ABSTRACT
ObjectiveTo explore the therapeutic effect and mechanism of Guipitang on rats with myocardial ischemia. MethodFifty SD rats were divided into five groups: a control group, a model group, low and high-dose Guipitang (7.52, 15.04 g·kg-1) groups, and a trimetazidine group (0.002 g·kg-1). By intragastric administration of vitamin D3 and feeding rats with high-fat forage and injecting isoproterenol, the rat model of myocardial ischemia was established. After drug treatment of 15 d, an electrocardiogram (ECG) was performed to analyze the degree of myocardial injury. A fully automatic biochemical analyzer was used to detect the changes in the serum levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C). Hematoxylin-eosin (HE) staining and Masson staining were used to observe myocardial histopathological changes. TdT-mediated dUTP nick end labeling (TUNEL) staining was used to detect cardiomyocyte apoptosis. Western blot was adopted to detect the protein levels of extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-ERK1/2 (p-ERK1/2), p38 mitogen-activated protein kinase (p38 MAPK), phospho-p38 MAPK (p-p38 MAPK), B-cell lymphoma-2 (Bcl-2)-associated X (Bax), Bcl-2, and cleaved cysteine aspartate proteolytic enzyme (cleaved Caspase-3). ResultCompared with the control group, the ECG S-T segment decreased in the model group. The serum levels of TC, TG, and LDL-C were increased significantly (P<0.05). The arrangement of myocardial tissue was disordered, and the proportion of cardiomyocyte apoptosis increased. The protein levels of cleaved Caspase-3, Bax, and p-p38 MAPK in the heart were increased, and the Bcl-2 expression was decreased (P<0.05). Compared with the model group, the S-T segment downward shift was restored in the low and high-dose Guipitang groups and trimetazidine group, and the levels of TC, TG, and LDL-C were decreased. The protein expression of cleaved Caspase-3 and Bax in the heart dropped, and p-p38 MAPK and p-ERK1/2 protein expressions increased significantly (P<0.05). The degree of myocardial injury was alleviated, and the proportion of cardiomyocyte apoptosis decreased. Bcl-2 protein expression was increased significantly in the low-dose Guipitang group (P<0.05). ERK1/2 and p38 MAPK proteins had no significant difference among different groups. ConclusionGuipitang could alleviate myocardial injury and inhibit cardiomyocyte apoptosis in rats by activating the expression of ERK1/2 and p38 MAPK.
ABSTRACT
ObjectiveTo investigate the regulatory effect of Danggui Shaoyaosan on adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/Unc-51-like kinase-1 (ULK1) signaling pathway in the rat model of metabolism-associated fatty liver disease (MAFLD). MethodSixty SD rats were randomized into control, model, western medicine (polyene phosphatidylcholine capsules,0.144 g·kg-1), and low-, medium-, and high-dose (2.44, 4.88, 9.76 g·kg-1, respectively) Danggui Shaoyaosan groups. After being fed with a high-fat diet for 8 weeks, the rats in each group were administrated with corresponding drugs for 4 weeks. At the end of drug treatment, serum and liver tissue were collected for subsequent determination of related indicators. ResultCompared with the control group, the model group showed increased contents of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum, increased contents of TC, TG, and free fatty acids (FFAs) in the liver (P<0.01), and decreased content of high-density lipoprotein cholesterol (HDL-C) in the serum (P<0.01). Furthermore, the model group showed down-regulated protein levels of p-AMPK, microtubule-associated protein 1 light chain 3B (LC3B) Ⅱ, Beclin1, and ULK1 (P<0.01) and up-regulated protein levels of p-mTOR and ubiquitin-binding protein p62 in the liver (P<0.01). The hepatic steatosis was obvious and the NAFLD activity score (NAS) and oil red O staining area increased in the model group, (P<0.05, P<0.01). Compared with the model group, Danggui Shaoyaosan reduced the contents of TC and TG and the activities of ALT and AST in the serum, lowered the levels of TC, TG, and FFA in the liver, down-regulated the protein levels of p-mTOR and p62 (P<0.01), elevated the serum HDL-C level, and up-regulated the protein levels of p-AMPK, LCBⅡ, Beclin1, and ULK1 in the liver (P<0.05, P<0.01). Moreover, it alleviated hepatic steatosis and decreased the NAS and oil red O staining area (P<0.05, P<0.01). ConclusionDanggui Shaoyaosan has therapeutic effect on MAFLD rats by regulating AMPK/mTOR/ULK1 signaling pathway to enhance autophagy.
ABSTRACT
ObjectiveTo explore the mechanism and pathway of Gandou Fumu decoction (GDFMD) in the development of liver fibrosis in Wilson's disease (WD). MethodFirst, 30 TX-j mice were randomly divided into the model group, high-dose, medium-dose, and low-dose GDFMD groups, and penicillamine group, with six mice in each group, and another six wild-type mice were used as the normal group. The high-dose, medium-dose, and low-dose GDFMD groups were intragastrically administered drugs of 13.92, 6.96, 3.48 g·kg-1. In the penicillamine group, 0.1 g·kg-1 of penicillamine was given by intragastric administration. The model group and the normal group were given equal volume of normal saline, once a day, for four consecutive weeks. Samples were collected four weeks after gavage, and enzyme-linked immunosorbent assay (ELISA) was used to detect type Ⅲ procollagen peptide (PCⅢ), collagen type Ⅳ (Col Ⅳ), hyaluronic acid (HA), and laminin (LN). Hematoxylin-eosin (HE), Masson, and picric acid-Sirus red collagen (Sirus Red) staining were used to observe the histopathological changes of liver fibrosis. Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR), immunohistochemistry, and Western blot were used to observe the expressions of α-smooth muscle actin (α-SMA) and collagen type Ⅰ (Col Ⅰ), which were related to the activation of hepatic stellate cells (HSCs). The expression of miR-29b-3p was observed by Real-time PCR. The expression of Unc-51-like kinase 1 (ULK1) and its downstream-related factors were observed by Western blot. The downstream genes of miR-29b-3p were verified by the dual luciferase reporter gene detection method. ResultCompared with the normal group, the four items of liver fibrosis (PCⅢ, Col Ⅳ, HA, and LN) in the model group were significantly abnormal (P<0.01), and the pathology was significantly abnormal. The expression of HSC activation-related indicators including α-SMA and Col Ⅰ, as well as α-SMA mRNA and Col Ⅰ mRNA was up-regulated (P<0.05, P<0.01), and miR-29b-3p expression was down-regulated (P<0.01). ULK1, p-ULK1, autophagy-related gene 13 (Atg13), p-Atg13, Beclin-1, FAK family kinase-interacting protein of 200 kDa (FIP200), activating molecule in BECN1-regulated autophagy protein 1 (AMBKA1), and microtubule-associated protein 1 light chain 3Ⅱ/Ⅰ(LC3Ⅱ/Ⅰ) were up-regulated (P<0.05, P<0.01). p62 protein expression was down-regulated (P<0.01). Compared with the model group, the four items of liver fibrosis in the high-dose, medium-dose, and low-dose GDFMD groups and the penicillamine group were significantly improve (P<0.01), and the pathological conditions were improved. The expression of HSC activation-related indicators including α-SMA and Col Ⅰ, as well as α-SMA mRNA and Col Ⅰ mRNA was down-regulated (P<0.05, P<0.01), and the expression of miR-29b-3p was up-regulated (P<0.01). ULK1, p-ULK1, Atg13, p-Atg13, Beclin-1, FIP200, AMBKA1, and LC3Ⅱ/Ⅰ were down-regulated (P<0.05, P<0.01), and p62 protein expression was up-regulated (P<0.01). The prediction software predicted that there was a binding site between miR-29b-3p and ULK1. The dual-luciferase reporter gene detection method indicated that the luciferase activity of the ULK1-WT plasmid-transfected cell group was reduced when miR-29b-3p mimics were co-cultured (P<0.01). ConclusionGDFMD can regulate ULK1-mediated autophagy by up-regulating miR-29b-3p and further exert its anti-hepatic fibrosis effect in Wilson's disease.
