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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.
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BACKGROUND:Clinical skin wound healing continues to be a significant concern,and tissue repair research has moved to the forefront with the development of biomaterials with immunomodulatory properties.Therefore,it is crucial to research wound dressings that have immunomodulatory properties. OBJECTIVE:To prepare chitosan hydrogels that have been modified by arginine with different configurations and assess their capacity to speed up wound healing in a rat animal model. METHODS:(1)In vitro trial:Chitosan modified by pure L-arginine,pure D-arginine,and L-arginine and D-arginine was synthesized by EDC/NHS system,which was then crosslinked with aldehyde-modified four-arm polyethylene glycol.Different chitosan-based hydrogels(CS-L,CS-D,and CS-DL)were finally formed via the Schiff base reaction.Three kinds of hydrogel extracts were co-cultured with fibroblasts respectively.Hydrogel cytocompatibility was assessed using the CCK-8 assay and live/dead staining.The effect of hydrogel on the migration capacity of fibroblasts was assessed by using a scratch test.Three kinds of hydrogels were incubated with rat erythrocyte suspension respectively to evaluate the hemocompatibility of the hydrogels.The hydrogel extract was co-cultured with RAW264.7 macrophages to test the hydrogels'capacity to enhance macrophage NO generation and polarize macrophage phenotype.(2)In vivo experiment:A total of 36 adult SD rats were divided into 4 groups with 9 rats in each group by the random number table method.Two full-layer skin defect wounds of 2 cm×2 cm were made on the back of each rat.Normal saline was added to the wounds of the control group,and corresponding hydrogel was added to the wounds of the CS-L,CS-D,and CS-DL groups,respectively,and then bandaged and fixed.The wound healing was observed regularly after operation.Hematoxylin-eosin staining was performed at 3,10,and 21 days after operation.The samples were collected 10 days after operation and M2 macrophage immunofluorescence staining was performed. RESULTS AND CONCLUSION:(1)In vitro experiments:Under scanning electron microscopy,the three kinds of hydrogels exhibited obvious interpenetrating network structures with pore sizes ranging from 70-200 μm.The three kinds of hydrogels have good swelling performance,degradation performance,self-healing performance,and suitable mechanical strength.The three kinds of hydrogels had good cytocompatibility and hemocompatibility and could promote the migration of fibroblasts.All three kinds of hydrogels had the ability to promote the polarization of macrophages,and CS-D hydrogels had the strongest ability to promote the polarization of macrophages.CS-L hydrogel could significantly promote the production of NO in macrophages.(2)In vivo experiment:3 and 10 days after operation,the wound healing rate in the CS-L and CS-D groups was higher than that in the control group(P<0.05).After 21 days,the wound healing rate of the three hydrogel groups was higher than that of the control group.Hematoxylin-eosin staining displayed that a large number of inflammatory cells were infiltrated in the wound tissue of rats in all groups,accompanied by neovessels and fibroblasts 3 days after operation.10 days after operation,there was still more inflammatory cell infiltration in the wound of the control group,and the inflammation of the other three groups was improved,especially the decrease of inflammatory cells in the CS-D group was more obvious.21 days after operation,the wound epithelium of each group was well repaired,and there was basically no inflammatory cell infiltration in the CS-L and CS-D groups,while there was still a small amount of inflammatory cell infiltration in the control group.Immunofluorescence staining revealed that the number of M2-type macrophages in the CS-D group was higher than that in the other three groups(P<0.05).(3)The results conclude that chitosan hydrogels modified by different configurations of arginine can promote wound healing through different mechanisms.
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Objective To observe the effects of Gegen Qinlian Decoction on pancreatic endoplasmic reticulum stress in mice with type 2 diabetes mellitus(T2DM);To explore its mechanism of action in the treatment of T2DM.Methods Totally 75 SPF male db/db mice were randomly divided into model group,metformin group,and Gegen Qinlian Decoction high-,medium-,low-dosage groups,with 15 mice in each group.15 db/m mice were set as the blank group.The administration groups received corresponding medicine for gavage for 12 weeks.Body mass,fasting blood glucose(FBG)and glycated hemoglobin(HbA1c)in mice were detected,HE staining was used to observe the pathological changes of pancreatic tissue,the apoptosis of islet cells was determined by TUNEL staining,Western blot was used to detect pancreatic tissue glucose regulatory protein 78(GRP78),protein kinase R-like endoplasmic reticulum kinase(PERK),p-PERK,activated transcription factor 4(ATF4)and C/EBP homologous protein(CHOP)protein expression,RT-PCR was used to detect pancreatic tissue PERK,ATF4,CHOP mRNA expressions.Results Compared with the blank group,the body mass,FBG and HbA1c contents in the model group significantly increased(P<0.01);the pancreatic tissue structure was incomplete,with blurry boundaries and vacuoles inside,leading to a significant increase in pancreatic islet cells apoptosis(P<0.01);the expressions of GRP78,p-PERK,ATF4,and CHOP proteins in pancreatic tissue significantly increased(P<0.01),and the mRNA expressions of PERK,ATF4 and CHOP significantly increased(P<0.01).Compared with the model group,the body mass,FBG and HbA1c contents of mice in each administration group significantly decreased(P<0.05,P<0.01);pathological changes in pancreatic tissue was reduced,and islet cells apoptosis decreased to varying degrees(P<0.05,P<0.01);the expressions of GRP78,p-PERK,ATF4 and CHOP proteins in pancreatic tissue significantly decreased(P<0.01)in Gegen Qinlian Decoction high-and medium-dosage groups and the metformin group,and the expressions of PERK,ATF4 and CHOP mRNA significantly decreased(P<0.05,P<0.01).Conclusion Gegen Qinlian Decoction may decreased pancreatic islet cells apoptosis,protect pancreatic cell function,and delay the progression of T2DM by inhibiting the endoplasmic reticulum stress PERK/ATF4/CHOP signaling pathway,and down-regulating the expressions of related genes and proteins.
