/p53 signaling abnormalities in POI mice. In vitro experiments showed that NTZF reduced the level of OS and alleviated the senescence of H2O2-induced KGN. In addition, NTZF activated the protein expression of Sirt1, inhibited the mRNA transcription and protein expression of p53 and p21. Alleviating OGCs senescence and protecting ovarian function through Sirt1/p53 is one of the potential mechanisms of NTZF in the treatment of POI.

Parental betaine supplementation promotes gosling growth with epigenetic modulation of IGF gene family in the liver.

Betaine is widely used as a feed additive in the chicken industry to promote laying performance and growth performance, yet it is unknown whether betaine can be used in geese to improve the laying performance of goose breeders and the growth traits of offspring goslings. In this study, laying goose breeders at 39 wk of age were fed basal (Control, CON) or betaine-supplemented diets at low (2.5 g/kg, LBT) or high (5 g/kg, HBT) levels for 7 wk, and the breeder eggs laid in the last week were collected for incubation. Offspring goslings were examined at 35 and 63 d of age. The laying rate tended to be increased (P = 0.065), and the feed efficiency of the breeders was improved by betaine supplementation, while the average daily gain of the offspring goslings was significantly increased (P < 0.05). Concentrations of insulin-like growth factor 2 (IGF-2) in serum and liver were significantly increased in the HBT group (P < 0.05), with age-dependent alterations of serum T3 levels. Concurrently, hepatic mRNA expression of the IGF gene family was significantly increased in goslings derived from betaine-treated breeders (P < 0.05). A higher ratio of proliferating cell nuclear antigen (PCNA)-immunopositive nuclei was found in the liver sections of the HBT group, which was confirmed by significantly upregulated hepatic expression of PCNA mRNA and protein (P < 0.05). Moreover, hepatic expression of thyroxine deiodinase type 1 (Dio1) and thyroid hormone receptor ß (TRß) was also significantly upregulated in goslings of the HBT group (P < 0.05). These changes were associated with significantly higher levels of global DNA 5-mC methylation, together with increased expression of methyl transfer genes (P < 0.05), including betaine-homocysteine methyltransferase (BHMT), glycine N-methyltransferase (GNMT), and DNA (cytosine-5-)-methyltransferase 1 (DNMT1). The promoter regions of IGF-2 genes, as well as the predicted TRß binding site on the IGF-2 gene, were significantly hypomethylated (P < 0.05). These results indicate that gosling growth can be improved by dietary betaine supplementation in goose breeders via epigenetic modulation of the IGF gene family, especially IGF-2, in the liver.

The goose industry plays important roles in economics, cultures, and ecosystems, yet the low laying and growth rates of many indigenous breeds hinders the development of the goose farming. Betaine, an important methyl donor, is commonly used as a feed additive in livestock and poultry to enhance animal growth. Dietary supplementation of betaine in laying hens or gestational sows has been reported to promote the growth of their offspring. Here, we sought to investigate whether and how dietary betaine supplementation affects the growth and development of offspring goslings. In this study, goose breeders, both male and female, were fed a basal diet supplemented respectively with 0, 2.5, or 5 g/kg betaine for 7 wk. Goslings hatched from the breeder eggs of different groups were raised under the same standard condition for assessing the growth performance. Parental betaine increases the growth rate of offspring goslings with decreased DNA methylation on the IGF-2 gene promoter and increased expression of the IGF-2 gene in the liver. These results provide scientific evidence for the inter-generational effect of betaine on gosling growth.

[Buyang Huanwu Decoction promotes arteriogenesis after hindlimb ischemia in mice by activating PDGF signaling pathway].

This study aims to investigate the effect of Buyang Huanwu Decoction on blood flow recovery and arteriogenesis after hindlimb ischemia in mice via the platelet-derived growth factor(PDGF) signaling pathway. Forty C57BL/6 mice were randomized into model(clean water, 10 mL·kg~(-1)·d~(-1)), beraprost sodium(positive control, 18 µg·kg~(-1)·d~(-1)), and low-, medium-, and high-dose(10, 20, and 40 g·kg~(-1)·d~(-1), respectively) Buyang Huanwu Decoction groups(n=8). The hindlimb ischemia model was established by femoral artery ligation. The mice were administrated with corresponding agents by gavage daily for 14 days after ligation. For laser Doppler perfusion imaging, the mice were anesthetized and measured under a Periscan PSI imager. The density of capillary and arterio-le in the ischemic gastrocnemius was measured using immunofluorescence staining of the frozen tissue sections. Western blot was employed to determine the expression of PDGF subunit B(PDGFB), phosphorylated mitogen extracellular kinase(p-MEK), MEK, phosphorylated extracellular signal-regulated kinase(p-ERK), and ERK. Real-time PCR was employed to determine the mRNA level of PDGFB. The Buyang Huanwu Decoction-containing serum was used to treat the vascular smooth muscle cells(VSMCs) in hypoxia at doses of 10% and 20%. The proliferation and migration of VSMCs was assessed in vitro. The results showed that compared with the model group, beraprost sodium and Buyang Huanwu Decoction enhanced the blood flow recovery, increased the capillary and arteriole density, and up-regulated the protein levels of PDGFB, p-MEK, p-ERK, and mRNA levels of PDGFB, with the medium-dose Buyang Huanwu Decoction demonstrating the most significant effect. The 10% Buyang Huanwu Decoction-containing serum enhanced the proliferation and migration of VSMCs. Our findings demonstrate that Buyang Huanwu Decoction up-regulates PDGFB transcription and activates PDGF signaling pathway to promote arteriogenesis and blood flow recovery in ischemic gastrocnemius.