ABSTRACT
ObjectiveTo investigate the effect of Yishen Tongluo prescription (YSTLP) on apoptosis of renal tubular epithelial cells and explore the mechanism based on endoplasmic reticulum stress pathway of protein kinase R-like endoplasmic reticulum kinase (PERK)/activating transcription factor 4 (ATF4)/transcription factor C/EBP homologous protein (CHOP). MethodThe db/db mice were randomly divided into model group, valsartan group (10 mg·kg-1), and low, middle, high-dose YSTLP groups (1, 2.5, 5 g·kg-1). Samples were collected after eight weeks of drug intervention. In addition, db/m mice in the same litter served as the control group. Human renal tubular epithelial cells (HK-2) were cultured in vitro and divided into the control group, advanced glycated end-product (AGE) group, and AGE + low, middle, and high-dose YSTLP groups (100, 200, 400 mg·L-1). TdT-mediated dUTP nick end labeling (TUNEL) staining was used to detect the apoptosis rate of HK-2 cells. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay was conducted to detect the viability of HK-2 cells. Calcium fluorescence probe staining and luciferase reporter gene method were adopted to detect the luciferase activity of folded protein response element (UPRE) and endoplasmic reticulum stress. Immunohistochemical (IHC) analysis was carried out to measure the protein expressions of phosphorylated PKR (p-PERK), CHOP, and ATF4. Real-time polymerase chain reaction (Real-time PCR) was used to measure the mRNA expression levels of CHOP and X-box binding protein 1 (XBP1) in mouse kidney and HK-2 cells. Western blot was used to detect the protein expression level of p-PERK, PERK, CHOP, ATF4, B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), and cleaved Caspase-3 in mouse kidney and HK-2 cells. ResultIn the cellular assay, HK-2 cell viability was significantly reduced, and the apoptosis rate was elevated in the AGE group compared with the control group (P<0.01). The mRNA and protein expression levels of apoptosis-related factor Bcl-2 were significantly reduced (P<0.01), and those of Bax were significantly increased (P<0.01). The protein expression level of cleaved Caspase-3 was significantly increased (P<0.01). Compared with the AGE group, YSTLP administration treatment resulted in elevated cell viability and reduced apoptosis rate (P<0.01). The mRNA and protein expression levels of Bcl-2 were significantly elevated in a time- and dose-dependent manner (P<0.01), and those of Bax were significantly reduced in a time- and dose-dependent manner. The protein expression level of cleaved Caspase-3 was significantly reduced in a time- and dose-dependent manner (P<0.01). The intracellular Ca2+ imbalance and UPRE luciferase fluorescence intensity were increased in the AGE group compared with the control group (P<0.01). The mRNA levels of endoplasmic reticulum stress-related factors CHOP and XBP1 were significantly increased (P<0.01), and the protein expression levels of p-PERK, CHOP, and ATF4 were significantly increased (P<0.05). Compared with the AGE group, YSTLP effectively improved intracellular Ca2+ imbalance in HK-2 cells and decreased UPRE luciferase fluorescence intensity in a dose-dependent manner (P<0.01). It reduced the mRNA levels of endoplasmic reticulum stress-related factors CHOP and XBP1 (P<0.01) and the protein expression levels of intracellular p-PERK, CHOP, and ATF4 in a dose- and time-dependent manner (P<0.01). In animal experiments, the protein expression level of Bcl-2 was significantly reduced(P<0.01), and that of cleaved Caspase-3 and Bax was significantly increased in the model group compared with the control group (P<0.05). The protein expression level of Bcl-2 was dose-dependently elevated, and that of cleaved Caspase-3 and Bax was dose-dependently decreased in the YSTLP groups compared with the model group (P<0.01). Compared with the control group, the mRNA expression levels of CHOP and XBP1 were significantly elevated in the model group (P<0.05, P<0.01), and the protein expression levels of p-PERK, CHOP, and ATF4 were significantly increased (P<0.05). Compared with the model group, YSTLP significantly decreased the mRNA expression levels of CHOP and XBP1 (P<0.01) and the protein expression levels of p-PERK, CHOP, and ATF4 (P<0.01). ConclusionYSTLP can effectively inhibit endoplasmic reticulum stress and improve apoptosis of renal tubular epithelial cells, and its mechanism may be related to the regulation of the PERK/AFT4/CHOP pathway.