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ObjectiveTo investigate the effect and mechanism of Shenqi Tangluo pill (SQTLP) on oxidative stress injury of skeletal muscle of type 2 diabetes mellitus (T2DM) mice based on nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)/NAD(P)H quinone oxidoreductase 1 (NQO1) pathway. MethodA total of 60 7-week-old male db/db mice [specific pathogen-free (SPF) grade] were selected and fed for one week for adaption. They were divided into the model control group, SQTLP low-, medium- and high-dose (19, 38, and 76 g·kg-1) groups and metformin group (0.26 g·kg-1) by gavage. Each group consisted of 12 mice. Twelve male db/m mice of the same age were selected as the blank group. The intervention was implemented continuously for 8 weeks. Fasting blood glucose (FBG) was detected. Fasting serum insulin (FINS) levels were detected by enzyme-linked immunosorbent assay (ELISA), and the homeostasis model assessment-insulin resistance (HOMA-IR) index and the homeostasis model assessment-insulin sensitivity index (HOMA-ISI) were calculated. Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were conducted. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and the contents of malondialdehyde (MDA) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) in skeletal muscle tissues were detected by biochemical kits. Hematoxylin-eosin (HE) staining was used to observe the pathological changes in skeletal muscle tissues. The levels of reactive oxygen species (ROS) and 4-hydroxynonenal (4-HNE) in skeletal muscle tissue were detected by immunofluorescence (IF). The expression levels of Nrf2, HO-1, NQO1 and glutamate-cysteine ligase catalytic subunit (GCLC) proteins in skeletal muscle tissues were detected by Western blot. ResultCompared with those in the blank group, FBG, FINS and HOMA-IR in the model group were significantly increased (P<0.05), while HOMA-ISI was decreased (P<0.05). The results of OGTT and ITT showed that blood glucose was significantly increased at all time points (P<0.05), and glucose tolerance and insulin tolerance were significantly impaired. SOD and GSH-Px activities in skeletal muscle tissues were significantly decreased (P<0.05), and MDA and NADPH contents were significantly increased (P<0.05). In skeletal muscle tissues, the arrangement of muscle fibers was loose, the nucleus was disordered, and inflammatory cells were infiltrated. The expression levels of ROS and 4-HNE in skeletal muscle tissues were significantly increased (P<0.05). The protein expression levels of Nrf2, HO-1, NQO1 and GCLC in skeletal muscle tissues were significantly decreased (P<0.05). Compared with those in the model group, FBG, FINS and HOMA-IR in the metformin group were significantly decreased (P<0.05), while HOMA-ISI was increased (P<0.05). The results of OGTT and ITT showed that blood glucose in the metformin group was significantly decreased at all time points (P<0.05). The activities of SOD and GSH-Px in skeletal muscle tissues were significantly increased (P<0.05), while the contents of MDA and NADPH were significantly decreased (P<0.05). No obvious abnormality was found in the skeletal muscle tissue of the metformin group. The expressions of ROS and 4-HNE in skeletal muscle tissues were decreased (P<0.05). The protein expression levels of Nrf2, HO-1, NQO1 and GCLC in skeletal muscle tissues were significantly increased (P<0.05). Compared with those in the model group, FBG, FINS and HOMA-IR in the SQTLP medium- and high-dose groups were significantly decreased (P<0.05), while HOMA-ISI was increased (P<0.05). The results of OGTT and ITT showed that the glucose tolerance and insulin tolerance of mice were improved in each dose group of SQTLP. The GSH-Px activity in the SQTLP low-dose group was significantly increased (P<0.05), and the NADPH content was decreased (P<0.05). The activities of SOD and GSH-Px in the SQTLP medium- and high-dose groups were significantly increased (P<0.05), while the contents of MDA and NADPH were significantly decreased (P<0.05). The skeletal muscle tissue injury of mice in each dose group of SQTLP was ameliorated to different degrees. In the SQTLP medium- and high-dose groups, the expressions of ROS and 4-HNE were decreased (P<0.05), and the protein expression levels of Nrf2, HO-1, NQO1 and GCLC were significantly increased (P<0.05). Compared with those in the SQTLP low-dose group, FBG and HOMA-IR in the SQTLP high-dose group were significantly decreased (P<0.05), while HOMA-ISI was increased (P<0.05). The results of OGTT and ITT showed that the SQTLP high-dose group significantly improved the glucose tolerance and insulin tolerance of mice. The activities of SOD and GSH-Px in skeletal muscle tissues were significantly increased (P<0.05), while the contents of MDA and NADPH were significantly decreased (P<0.05). No obvious abnormality was found in the skeletal muscle tissue, the expressions of ROS and 4-HNE were decreased (P<0.05), and the protein expression levels of Nrf2, HO-1, NQO1 and GCLC were significantly increased (P<0.05) in the skeletal muscle tissue of the SQTLP high-dose group. ConclusionSQTLP can significantly improve IR in T2DM mice, and the mechanism is related to SQTLP activating the Nrf2/HO-1/NQO1 signaling pathway, promoting the expression of antioxidant enzymes, and thus improving the oxidative stress injury in the skeletal muscle.