ABSTRACT
ObjectiveTo investigate the effects of Jiaohong pills (JHP) and its prescription, Pericarpium Zanthoxyli (PZ) and Rehmanniae Radix (RR) cognitive dysfunction in scopolamine-induced Alzheimer's disease (AD) mice and its mechanism through pharmacodynamic and metabolomics study. MethodThe animal model of AD induced by scopolamine was established and treated with PZ, RG and JHP, respectively. The effects of JHP and its formulations were investigated by open field test, water maze test, object recognition test, avoidance test, cholinergic system and oxidative stress related biochemical test. Untargeted metabolomics analysis of cerebral cortex was performed by ultra-performance liquid chromatography-Quadrupole/Orbitrap high resolution mass spectrometry (UPLC Q-Exactive Orbitrap MS). ResultThe behavioral data showed that, compared with the model group, the discrimination indexes of the high dose of JHP, PZ and RR groups was significantly increased (P<0.05). The staging rate of Morris water maze test in the PZ, RR, high and low dose groups of JHP was significantly increased (P<0.05, P<0.01), the crossing numbers in the PZ, JHP high and low dose groups were significantly increased (P<0.05, P<0.01); the number of errors in the avoidance test were significantly reduced in the PZ and high-dose JHP groups (P<0.01), and the error latencies were significantly increased in the JHP and its prescription drug groups (P<0.01). Compared with the model group, the activities of acetylcholinesterase in the cerebral cortex of the two doses of JHP group and the PZ group were significantly increased (P<0.05, P<0.01), and the activity of acetylcholinesterase in the high-dose JHP group was significantly decreased (P<0.05), and the level of acetylcholine was significantly increased (P<0.01). At the same time, the contents of malondialdehyde in the serum of the two dose groups of JHP decreased significantly (P<0.05, P<0.01). The results of metabolomics study of cerebral cortex showed that 149 differential metabolites were identified between the JHP group and the model group, which were involved in neurotransmitter metabolism, energy metabolism, oxidative stress and amino acid metabolism. ConclusionJHP and its prescription can antagonize scopolamine-induced cognitive dysfunction, regulate cholinergic system, and reduce oxidative stress damage. The mechanism of its therapeutic effect on AD is related to the regulation of neurotransmitter, energy, amino acid metabolism, and improvement of oxidative stress.
ABSTRACT
ObjectiveTo investigate the different effects of Yunvjian with or without Achyranthis Bidentatae Radix on glucose and lipid metabolism and inflammatory response in diabetic rats with the syndrome of Yin deficiency and internal heat. MethodThe rat model of diabetes due to Yin deficiency and internal heat was established by feeding with a high-sugar and high-fat diet and injection of thyroxine and streptozotocin. The successfully modeled rats were randomized into model control, Yunvjian without Achyranthis Bidentatae Radix (11.8 g·kg-1), Yunvjian with Achyranthis Bidentatae Radix (12.8 g·kg-1), and Achyranthis Bidentatae Radix (1.0 g·kg-1) groups (n=10), and another 10 rats were taken as the normal control group. Each group was administrated with corresponding drugs or saline by gavage for 28 days. The fasting blood glucose (FBG), fasting insulin (FINS), total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in rats were measured. Enzyme-linked immunosorbent assay was employed to determine the levels of cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), triiodothyronine (T3), thyroxine (T4), interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and C-reactive protein (CRP) in the serum. The histopathological changes of the liver were observed. The expression of lipoxygenase-2 (COX-2) was detected by immunofluorescence. The mRNA levels of nuclear transcription factors-κB (NF-κB), monocyte chemotactic protein-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) were determined by real-time polymerase chain reaction (Real-time PCR).Western blot was employed to determine the protein levels of NF-κB in hibitory protein(IκB) kinase β (IKKβ), IκBα, and phosphorylated IκBα (p-IκBα) in the liver and the protein levels of NF-κB in the cytoplasm and nucleus. ResultCompared with the normal group, the model group showed elevated levels of FBG, FINS, insulin resistance index, TC, TG, LDL-C, cAMP, T3, T4, IL-1β, IL-6, TNF-α, and CRP, up-regulated mRNA levels of NF-κB, MCP-1, and ICAM-1, and up-regulated protein levels of COX-2, p-IκBα, and nuclear NF-κB (P<0.01). Compared with the model group, Yunvjian without Achyranthis Bidentatae Radix lowered the levels of FBG, FINS, insulin resistance index, TC, TG, LDL-C, cAMP, T3, T4, IL-1β, IL-6, TNF-α, and CRP, down-regulated the mRNA levels of NF-κB, MCP-1, and ICAM-1, and down-regulated the protein levels of COX-2, p-IκBα and nuclear NF-κB (P<0.05, P<0.01). Compared with the Yunvjian without Achyranthis Bidentatae Radix, Yunvjian with Achyranthis Bidentatae Radix showed lowered levels of FBG, FINS, insulin resistance index, and inflammatory cytokines, down-regulated mRNA levels of NF-κB, MCP-1, and ICAM-1, and down-regulated protein levels of p-IκBα and nuclear NF-κB (P<0.05, P<0.01). ConclusionAchyranthis Bidentatae Radix can enhance the performance of Yunvjian in reducing blood glucose and inhibiting inflammation in diabetic rats with the syndrome of yin deficiency and internal heat by down-regulating the IKK/IκB/NF-κB signaling pathway.
ABSTRACT
ObjectiveBy observing the effect of Qianyang Yuyin granules on the phenotype of renal tubule epithelial cells, the intervention of Qianyang Yuyin granule on renal interstitial fibrosis was investigated. MethodThe renal tubular epithelial cells (HK-2) were treated with different concentrations of transforming growth factor (TGF)-β1 (5, 10, 15, 20, 25 μg·L-1) for 24 hours, and cell morphology and growth state were observed with an inverted phase contrast microscope. The 20 μg·L-1 was selected as the most appropriate concentration of TGF-β1 according to Western blot results for subsequent experiments. HK-2 cells were divided into six groups: blank group, TGF-β1 group (concentration of 20 μg·L-1), low, medium, and high dose Qianyang Yuyin granule groups (concentration of 0.5, 1, 2 g·L-1), and valsartan group (1 × 10-5 mol·L-1). The cell activity was measured by cell proliferation and cell counting kit-8 (CCK-8). The cell migration ability was detected by scratch test. The Transwell method was used to detect the invasiveness of cells. Western blot was used to detect levels of fibronectin (FN), E-cadherin, α-smooth muscle activator (α-SMA), Vimentin, collagen type Ⅰ(Col Ⅰ), collagen type Ⅳ(Col Ⅳ), and other related proteins. ResultTGF-β1 stimulating epithelial-mesenchymal transition (EMT) in renal tubular epithelial cells was time- and concentration-dependent. Compared with the blank group, higher concentration in the TGF-β1 group indicates longer intervention time and more obvious long spindle change of cells, and the migration and invasion ability of the cells was significantly enhanced. The protein expression level of FN, α-SMA, Vimentin, Col Ⅰ, and Col Ⅳ increased significantly (P<0.05, P<0.01), while the expression level of E-cadherin protein decreased (P<0.05). Compared with the TGF-β1 group, Qianyang Yuyin granule groups could maintain normal cell morphology, and the migration and invasion ability of the cells was inhibited. The protein expression level of FN, α-SMA, Vimentin, Col Ⅰ, and Col Ⅳ decreased (P<0.05, P<0.01), and the expression of E-cadherin protein was significantly restored (P<0.05). ConclusionQianyang Yuyin granule can reverse TGF-β1-induced interstitial transformation of renal tubular epithelial cells by reducing the phenotypic expression of mesenchymal cells and increasing the phenotypic expression of epithelial cells.
ABSTRACT
ObjectiveTo explore the protective effect and mechanism of Zingiberis Rhizoma Recens alcohol extract on lipopolysaccharide (LPS)-induced acute lung injury in mice. MethodBalb/c mice were randomly divided into normal group, model group, dexamethasone group, and low- and high-dose Zingiberis Rhizoma Recens groups. Mice in the normal group were instilled with normal saline through the nose, and the other groups were instilled with normal saline containing LPS (50 μg). After 30 minutes of modeling, the dexamethasone group was gavaged with 5 mg·kg-1 of dexamethasone acetate solution, the low- and high-dose Zingiberis Rhizoma Recens groups were gavaged with different doses of (7, 14 g·kg-1) of Zingiberis Rhizoma Recens alcohol extract, and the normal group and the model group were gavaged with the same volume of water. After 24 hours of modeling, the total number of white blood cells in bronchoalceolar lavage fluid (BALF) was detected by cell counter, and the levels of the inflammatory factors including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and superoxide dismutase (SOD), and myeloperoxidase (MPO) was detected by enzyme-linked immunosorbent assay (ELISA). Haematoxylin-eosin (HE) staining method was used to observe the pathological changes of lung tissue in each group, and the Western blot was used to detect the protein expression of nuclear transcription factor (NF)-κB p65, phosphorylation (p)-NF-κB p65, and Toll-like receptor 4 (TLR4) in lung tissue. ResultCompared with the normal group, the white blood cell count in BALF and the levels of TNF-α, IL-1β, IL-6, and MPO in the model group was increased (P<0.01), and the level of SOD was decreased (P<0.05). Pathological damage of lung tissue was obvious, and the protein expression of NF-κB p65, p-NF-κB p65, and TLR4 in lung tissue was increased (P<0.01). Compared with the model group, the white blood cell count in BALF and the levels of TNF-α, IL-1β, IL-6, and MPO in the treatment group was decreased (P<0.05,P<0.01), and the level of SOD was increased (P<0.05,P<0.01). Pathological damage of lung tissue was alleviated, and the protein expression of NF-κB p65, p-NF-κB p65, and TLR4 in lung tissue was decreased (P<0.01). ConclusionZingiberis Rhizoma Recens alcohol extract may play a protective role in LPS-induced acute lung injury in mice by inhibiting the TLR4/NF-κB signaling pathway.
ABSTRACT
ObjectiveTo investigate the effect and mechanism of total saponins from Panax japonicus (TSPJ) on white adipose tissue (WAT) browning/brown adipose tissue (BAT) activation in C57BL6/J male mice fed on a high-fat diet (HFD). MethodThirty-two C57BL6/J male mice (8-week-old) were randomly divided into a normal group, a model group, a low-dose TSPJ group, and a high-dose TSPJ group. The mice in the low-dose and high-dose TSPJ groups were given TSPJ for four months by gavage at 25, 75 mg·kg-1·d-1, respectively, and those in the other groups were given 0.5% sodium carboxymethyl cellulose (CMC-Na) accordingly. After four months of feeding, all mice were placed at 4 ℃ for acute cold exposure, and the core body temperature was monitored. Subsequently, all mice were sacrificed, and BAT and inguinal WAT (iWAT) were separated rapidly to detect the corresponding indexes. Hematoxylin-eosin (HE) staining was used to observe the morphological changes in each group. The effect of TSPJ on the mRNA expression of uncoupling protein 1 (UCP1), fatty acid-binding protein 4 (FABP4), cytochrome C (CytC), PR domain-containing protein 16 (PRDM16), elongation of very long chain fatty acids protein 3 (ELOVL3), peroxisome proliferator-activated receptor γ (PPARγ), and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) in iWAT and BAT was detected by Real-time polymerase chain reaction (Real-time PCR). Western blot was also used to detect the protein expression of UCP1, PRDM16, PPARγ, and PGC-1α in BAT and iWAT of each group. The effect of TSPJ on UCP1 expression in BAT and iWAT was detected by immunohistochemistry. Result① Compared with the model group, TSPJ could decrease the body weight and proportions of iWAT and BAT in the HFD-induced mice (P<0.05, P<0.01). ② The body temperature of mice in the model group decreased compared with that in the normal group in the acute cold exposure tolerance test (P<0.05). The body temperature in the high-dose TSPJ group increased compared with that in the model group (P<0.01). ③ Compared with the normal group, the model group showed increased adipocyte diameter in iWAT and BAT and decreased number of adipocytes per unit area. Compared with the model group, the TSPJ groups showed significantly reduced cell diameter and increased number of cells per unit area, especially in the high-dose TSPJ group. ④ Compared with the normal group, the model group showed decreased mRNA expression of FABP4, UCP1, CytC, PRDM16, ELOVL3, PGC-1α, and PPARγ in adipose tissues of mice (P<0.05, P<0.01). Compared with the model group, after intervention with TSPJ, the mRNA expression was significantly up-regulated (P<0.05, P<0.01). ⑤ Compared with the normal group, the model group showed decreased protein expression of UCP1, PRDM16, PPARγ, and PGC-1α in adipose tissues of mice (P<0.05, P<0.01). Compared with the model group, after intervention with TSPJ, the protein expression increased significantly (P<0.05, P<0.01). ConclusionTSPJ could induce the browning of iWAT/BAT activation and enhance adaptive thermogenesis in obese mice induced by HFD. The underlying mechanism may be attributed to the activation of the PPARγ/PGC-1α signaling pathway.