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AIM: To explore the intervention effect of Dahuangtang pellets (DHT) on diabetic nephropathy (DN) based on the AMP-activated protein kinase/mammalian target of rapamycin/unc-51-like kinase 1 (AMPK/mTOR/ULK1) signaling pathway. METHODS: Eight mice were randomly assigned to the model group, the dapagliflozin group, and the DHT (high, medium, and low dosage) group out of a total of 40 C57BL/KSJ-db/db (hereafter referred to as db/db) mice; another 10 C57BL/KSJ-db/dm mice were used as the normal group, saline was provided to the normal and model groups, and the mice in the treatment group received the appropriate medications. The medications were given for 10 consecutive weeks, once per day, to the mice in the treatment group. At weeks 0, 4, 8, and 10 of administration, fasting blood glucose (FBG) was assessed by drawing blood at a predetermined time from the tail vein; Urine samples were taken at 0, 5, and 10 weeks after treatment to evaluate the levels of albumin and creatinine, and the urinary albumin-creatinine ratio (ACR) was computed. After 10 weeks, mice in each group were assayed for 24 h total urine protein, serum creatinine (Scr), urea nitrogen (BUN) levels; Western blotting analysis was conducted to detect the expression of p-AMPK, p-mTOR, and p-ULK1, as well as the expression of autophagy related proteins homolog of yeast Atg6 (Beclin-1), autophagy-related proteins microtubule-associated protein 1 light chain 3 (LC3), P62 in renal tissue; Immunohistochemistry was used to measure the expression of podocyte lacunar membrane proteins (Nephrin, Podocin) in renal tissues; The pathological morphology of renal tissue was observed by light microscopy and transmission electron microscopy. RESULTS: Compared with the model group, FBG, ACR, and 24 h total urine protein were reduced in the dapagliflozin group and DHT groups of mice, and there was no statistically significant difference in Scr and BUN; In renal tissues, there is increased expression of p-AMPK and p-ULK1, decreased expression of p-mTOR, increased expression of LC3II / LC3I and Beclin-1, and decreased expression of P62 (P<0.01, P< 0.05); differentially upregulated in glomeruli are the podocyte lacunar membrane proteins Nephrin and Podocin (P<0.01, P<0.05); renal pathologic damage was reduced to varying degrees; transmission electron microscopy showed an increase in the number of autophagic vesicles and autophagic lysosomes. CONCLUSION: DHT can delay the development of DN by regulating the AMPK / mTOR / ULK1 signaling pathway, enhancing podocyte autophagy, and protecting glomeruli.
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ObjectiveTo validate the alleviating effect of Gegen Qinliantang (GGQLT) on insulin resistance in db/db diabetic mice by regulating the silent information regulator 1 (SIRT1)/forkhead transcription factor O1 (FoxO1) autophagy pathway. MethodSeventy-five SPF-grade spontaneous type 2 diabetic db/db mice and 15 control db/m mice were selected and maintained on regular feed for one week before measuring blood glucose. They were randomly divided into six groups, with 15 mice in each group. The groups included a normal group (physiological saline, 0.2 g·kg-1), a metformin group (0.2 g·kg-1), high-, medium-, and low-dose GGQLT groups (31.9, 19.1, 6.9 g·kg-1), and a model group (physiological saline, 0.2 g·kg-1). They were orally treated with corresponding drugs for eight weeks, once daily. Fasting blood glucose (FBG) was measured using a Roche glucometer. Serum levels of high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), and total cholesterol (TC) were measured using an automated biochemical analyzer. Fasting serum insulin (INS) levels were determined using enzyme-linked immunosorbent assay (ELISA), and the homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. Western blot was used to detect the expression of Beclin-1, microtubule-associated protein 1 light chain 3 (LC3), and SIRT1/FoxO1 autophagy pathway-related proteins in liver tissues. Immunohistochemistry was performed to assess the expression of SIRT1, FoxO1, Beclin-1, and LC3B proteins in liver tissues. Transmission electron microscopy was used to observe the formation of autophagosomes in the liver. ResultCompared with the normal group, the model group showed significant increases in FBG, FINS, HOMA-IR, TC, TG, LDL-C, and HDL-C levels (P<0.01), and significant increases in the expression of SIRT1, Beclin-1, LC3, and FoxO1 proteins in liver tissues (P<0.01). Transmission electron microscopy revealed the highest number of autophagosomes in the model group. Compared with the model group, the metformin group and the low-, medium-, and high-dose GGQLT groups showed significant decreases in serum FBG, FINS, HOMA-IR, TC, TG, LDL-C, and HDL-C levels (P<0.05, P<0.01), significant decreases in the expression of SIRT1, Beclin-1, LC3 (P<0.05, P<0.01), and up-regulated FoxO1 protein (P<0.01). Transmission electron microscopy showed a reduction in the degree of autophagy in the treatment groups. Compared with the metformin group, the medium- and high-dose GGQLT groups showed significant decreases in FBG, FINS, and TG levels (P<0.01), significant decreases in the expression of SIRT1, Beclin-1, and LC3 in liver tissues (P<0.05, P<0.01), and reduced FoxO1 protein (P<0.01). The high-dose GGQLT group showed reduced HOMA-IR, TC, LDL-C, and HDL-C levels (P<0.05, P<0.01). Transmission electron microscopy revealed a significant reduction in autophagosomes in the medium- and high-dose GGQLT groups. ConclusionGGQLT can significantly improve glucose and lipid metabolism disorders, alleviate insulin resistance in db/db mice, and prevent and treat type 2 diabetes by activating the SIRT1/FoxO1 autophagy pathway.