ABSTRACT
ObjectiveTo explore the mechanism of Bushen Huoxue enema in treating the rat model of kidney deficiency and blood stasis-thin endometrium (KDBS-TE) by transcriptome sequencing. MethodThe rat model of KDBS-TE was established by administration of tripterygium polyglycosides tablets combined with subcutaneous injection of adrenaline. The pathological changes of rat endometrium in each group were then observed. Three uterine tissue specimens from each of the blank group, model group, and Bushen Huoxue enema group were randomly selected for transcriptome sequencing. The differentially expressed circRNAs, lncRNAs, and miRNAs were screened, and the disease-related specific competitive endogenous RNA (ceRNA) regulatory network was constructed. Furthermore, the gene ontology (GO) functional annotation and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were performed for the mRNAs in the network. ResultCompared with the blank group, the model group showed endometrial dysplasia, decreased endometrial thickness and endometrial/total uterine wall thickness ratio (P<0.01), and differential expression of 18 circRNAs, 410 lncRNAs, and 7 miRNAs. Compared with the model group, the enema and estradiol valerate groups showed improved endometrial morphology and increased endometrial thickness and ratio of endometrial to total uterine wall thickness (P<0.05). In addition, 21 circRNAs, 518 lncRNAs, and 17 miRNAs were differentially expressed in the enema group. The disease-related specific circRNA-miRNA-mRNA regulatory network composed of 629 nodes and 664 edges contained 2 circRNAs, 34 miRNAs, and 593 mRNAs. The lncRNA-miRNA-mRNA regulatory network composed of 180 nodes and 212 edges contained 5 lncRNAs, 10 miRNAs, and 164 mRNAs. The mNRAs were mainly enriched in Hippo signaling pathway, autophagy-animal, axon guidance, etc. ConclusionBushen Huoxue enema can treat KDBS-TE in rats by regulating specific circRNAs, lncRNAs, and miRNAs in the uterus and the ceRNA network.
ABSTRACT
ObjectiveTo observe the effects and underlying mechanisms of Fagopyri Dibotryis Rhizoma extract on the proliferation, apoptosis, and autophagy of human colorectal cancer HCT-116 cells. MethodFirstly, cellular activity was detected by the cell proliferation and activity-8 (CCK-8) assay, and the half inhibition rate (IC50) was calculated. Blank group and Fagopyri Dibotryis Rhizoma group (2, 4, 8 mg·L-1) were set. The effect of Fagopyri Dibotryis Rhizoma on the proliferation of HCT-116 cells was observed by cloning experiments, and that of Fagopyri Dibotryis Rhizoma on apoptosis was observed by flow cytometry. The expressions of autophagy-related proteins, p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), phosphorylated (p)-p38, p-ERK, p-JNK, and other proteins were detected by Western blot. Finally, flow cytometry instrumentation and fluorescence microscopy were used to analyze the changes in reactive oxygen species (ROS), and a ROS scavenger (NAC) was adopted for verification. ResultCompared with the blank group, the activity of HCT-116 cells was significantly decreased in the Fagopyri Dibotryis Rhizoma group (P<0.05, P<0.01). The apoptosis rate of HCT-116 cells in the Fagopyri Dibotryis Rhizoma group was significantly increased (P<0.01). The expression of autophagy-related protein ubiquitin-binding protein (p62) was decreased, but that of autophagy-specific genes (Beclin1) and autophagic microtubule-associated protein 1 light-chain 3B (LC3B) was enhanced (P<0.05, P<0.01). Compared with the Fagopyri Dibotryis Rhizoma group, the apoptosis rate of HCT-116 cells in the Fagopyri Dibotryis Rhizoma + NAC group was significantly reduced (P<0.01). The expression of related autophagy protein Beclin1 was significantly reduced (P<0.01), and that of LC3B protein was reduced (P<0.01). In addition, the expression of MAPK pathway-related proteins ERK and JNK was decreased in the Fagopyri Dibotryis Rhizoma group (P<0.05, P<0.01), and that of p-ERK and p-JNK was enhanced (P<0.05, P<0.01). ConclusionFagopyri Dibotryis Rhizoma can inhibit the proliferation of HCT-116 cells and induce apoptosis and autophagy through the ROS/MAPK signaling pathway.
ABSTRACT
ObjectiveTo investigate the effect of Tangzhi pills on the improvement of insulin resistance (IR) in the liver with type 2 diabetes (T2DM) by regulating phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway based on differential genes and its possible molecular mechanism. MethodT2DM rat models were prepared by high fat (HFD) diet combined with streptozotocin (STZ) intraperitoneal injection. The experiment was divided into blank group, model group, metformin hydrochloride group (0.18 g·kg-1), Tangzhi pills high (1.08 g·kg-1), medium (0.54 g·kg-1) and low (0.27 g·kg-1) dose groups. Rat serum, liver, and pancreatic tissue were collected, and the pathological tissue of the liver and pancreas was observed using hematoxylin-eosin (HE) staining. The fasting blood glucose level (FBG) was detected, and oral glucose tolerance (OGTT) tests were conducted. Enzyme-linked immunosorbent assay (ELISA) was used to detect fasting serum insulin (FINS) and glycated hemoglobin (GHb) levels in rats. IR homeostasis model index (HOMA-IR), β cellular homeostasis index (HOMA-β), and insulin sensitivity index (ISI) were calculated. Biochemical methods were used to determine the levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL-C), and high-density lipoprotein (HDL-C) in rat serum. Transcriptomics obtained differentially expressed mRNA from liver tissue and enriched differentially expressed pathways. Real-time reverse transcriptase polymerase chain reaction (Real-time PCR) was used to detect the mRNA expression of cyclic adenylate responsive element binding protein 3-like protein 2 antibody (CREB3l2), B-lymphocyte tumor 2 (Bcl-2), Toll-like receptor 2 (TLR2), cyclin-dependent kinase inhibitor 1A (CDNK1A), and DNA damage induced transcription factor 4-like protein (DDIT4) in liver tissue. Western blot was used to detect the protein expression of phosphorylated phosphatidylinositol 3-kinase (p-PI3K), phosphorylated protein kinase B (p-Akt), glucose transporter 4 (GLUT4), insulin receptor (INSR), and insulin receptor substrate 2 (IRS2). ResultThe pharmacodynamic experiment results showed that compared with model group, Tangzhi pills groups repaired liver and pancreatic tissue to varying degrees, reduced blood sugar (P<0.01), and promoted a decrease in serum FINS, GHb, and HOMA-IR (P<0.05, P<0.01). In addition, HOMA-β and ISI increased (P<0.05, P<0.01). The levels of TC, TG, and LDL-C decreased (P<0.05, P<0.01), while the levels of HDL-C increased (P<0.05, P<0.01). The transcriptomics experimental results confirmed that the PI3K/Akt signaling pathway was significantly expressed in both the blank group and model group, as well as in the high-dose Tangzhi pills group and model group. CDNK1A, DDIT4, CREB3l2, Bcl-2, and TLR2 were significantly differentially expressed mRNA during TG intervention in T2DM. Compared with the model group, the protein expression of p-PI3K, p-Akt, GLUT4, INSR, and IRS2 increased in all Tangzhi pills groups (P<0.01). The mRNA expression of CREB3l2, Bcl-2, and TLR2 increased (P<0.01), while that of CDNK1A and DDIT4 decreased (P<0.01). ConclusionTangzhi pills may regulate the PI3K/Akt signaling pathway based on the differential mRNA expression of CREB3l2, Bcl-2, TLR2, CDNK1A, and DDIT4, thereby improving IR in the liver with T2DM.
ABSTRACT
ObjectiveTo observe the effects of Hirudo, Notoginseng Radix et Rhizoma, and drug pair on renal pathological morphology and protein phosphatase 2A (PP2A)/adenylate activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signal pathway in rats with chronic renal failure (CRF). MethodThe 55 male SD rats were randomly divided into a normal group (n=11) and a modeling group (n=44). The normal group was fed conventionally, and the modeling group was given 0.25 g·kg-1·d-1 adenine by gavage for 28 days to replicate the CRF model. After successful modeling, rats were randomly divided into model group, Hirudo group (3 g·kg-1·d-1), Notoginseng Radix et Rhizoma group (3 g·kg-1·d-1), and Hirudo + Notoginseng Radix et Rhizoma group (3 g·kg-1·d-1), with 9 rats in each group. The normal group and model group were given a constant volume of normal saline by intragastric administration for 30 days. At the end of the experiment, the levels of serum creatinine (SCr) and urea nitrogen (BUN) in all groups were measured. The renal pathological morphology changes were observed by hematoxylin-eosin (HE) staining, Masson staining, and electron microscopy. The mRNA expressions of PP2A, AMPK, and mTOR were detected by Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR). The protein expression levels of PP2A, AMPK, phosphorylation(p)-AMPK, mTOR, and p-mTOR in renal tissue were detected by Western blot. ResultCompared with the normal group, the renal pathological structure changes were obvious, and the levels of SCr and BUN were significantly increased. The mRNA expression of PP2A, protein expression of PP2A, and p-mTOR/mTOR expression were significantly increased, and the p-AMPK/AMPK was significantly decreased in the model group (P<0.05). Compared with the model group, the renal pathological morphology changes were significantly improved, and the levels of SCr and BUN were significantly decreased. The mRNA expression of PP2A, protein expression of PP2A, and p-mTOR/mTOR expression in the renal tissue were significantly decreased, and the p-AMPK/AMPK was significantly increased (P<0.05) in all groups after drug intervention. In addition, the effect in the Hirudo+Notoginseng Radix et Rhizoma group was better. The mRNA expression levels of AMPK and mTOR in the renal tissue were not significantly different among the normal group, model group, and other groups. ConclusionThe efficacy of Hirudo and Notoginseng Radix et Rhizoma pairs in improving renal fibrosis in rats with CRF is significantly better than that of the single drug, and its improvement on renal fibrosis in rats with CRF may be related to the regulation of PP2A/AMPK/mTOR signaling pathway.
ABSTRACT
ObjectiveTo observe the effect of earthworm protein on the expression of phosphatidylinositol 3-kinase/protein kinase B/nuclear factor E2-related factor 2 (PI3K/Akt/Nrf2) pathway in the aorta of spontaneously hypertensive rats (SHR) and explore mechanism of earthworm protein in treating hypertensive vascular endothelial dysfunction (VED). MethodTen 10-week-old Wistar Kyoto (WKY) rats and fifty SHR rats were selected for a week of adaptive feeding. WKY rats were selected as the normal group, and fifty SHR rats were randomized according to body weight into model, valsartan (8×10-3 g·kg-1·d-1), and high-, medium-, and low-dose (0.2, 0.1, 0.05 g·kg-1·d-1, respectively) earthworm protein groups. The normal and model groups were administrated with equal volume of double distilled water by gavage. During the drug intervention period, the general situations of rats in each group were observed and their blood pressure was monitored at specific time points every other week before and after administration. After 8 weeks of drug intervention, enzyme-linked immunosorbent assay was employed to measure the levels of angiotensin-Ⅱ (Ang-Ⅱ) and endothelin-1 (ET-1) in the serum of rats in each group. The corresponding kits were used to determine the levels of nitric oxide (NO), malondialdehyde (MDA), glutathione peroxidase (GPX), superoxide dismutase (SOD), and ferrous ion (Fe2+). Hematoxylin-eosin (HE) staining was employed to observe the changes in the intima of the aorta. Fluorescence quantitative polymerase chain reaction (Real-time PCR) was employed to measure the mRNA levels of PI3K, Akt, Nrf2, heme oxygenase-1 (HO-1), and glutathione peroxidase 4 (GPX4) in the aortic tissue. Western blotting was used to determine the protein levels of p-PI3K (Tyr467/199), PI3K, p-Akt (Ser473), Akt, Nrf2, HO-1, and GPX4 in the thoracic aorta. ResultCompared with the normal group, the model group had decreased body mass, increased irritability, severe endothelial damage, elevated blood pressure and serum levels of Ang-Ⅱ, ET1, MDA, and Fe2+ (P<0.01), lowered NO level (P<0.01), and down-regulated mRNA and protein levels of p-PI3K (Tyr467/199), PI3K, p-Akt (Ser473), Akt, Nrf2, HO-1, and GPX4 in the aortic tissue (P<0.01). Compared with the model group, drug intervention caused no significant change in the body mass, calmed the rats, alleviated the endothelial damage, lowered blood pressure and serum levels of Ang-Ⅱ, ET1, MDA, and Fe2+ (P<0.01), elevated the NO level (P<0.05), and up-regulated the mRNA and protein levels of p-PI3K (Tyr467/199), PI3K, p-Akt (Ser473), Akt, Nrf2, HO-1, and GPX4 (P<0.05). ConclusionThe earthworm protein can exert antihypertensive effects by ameliorating VED in SHR. Specifically, it may regulate the PI3K/Akt/Nrf2 signaling pathway to inhibit oxidative stress and ferroptosis.