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ObjectiveTo observe the effect of modified Gegen Qinliantang on the expression levels of proteins related to the farnesoid X receptor/small heterodimer partner/peroxisome proliferator-activated receptor α (FXR/SHP/PPARα) signaling pathway in the liver tissue of db/db model mice with type 2 diabetes mellitus (T2DM) and explore the underlying mechanism of action of modified Gegen Qinliantang. MethodThirty db/db mice were randomly divided into model group, metformin group (0.2 g·kg-1), and high-, medium-, and low-dose modified Gegen Qinliantang groups (31.9, 19.1, 6.4 g·kg-1), with 6 mice in each group. An additional six m/m mice were assigned to the blank group. Respective drugs were administered via oral gavage for 12 weeks. Mouse body weight, fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels were measured. Oil red O staining was used to observe hepatic lipid accumulation and periodic acid-schiff (PAS) staining was used to assess hepatic glycogen deposition. Ammonium ferric sulfate staining was used to observe cholesterol deposition in intestinal tissues. Western blot was employed to detect the expression of FXR, cholesterol 7α-hydroxylase (CYP7A1), SHP, and PPARα proteins in liver tissues, and enzyme-linked immunosorbent assay (ELISA) was used to measure serum free fatty acid (FFA) levels. ResultAt the end of the treatment, compared with the blank group, the model group exhibited significant increases in mouse body weight, FBG, FFA, TC, TG, and LDL-C levels (P<0.01), along with significant hepatic lipid droplets, reduced hepatic glycogen, noticeable cholesterol accumulation in intestinal tissues, significantly decreased expression of FXR, SHP, PPARα proteins, and significantly increased expression of CYP7A1 protein in liver tissues (P<0.01). Compared with the model group, the metformin group and the high- and medium-dose modified Gegen Qinliantang groups demonstrated significant reductions in mouse body weight, FBG, FFA, TC, TG, LDL-C levels (P<0.05, P<0.01), significant increases in HDL-C levels (P<0.05, P<0.01), decreased hepatic lipid accumulation, increased hepatic glycogen, reduced intestinal cholesterol accumulation, significantly increased expression of FXR, SHP, PPARα proteins, and significantly decreased expression of CYP7A1 protein in liver tissues (P<0.01). ConclusionModified Gegen Qinliantang may regulate the FXR/SHP/PPARα signaling pathway to suppress FFA levels and improve lipid metabolism in T2DM mice.
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Objective:To observe the effects of different doses of Gegen Qinlian Decoction on nucleotide oligomeric domain-like receptor protein 3 (NLRP3)/Caspase-1/IL1β inflammatory signaling pathway in liver of db/db mice with type 2 diabetes mellitus (T2DM).Methods:Totally 75 SPF male db/db mice were randomly divided into model group, metformin group (0.2 g/kg), Gegen Qinlian Decoction high-, medium-, and low-dosage groups (61.80, 30.90, 15.45 g/kg), with 15 mice in each group. Another 15 db/m male mice were selected as blank control group. Each administration group was given relevant medicine for gavage, while the blank group and model group were given 0.9% sodium chloride solution for gavage, once a day, for 12 weeks. The body weight, fasting blood glucose (FBG) and glycosylated hemoglobin (HbA1c) contents of each group were measured after treatment. The mRNA expression levels of NLRP3, Caspase-1 and interleukin-1β (IL-1β) in liver were detected by real-time quantitative PCR. The expression levels of NLRP3, Caspase-1, IL-1β and IL-18 in liver tissues were detected by Western Blot. HE staining was used to observe the morphology of liver tissues.Results:Compared with model group, body weight, fasting blood glucose and HbA1c contents of mice in Gegen Qinlian Decoction high- and medium-dosage groups and metformin groups decreased ( P<0.05), the body weight and fasting blood glucose levels of mice in Gegen Qinlian Decoction low-dosage group decreased ( P<0.05); the mRNA levels of NLRP3 and IL-1β in liver tissues of all treatment groups decreased ( P<0.05), the mRNA level of Caspase-1 in liver tissue decreased in Gegen Qinlian Decoction high- and medium-dosage groups ( P<0.05); the expression of NLRP3, Caspase-1, and IL-18 in liver tissue of each treatment group decreased ( P<0.05), while the expression of IL-1β in Gegen Qinlian Decoction high- and medium-dosage groups and the metformin group decreased ( P<0.05); compared with the metformin group, the body weight and fasting blood glucose of mice in the Gegen Qinlian Decoction high-dosage decreased ( P<0.05), while the HbA1c levels in the Qinlian Decoction high- and medium-dosage decreased ( P<0.05); the expressions of NLRP3, Caspase-1 and IL-18 in liver tissues of Gegen Qinlian Decoction high-dosage group decreased ( P<0.05), the expression of IL-1β, NLRP3, Caspase-1, IL-1β, and IL-18 decreased ( P<0.05); HE staining showed that the pathological changes of liver tissue were reduced in all treatment groups. Conclusion:Gegen Qinlian Decoction may reduce blood sugar by inhibiting the activation of NLRP3/Caspase-1/IL-1β inflammatory signaling pathway in liver of db/db mice, thereby improving the inflammatory damage of T2DM.