ABSTRACT
ObjectiveTo investigate the clinical efficacy and safety of iguratimod combined with the Chinese medicine Runzaoling in the treatment of primary Sjögren's syndrome (pSS). MethodSeventy-two patients treated in the Department of Rheumatology and Immunology of the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine(TCM) from January 2021 to June 2022 who met the Western medical diagnosis of pSS and had the TCM syndrome of Yin deficiency and heat toxin syndrome were randomly assigned into an observation group and a control group, with 36 patients in each group. The observation group was treated with iguratimod combined with Runzaoling, and the control group was treated with iguratimod. The treatment in both groups lasted for 12 weeks. The clinical symptoms, EULAR Sjogren's syndrome patient reported index (ESSPRI), EULAR Sjögren's syndrome disease activity index (ESSDAI), erythrocyte sedimentation Rate (ESR), C-reactive protein (CRP), immunoglobulin (IgG), Schirmer score, and saliva flow of the two groups were determined before and after treatment. Furthermore, the incidence of adverse reactions was compared between the two groups. ResultThe total response rate in the observation group was 75.0% (27 patients with response and 9 patients with no response), which was higher than that (61.11%, 22 patients with response and 14 patients without response) in the control group (P<0.05). After treatment, the ESSPRI, ESSDAI, and TCM syndrome scores in both groups decreased and the decreases were more obvious in the observation group than in the control group (P<0.05). The treatment in both groups recovered the ESR, CRP, IgG, Schirmer score, and saliva flow (P<0.05). Moreover, the observation outperformed the control group in terms of the ESR, CRP, IgG, and saliva flow (P<0.05) and had no significant difference in the Schirmer score compared with the control group. During the treatment period, 2 patients in the observation group had nausea, and 1 patient had an abnormal liver function, which were relieved after symptomatic treatment and did not affect the treatment. In the control group, 1 patient withdrew from the study due to rashes and showed no special discomfort in the follow-up 4 weeks, and 1 patient had nausea, which was relieved after symptomatic treatment. ConclusionIguratimod combined with Runzaoling has good clinical efficacy and safety in the treatment of pSS.
ABSTRACT
ObjectiveTo evaluate the clinical efficacy of modified Banxia Xiexintang combined with vedolizumab (VDZ) in the treatment of active moderate to severe Crohn's disease (CD) with the syndrome of cold and heat in complexity and the effect of the therapy on intestinal flora. MethodEighty patients were randomized based on the random number table method into a control group (40 cases) and an observation group (40 cases). The control group was treated with VDZ, and the observation group was treated with modified Banxia Xiexintang (1 bag per day) combined with VDZ. The treatment in both groups lasted for 14 weeks and the follow-up lasted until the 52th weeks. The CD activity index (CDAI), CD simplified endoscopic score (SES-CD), inflammatory bowel disease questionnaire (IBDQ) score, and syndrome score of cold and heat in complexity were assessed before treatment, after treatment, and at the end of follow-up. The levels of hemoglobin (HGB), hematocrit (HCT), albumin (ALB), C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-17 (IL-17), and fecal calprotectin (FC) were measured before and after treatment. Intestinal flora was examined before and after treatment. The safety of the therapy was evaluated. ResultCompared with those before treatment, the scores of CDAI, SES-CD, and the syndrome of cold and heat in complexity decreased (P<0.05) and the IBDQ score increased after treatment (P<0.05). Compared with those before treatment, the scores of CDAI, SES-CD, and the syndrome of cold and heat in complexity increased (P<0.05) and the IBDQ score decreased (P<0.05) at the end of follow-up. After treatment and at the end of follow-up, the observation group had lower scores of CDAI, SES-CD, and syndrome of cold and heat (P<0.05) and higher IBDQ score (P<0.05) than the control group. Moreover, the observation group had higher clinical remission rate(χ2=4.381,3.962) and response rate(χ2=5.541,4.306) and lower non-response rate(χ2=6.646,4.306) than the control group at the two time points (P<0.05). The endoscopic remission rate(χ2=4.072,3.985) and response rate(χ2=4.528,5.161) in the observation group were higher than those in the control group (P<0.05). After treatment, the HGB, HCT, and ALB levels in both groups elevated, and the observation group had higher levels than the control group (P<0.05). The treatment in both groups lowered the levels of CRP, IL-6, TNF-α, IL-17, and FC (P<0.05), and the observation group had lower levels of CRP, IL-6, TNF-α, IL-17, and FC than the control group after treatment (P<0.05). The relative abundance of Bifidobacterium, Lactobacillus, and Prevotella increased (P<0.05), while that of Proteus, Klebsiella, and Enterococcus decreased (P<0.05) in the two groups after treatment. Moreover, the changes in the relative abundance of these bacteria in the observation group were more obvious than those in the control group (P<0.05). No adverse reactions related to the modified Modified Banxia Xiexintang were observed during the study period. ConclusionModified Banxia Xiexintang combined with VDZ can play a synergistic role and has good short-term and long-term efficacy. This therapy can improve the nutritional status, regulate intestinal flora, and reduce inflammatory injury in the treatment of moderate to severe active CD patients with the syndrome of cold and heat in complexity without causing severe adverse reactions.
ABSTRACT
ObjectiveTo systematically evaluate the safety of Tianzhi granules used in the treatment of mild-to-moderate vascular dementia. MethodA randomized, double-blind, double-simulated, positive drug/placebo parallel controlled multi-center phase Ⅳ clinical trial and an open multi-center phase Ⅳ clinical trial of Tianzhi granules in the treatment of mild-to-moderate vascular dementia were conducted. Safety data of 1 492 patients were included and analyzed according to inclusion and exclusion criteria. The main evaluation measures were the incidence rate of adverse events/adverse reactions, laboratory indicators, vital signs, and electrocardiogram (ECG) results. ResultA total of six adverse events possibly related to the test drug occurred in 520 patients of the double-blind trial, and the symptoms were all mild and recovered. The incidence of adverse events was not statistically different among Tianzhi granules, donepezil, and placebo groups. Nine adverse events possibly related to the test drug were observed in 972 patients of the open trial, and the symptoms were mild and recovered. Laboratory tests (blood routine, urine routine, liver function, kidney function, and coagulation) and vital signs were compared before treatment (baseline) and after treatment of 12 and 24 weeks, respectively. There was no statistical significance in the main indicators before and after treatment. In the double-blinded trial, there was no significant difference in safety indicators between different groups before and after treatment. The most frequent adverse reaction was gastrointestinal discomfort, with an incidence rate of 6.64‰. ConclusionAdverse reactions occasionally occur in patients using Tianzhi granules, and it is safe to use Tianzhi granules to treat mild-to-moderate vascular dementia clinically.