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ObjectiveTo discuss the effect of modified Gegen Qinliantang (MGQT) on blood glucose and lipids and Takeda G protein-coupled receptor 5 (TGR5)-related pathways in pancreatic tissue of obese type 2 diabetes mellitus (T2DM) mice. MethodA total of 10 male specific pathogen free (SPF) m/m mice (7 weeks old) and 50 male SPF (7 weeks old) were adaptively fed for one week in SPF laboratory. The m/m mice were included in the blank group. T2DM was induce d in the 50 db/db mice. The model mice were randomized into the model group, metformin group (0.2 g·kg-1), high-dose, medium-dose, and low-dose (31.9, 19.1, 6.4 g·kg-1) MGQT groups, with 10 in each group, and the drug dose was10 mL·kg-1. The model group and the blank group received distilled water of the same volume. The administration lasted 12 weeks (once/day). Fasting blood glucose (FBG) was detected regularly. After 12 weeks of administration, serum levels of glycated serum protein (GSP), serum glucose (GLU), total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were detected. Pathological changes in the pancreatic tissue were based on hematoxylin-eosin (HE) staining. Western blot was used to determine the protein expression of TGR5, protein kinase A (PKA), phosphorylated (p)-PKA, cyclic-AMP response element binding protein (CREB), p-CREB, proprotein convertase 1/3 (PC1/3), and glucagon-like peptide-1 (GLP-1) in pancreatic tissues. The level of cyclic adenosine monophosphate (cAMP) in pancreatic tissue was determined by enzyme-linked immunosorbent assay (ELISA). ResultCompared with the blank group, the model group had pathological changes in pancreatic tissue, high levels of FBG, GSP, GLU, TC, TG, and LDL-C (P<0.01), low level of HDL-C (P<0.05), low protein expression of TGR5, p-PKA (Thr197)/PKA, p-CREB (Ser133)/CREB, PC1/3, and GLP-1 in pancreatic tissue (P<0.01), and low content of cAMP in the pancreas (P<0.01). Pancreatic tissue lesion in the treatment groups were milder than that in the model group. Both the high-dose MGQT and metformin can reduce the levels of FBG, GSP, GLU, TC, TG, and LDL-C in db/db mice (P<0.05, P<0.01) and increase the level of HDL-C (P<0.01). Except the GLP-1 protein in the medium-dose MGQT group, the protein expression of TGR5, p-PKA (Thr197)/PKA, p-CREB (Ser133)/CREB, PC1/3, and GLP-1 in the high-dose and medium-dose MGQT groups and the metformin group increased compared with that in the model group (P<0.05, P<0.01). The content of cAMP in the pancreatic tissue of the high-dose and medium-dose MGQT groups and the metformin group was raised compared with that in model group (P<0.05, P<0.01). ConclusionMGQT can improve the glucose homeostasis in db/db mice with T2DM by regulating TGR5/cAMP/GLP-1 signaling pathway-related protein expression.
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ObjectiveTo explore the mechanism of Gegen Qinliantang (GQT) in improving ectopic lipid accumulation in the liver of db/db mice with type 2 diabetes mellitus (T2DM) by regulating the adenosine monophosphate-activated protein kinase (AMPK)-forkhead box O3a (FoxO3a) autophagy axis, to provide a scientific basis for clarifying the hypoglycemic mechanism of GQT and its clinical application. MethodSeventy-five spontaneous T2DM db/db mice and 15 normal db/m mice were selected and maintained on a regular diet for one week, followed by the measurement of blood glucose. They were then randomly divided into six groups, with 15 mice in each group, including normal group (0.2 g·kg-1 saline), metformin group (0.2 g·kg-1), high-, medium, and low-dose GQT group (31.9, 19.1, 6.9 g·kg-1), and model group (0.2 g·kg-1 saline). The mice were orally administered the corresponding drugs once daily for 12 weeks. Fasting blood glucose (FBG) and glycated hemoglobin (HbA1c) were detected. Fasting insulin (FINS) and free fatty acid (FFA) levels were measured by enzyme-linked immunosorbent assay (ELISA). Pathological changes in liver tissues were observed by hematoxylin-eosin (HE) staining. The protein expression levels of phosphorylated (p)-AMPK, p-FoxO3a, and autophagy-related proteins microtubule-associated protein 1 light chain 3 Ⅱ (LC3Ⅱ) and p62 were detected using Western blot. Immunofluorescence was used to detect the expression of hypoxia-inducible factor-1α (HIF-1α) in liver tissues. Real-time polymerase chain reaction (Real-time PCR) was performed to detect the mRNA expression of AMPK, FoxO3a, and LC3 in liver tissues. ResultCompared with the normal group, the model group showed pathological changes in liver tissues, increased FBG, HbA1c, FINS, and FFA levels (P<0.01), increased protein expression levels of p-AMPK, p62, and HIF-1α, decreased protein expression levels of p-FoxO3a and LC3Ⅱ in liver tissues (P<0.01), decreased mRNA expression of AMPK, and increased expression of FoxO3a (P<0.01). Compared with the model group, the treatment groups showed relieved liver tissue lesions and decreased FBG, HbA1c, FINS, and FFA levels (P<0.01). The expression of p-AMPK, p62, and HIF-1α increased, while the expression of p-FoxO3a showed a dose-dependent decrease in the high-dose GQT group. The expression of LC3Ⅱ increased in the metformin group and the high-dose GQT group (P<0.01). The mRNA expression of AMPK showed a dose-dependent increase, and the expression of FoxO3a showed a dose-dependent decrease in the treatment groups (P<0.01). ConclusionGQT can improve ectopic lipid accumulation in the liver of T2DM db/db mice, which may be related to the regulation of the AMPK-FoxO3a autophagy axis.