ABSTRACT
ObjectiveBased on ultra performance liquid chromatography-mass spectrometry(UPLC-MS) and non-targeted metabolomics technology to discuss the central regulatory effect of Chaishao Liujuntang on chronic atrophic gastritis(CAG) rats with liver-depression and spleen-deficiency, and to look for the correlation between cerebral cortex, hypothalamus and metabolic status of gastric tissues. MethodA CAG rat model with liver-depression and spleen-deficiency was established by chemical induction, hunger and satiety disorders, chronic restraint and tail clamping stimulation, lasting for 16 weeks. Twenty-eight Wistar rats were randomly divided into a blank group of 8 rats and a model group of 20 rats. After the completion of modeling, 4 rats in the model group were taken to observe the pathological changes of gastric mucosa. The remaining model rats were randomly divided into a model group of 8 rats and a Chaishao Liujuntang group of 8 rats. Chaishao Liujuntang group rats were given 5.1 g·kg-1 by gavage, and the remaining rats were given equal volume sterilized water by gavage for 4 weeks. Macroscopic characteristics, behavioral indicators and histopathological changes of the gastric mucosa of rats in each group were observed and compared. UPLC-MS non-targeted metabolomics was used to explore the metabolic regulation effect of Chaishao Liujuntang on the cerebral cortex, hypothalamus and stomach tissues of CAG rats with liver-depression and spleen-deficiency. Pearson correlation coefficient method was used to analyze the correlation between different tissue metabolites. ResultCompared with the model group, the macroscopic characteristics of rats in Chaishao Liujuntang group were improved, such as hair color, mental state and stool properties, and the number of times of crossing and standing in the open field experiment was significantly increased, and the static time of forced swimming was significantly reduced(P<0.01), and the gastric mucosa atrophy was reduced. The metabolic data from the cerebral cortex of rats in each group identified a total of 3 common potential biomarkers, but not enriched in pathways, 26 common potential biomarkers were identified in the hypothalamus, and the key metabolic pathways involved were mainly enriched in purine metabolism, glycerol phospholipid metabolism, D-glutamine and D-glutamic acid metabolism. Seventeen common potential biomarkers were identified in the stomach, and the key metabolic pathways involved were mainly enriched in thiamine metabolism, valine, leucine and isoleucine biosynthesis, and taurine and taurine metabolism. Correlation analysis of metabolites in different tissues revealed that multiple amino acids and their derivatives mediated metabolic connections between the cerebral cortex, hypothalamus and stomach of rats. ConclusionThe metabolic disorders in the cerebral cortex, hypothalamus and stomach of CAG rats with liver-depression and spleen-deficiency have their own characteristics, mainly manifested by changes in the content of glycerol phospholipids, fatty acids and bile acid metabolites. Moreover, Chaishao Liujuntang may play a central regulatory role in CAG rats with liver-depression and spleen-deficiency by correcting the metabolic disorders of amino acids.
ABSTRACT
ObjectiveTo screen the differential markers by analyzing volatile components in Dalbergia odorifera and its counterfeits, in order to provide reference for authentication of D. odorifera. MethodThe volatile components in D. odorifera and its counterfeits were detected by headspace gas chromatography-mass spectrometry(HS-GC-MS), and the GC conditions were heated by procedure(the initial temperature of the column was 50 ℃, the retention time was 1 min, and then the temperature was raised to 300 ℃ at 10 ℃ for 10 min), the carrier gas was helium, and the flow rate was 1.0 mL·min-1, the split ratio was 10∶1, and the injection volume was 1 mL. The MS conditions used electron bombardment ionization(EI) with the scanning range of m/z 35-550. The compound species were identified by database matching, the relative content of each component was calculated by the peak area normalization method, and principal component analysis(PCA), orthogonal partial least squares-discrimination analysis(OPLS-DA) and cluster analysis were performed on the detection results by SIMCA 14.1 software, and the differential components of D. odorifera and its counterfeits were screened out according to the variable importance in the projection(VIP) value>2 and P<0.05. ResultA total of 26, 17, 8, 22, 24 and 7 volatile components were identified from D. odorifera, D. bariensis, D. latifolia, D. benthamii, D. pinnata and D. cochinchinensis, respectively. Among them, there were 11 unique volatile components of D. odorifera, 6 unique volatile components of D. bariensis, 3 unique volatile components of D. latifolia, 6 unique volatile components of D. benthamii, 8 unique volatile components of D. pinnata, 4 unique volatile components of D. cochinchinensis. The PCA results showed that, except for D. latifolia and D. cochinchinensis, which could not be clearly distinguished, D. odorifera and other counterfeits could be distributed in a certain area, respectively. The OPLS-DA results showed that D. odorifera and its five counterfeits were clustered into one group each, indicating significant differences in volatile components between D. odorifera and its counterfeits. Finally, a total of 31 differential markers of volatile components between D. odoriferae and its counterfeits were screened. ConclusionHS-GC-MS combined with SIMCA 14.1 software can systematically elucidate the volatile differential components between D. odorifera and its counterfeits, which is suitable for rapid identification of them.
ABSTRACT
ObjectiveTo clarify the scientific validity of in vivo pharmacokinetic determination of the whole drug composition in Shenbai nanosuspension in rats, and to provide methodological guidance and theoretical basis for the in vivo study of multi-component complex system of traditional Chinese medicine(TCM) preparations. MethodThe concentration of the overall components, mainly total saponins and total polysaccharides in Shenbai decoction and Shenbai nanosuspension, was determined in rat plasma at different times by area under the absorbance-wavelength curve method(AUAWC), and the concentration of individual ginsenoside Rg1 was determined by high performance liquid chromatography(HPLC), and the methodology was verified. The pharmacokinetic parameters of the whole component were compared with those of ginsenoside Rg1 to evaluate the in vivo operational characteristics of the two preparations. ResultThe methodological investigations of AUAWC and HPLC were in accordance with the requirements. AUAWC analysis showed that the overall components in both the decoction group and the nanosuspension group showed a one-compartment model, with half-life(t1/2) of 2.43 h and 2.04 h, respectively. The relative bioavailability of Shenbai nanosuspension was 138.99%. HPLC assay showed that ginsenoside Rg1 in the decoction group and the nanosuspension group showed a two-compartment model, with distribution half-life(t1/2α) of 0.13 h and 2.55 h, and elimination half-life(t1/2β) were 14.28 h and 3.85 h, respectively. The relative bioavailability of Shenbai nanosuspension was 127.49%. Compared with Shenbai decoction, the time to peak(tmax), peak concentration(Cmax) and area under the drug-time curve(AUC) of the overall components and ginsenoside Rg1 in Shenbai nanosuspension were increased. ConclusionThe established AUAWC can be used for the pharmacokinetic study of the overall components of TCM preparations, which is complementary to the results of individual components measured by HPLC, and can provide useful reference for the in vivo study of new dosage forms of TCM.