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OBJECTIVE: To investigate the therapeutic effect of Sishen Wan (, SSW) on ulcerative colitis (UC) induced by dinitrobenzene sulfonic acid and its effect on toll-like receptor 2/interleukin-1 receptor-associated kinase-4/nuclear factor-κB (TLR2/IRAK4/NF-κB) sig-naling pathway in colonic tissue. METHODS: In this study, 120 Sprague-Dawley rats were randomly divided into blank and model groups. The experimental UC model in rats was established by subcutaneous injection of hydrocortisone + senna gavage for 21 d + dinitrobenzene sulfonic acid (DNBS)/ ethanol solution enema. The successful model rats were randomly divided into the model group; mesalazine (0.36 g/kg) group; and high-, medium-, and low- dose SSW (24, 12, and 6 g/kg) groups. The model and blank groups were gavaged with equal volumes of distilled water once a day for 21 d. The general condition of the rats was observed, and the body mass, fecal properties, and occult blood were recorded for calculating the disease activity index (DAI) score. The colonic tissue of the rats was collected, and its general morphology and pathological form were noted for obtaining the colonic mucosal injury index (CMDI) score. Hematoxylin-eosin staining was used to view the pathological changes of the colon tissue in each group, apoptosis of the cells was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and quantitative real-time polymerase chain reaction was used to measure the expressions of TLR2, myeloid differentiation primary response gene 88 (MyD88), IRAK4, and NF-κB p65 mRNA in the colon tissue. The expressions of TLR2, MyD88, IRAK4, and NF-κB p65 protein were detected using western blotting and immunohistochemistry assay, and the levels of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in the colon tissue were determined using enzyme linked immunosorbent assay. RESULTS: Compared with the blank group, the general condition of the model group was relatively poor. The DAI and CMDI scores of the model group increased significantly (< 0.01), the glands and intestinal mucosa disappeared partially, and several inflammatory cells infiltrated and gathered in the mucosal layer and base layer of the rats in the model group. Furthermore, the cell apoptosis and expression levels of TLR2, MyD88, IRAK4, and NF-κB p65 mRNA and protein in the colon tissue of rats in the model group increased significantly (< 0.01). The levels of IL-1ß and TNF-α increased significantly in the colon tissue of rats in the model group (< 0.01). After treatment with SSW, compared with the model group, the general condition of the UC rats improved. Moreover, the DAI and CMDI scores of the UC rats decreased significantly (< 0.05), and the pathological changes in the colon tissue of the UC rats tended to be normal. The cell apoptosis and expression levels of TLR2, MyD88, IRAK4, and NF-κB p65 mRNA and protein in the colon tissue of the UC rats decreased gradually ( < 0.01), and the levels of IL-1ß and TNF-α decreased significantly (< 0.01). CONCLUSION: SSW can improve the general condition and alleviate the intestinal mucosal injury of UC model rats. Additionally, SSW can inhibit the TLR2/IRAK4/ NF-κB signaling pathway, but further studies are required to confirm it.
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Colite Ulcerativa , NF-kappa B , Animais , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/genética , Dinitrobenzenos , Medicamentos de Ervas Chinesas , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Quinases Associadas a Receptores de Interleucina-1/farmacologia , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Mensageiro , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Ácidos Sulfônicos/metabolismo , Ácidos Sulfônicos/farmacologia , Ácidos Sulfônicos/uso terapêutico , Receptor 2 Toll-Like/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BackgroundSince mid-December 2019, a cluster of pneumonia-like diseases caused by a novel coronavirus, now designated COVID-19 by the WHO, emerged in Wuhan city and rapidly spread throughout China. Here we identify the clinical characteristics of COVID-19 in a cohort of patients in Shanghai. MethodsCases were confirmed by real-time RT-PCR and were analysed for demographic, clinical, laboratory and radiological features. ResultsOf 198 patients, the median duration from disease onset to hospital admission was 4 days. The mean age of the patients was 50.1 years, and 51.0% patients were male. The most common symptom was fever. Less than half of the patients presented with respiratory systems including cough, sputum production, itchy or sore throat, shortness of breath, and chest congestion. 5.6% patients had diarrhoea. On admission, T lymphocytes were decreased in 45.8% patients. Ground glass opacity was the most common radiological finding on chest computed tomography. 9.6% were admitted to the ICU because of the development of organ dysfunction. Compared with patients not treated in ICU, patients treated in the ICU were older, had longer waiting time to admission, fever over 38.5{degrees} C, dyspnoea, reduced T lymphocytes, elevated neutrophils and organ failure. ConclusionsIn this single centre cohort of COVID-19 patients, the most common symptom was fever, and the most common laboratory abnormality was decreased blood T cell counts. Older age, male, fever over 38.5{degrees}C, symptoms of dyspnoea, and underlying comorbidity, were the risk factors most associated with severity of disease.
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To screen strains with antibacterial and antitumor activity, pregnenolone was used as the sole carbon source for screening bacteria from soil. Based on bacteriostatic activity assay, Pseudomonas aeruginosa HBD-12 was found to be effectively inhibiting the growth of Escherichia coli, Bacillus thuringiensis, Penicillium digitatum and Penicillium italicum, and its fermentation broth was separated and purified using column chromatography. Then, structure of the obtained monomeric compounds was analyzed by spectrum analysis, and their antitumor activity was measured using HTRF kinase detection kit. The isolated monomeric compounds 1-hydroxy-9,10-phenanthroline and 3-hydroxy-9,10-dihydrophenanthroline had significant antitumor activity. At 20 μg/mL, 1-hydroxy-9,10-phenanthroline and 3-hydroxy-9,10-dihydrophenanthroline inhibited 78.39±2.29% and 60.34±8.35% Aurora kinase A, respectively. Therefore, the secondary metabolites of Pseudomonas aeruginosa HBD-12 have the potential to develop antibacterial and antitumor drugs.
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Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Penicillium , Pseudomonas aeruginosaRESUMO
Objective To observe the change rules of expressions of IL-1β, IL-4, IFN-γ and TGF-β1 in serum and colon tissue in rats with ulcerative colitis of spleen-kidney yang deficiency type; To explore the action in the progress of ulcerative colitis of spleen-kidney yang deficiency type.MethodsComposite method was used to establish rat models with ulcerative colitis of spleen-kidney yang deficiency type. 75 Wistar rats were randomly divided into blank group, model 3 d group, model 7 d group and model 21 d group. ELISA was used to test the levels of IL-1β, IL-4, IFN-γ and TGF-β1 in serum and colon tissue.Results Compared with the blank group, contents of IL-1β and IFN-γ in model group increased significantly (P<0.05,P<0.01), and the contents of IL-4 and TGF-β1 content decreased (P<0.05,P<0.01),especially in the model 7 d group (P<0.01).ConclusionThe pro-inflammatory factor IL-1β, IFN-γ and anti-inflammatory factor IL-4 and TGF-β1 play an important role in rats with ulcerative colitis of spleen-kidney yang deficiency type.
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Objective To explore the effect of Baihe Zhimu Tang(百合知母汤,BZT) on the airway inflammation in bronchial asthma rats.Methods Forty Wistar rats were randomized into control group,asthma model group,BZT group and dexamethasone (DX) group with 10 rats in each group.The bronchial asthma rat model was built by intraperitoneal injection of 10% ovalbumin (OVA) and ultrasonic atomizing inhalation of 2% OVA except for the control group.Thirty minutes before every ultrasonic atomizing inhalation,the BZT group was given BZT 3.68 g/(kg · d) by gastric perfusion;the DX group was administered DX injection 1.2 mg/(kg · d) by gastric perfusion;as for the control group and asthma model group,they were given normal saline of the same volume;all groups were intervened for 14 days.After that,HE staining was used to observe the pathological changes of lung tissue,the levels of interleukin-2 (IL-2) and interleukin-4 (IL-4) were measured,and the cell cycle and calcineurin (CaN) activity were observed in separated peripheral blood lymphocytes.Results For the asthma model group,there was a lot of inflammatory cell infiltration in the bronchial wall under light microscope,the content of IL-4 in the plasma was higher than that in the control group,while the content of IL-2 decreased,the proportion of G0/G1 phase lymphocytes decreased,the proportion of S phase and G2/M increased,and the CaN activity of lymphocytes increased significantly (P < 0.05).For the BZT group,there was only a small amount of inflammatory cell infiltration in the lung tissue under light microscope,the IL-4 level in the plasma was lower than that in the asthma model group,while the IL-2 content increased,the proportion of G0/G1 phase lymphocytes increased,the proportion of S phase and G2/M decreased significantly,and the CaN activity of lymphocytes decreased significantly (P < 0.05).Conclusion BZT could reduce lymphocyte proliferation,decrease IL-4 level and reduce airway inflammation by reducing CaN activity in peripheral blood lymphocytes of bronchial asthma rats.
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Objective To observe the effects of Sishen Pills on gene and protein expressions of toll-like receptor 2 (TLR-2) and toll-like receptor 4 (TLR-4) of colonic tissue in spleen-kidney yang deficiency rats with ulcerative colitis (UC); To discuss its mechanism of action for spleen-kidney yang deficiency UC. Methods Lavage of senna +intraperitoneal injection of hydrocortisone injection + enema of 2,4,6-trinitrobenzene sulfonic acid/ethanol were used to establish the model of UC of spleen-kidney yang deficiency type. 90 Wister rats were randomly divided into blank group, model group, Salazosulfadimidine group and the Sishen Pills high-, medium- and low-group. Each medication group was intervened by relevant medicine. RT-qPCR was used to detect the expressions of TLR-2 and TLR-4, and Western blot was used to detect the protein expressions of TLR-2 and TLR-4. SOD and MDA in serum were detected. Results Compared with the blank group, SOD activity decreased and MDA content increased in the model group (P<0.01); gene and protein expressions of TLR-2 and TLR-4 increased significantly (P<0.05). Compared with model group, SOD increased and MDA content decreased in all the medication groups (P<0.05, P<0.01); gene and protein expressions of TLR-2 and TLR-4 decreased (P<0.05, P<0.01). Conclusion Sishen Pills can achieve the treatment of UC by improving the metabolism of oxygen radicals in colonic tissue and regulating the immune system of the intestinal epithelium.
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Objective To observe the effects ofLinggan Wuwei Jiangxin Decoction on contents of cAMP, cAMP dependent PKA and AQP5 of model rats with clod phlegm-fluid lying latent in the lung asthma;To discuss its mechanism.Methods Ovalbumin intraperitoneal injection and atomization motivation + cold stimulation were used to establish models. Ninety rats were randomly divided into blank control group, model group, hexadecadrol group, andLinggan Wuwei Jiangxin Decoction low-, medium-, high-dose groups. All administration groups were treated with relevant medicine. The levels of cAMP, PKA, and AQP5 were detected by ELISA.Results Compared with the blank control group, the levels of cAMP, PKA, and AQP5 of rats in model group decreased obviously (P<0.05). Compared with the model group, the levels of cAMP, PKA, and AQP5 of rats in all administration groups increased (P<0.05), which increased dramatically inLinggan Wuwei Jiangxin Decoction high-dose group.ConclusionLinggan Wuwei Jiangxin Decoction can regulate the normal secretion of cAMP, PKA, and AQP5 in serum of rats with clod phlegm-fluid lying latent in the lung asthma, increase secretion of air passage liquid and decrease mucoprotein concentration, through which plays the role of treating asthma.
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Objective To observe the influence of Sishen Pill on the NF-κB p65 mRNA and protein expressions of colonic mucosa in rats with experimental ulcerative colitis (UC), and identify its underlying mechanism of action. Methods The experimental rats were divided into blank group, model group, Sishen Pill group and SASP group. The models were prepared by TNBS/ethanol enema. Sishen Pill group was intragastrically administrated by Sishen Pill extract 5 g/kg, SASP group by SASP 0.3 g/kg, and blank group and model group by equal volume of normal saline. The morphological injury of colonic mucosa was observed and scored with the naked eyes, and NF-κB p65 gene and protein expression were detected by RT-PCR and immunohistochemical method. Results Inflammation and ulceration on the colonic mucous membrane were found in the model group by naked eyes, and had significant difference with the blank group (P<0.05). The relative expression amount of NF-κB p65 gene and protein of colonic tissues were increased in the model group compared with the blank group (P<0.01). Compared with the model group, the relative expression amount of NF-κB p65 gene and protein in Sishen Pill group were decreased (P<0.05, P<0.01). Conclusion Sishen Pill has effect for treating UC, which is probably related to the activation of NF-κB signal transduction pathway.
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Objective To explore the effects of Kangshuaiyizhi capsule on ChAT/AchE and NE, DA and 5-HT levels in the brain tissue of aging model rats, and explore its effect of protecting cerebral function. Methods Totally 72 rats were randomly divided into normal group and model group. The subacutely aging model rats were made by injecting D-gal (0.125 g/kg) into abdominal cavity continually, then aging rats were divided by random number table into model group, Naofukang group and Kangshuaiyizhi high-, low-dose group. After intervented with correspongding drugs for 60 days, activity of ChAT and AchE, cerebral cortex NE, DA, and 5-HT levels were detected. Results Activity of AchE was much higher (P<0.05), but level of ChAT, NE, DA and 5-HT in model group were significantly downregulated compared with normal group (P<0.05). After treated with Kangshuaiyizhi capsule, activity of AchE was downregulated, ChAT, NE, DA and 5-HT levels were significantly upregulated (P <0.01, P <0.05). Conclusion Kangshuaiyizhi capsule can regulate cholinergic and monoamine neurotransmitter levels in the brain tissue of aging model rats, and play a very important role in protecting cerebral function.
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Objective To observe the effect of Sishen Pill on ICAM-1 mRNA and protein expression of colonic mucosa in rats with ulcerative colitis (UC), and identify its mechanism. Methods Taolly 40 SPF Wistar rats were randomly divided into blank group, model group, Pill group and SASP group. Except the blank group, UC model was prepared with TNBS/ethanol enema. Pill group was given Sishen Pill 5 g/kg, and SASP group was given SASP 0.3 g/kg by gavage, blank group and model group was given the same volume physiological saline for three weeks. Morphological injury of colonic mucosa was observed and scored. ICAM-1 gene and protein expression were detected by RT-PCR and immunohistochemical method. Results Inflammation and ulceration were found on the colonic mucous membrane of rats in the model group. The expression of ICAM-1 gene and protein of colonic tissues of rats in the model group increased compared with that of the blank group (P<0.01). Compared with the model group, the expression of ICAM-1 in Pill group decreased (P<0.05, P<0.01). Conclusion Sishen Pill can decrease the expression of ICAM-1 mRNA and ICAM-1 protein, inhibit the infiltration of inflammation cells, prevent and reduce colon tissue damage, and play a vital role in the treatment of UC.