Jinlida granules ameliorate the high-fat-diet induced liver injury in mice by antagonising hepatocytes pyroptosis.

CONTEXT: Jinlida (JLD) as a traditional Chinese medicine formula has been used to treat type 2 diabetes mellitus (T2DM) and studies have shown its anti-obesity effect. OBJECTIVE: To investigate the therapeutic effects of JLD in a mouse model of non-alcoholic fatty liver (NAFL). MATERIALS AND METHODS: C57BL/6J mice were divided into three groups and fed a low-diet diet (LFD), high-fat diet (HFD), or HFD + JLD (3.8 g/kg) for 16 weeks, respectively. The free fatty acids-induced lipotoxicity in HepG2 cells were used to evaluate the anti-pyroptotic effects of JLD. The pharmacological effects of JLD on NAFL were investigated by pathological examination, intraperitoneal glucose and insulin tolerance tests, western blotting, and quantitative real-time PCR. RESULTS: In vivo studies showed that JLD ameliorated HFD-induced liver injury, significantly decreased body weight and enhanced insulin sensitivity and improved glucose tolerance. Furthermore, JLD suppressed both the mRNA expression of caspase-1 (1.58 vs. 2.90), IL-1ß (0.93 vs. 3.44) and IL-18 (1.34 vs. 1.60) and protein expression of NLRP3 (2.04 vs. 5.71), pro-caspase-1 (2.68 vs. 4.92) and IL-1ß (1.61 vs. 2.60). In vitro, JLD inhibited the formation of lipid droplets induced by 2 mM FFA (IC50 = 2.727 mM), reduced the protein expression of NLRP3 (0.74 vs. 2.27), caspase-1 (0.57 vs. 2.68), p20 (1.67 vs. 3.33), and IL-1ß (1.44 vs. 2.41), and lowered the ratio of p-IKB-α/IKB-α (0.47 vs. 2.19). CONCLUSION: JLD has a protective effect against NAFLD, which may be related to its anti-pyroptosis, suggesting that JLD has the potential as a novel agent in the treatment of NAFLD.

Long non-coding RNA HOXA11-AS accelerates the progression of keloid formation via miR-124-3p/TGFßR1 axis.

Emerging evidence reveals the importance of long non-coding RNAs (lncRNAs) in the development and progression of keloid formation, whereas the underlying mechanisms are not well understood. In the present study, we investigated the biological effects and molecular mechanisms of lncRNA HOXA11-AS in keloid formation. First, the expression levels of HOXA11-AS, miR-124-3p, and transforming growth factor ß receptor type I (TGFßR1) were measured in both keloid tissues and human keloid fibroblasts (HKFs) using qRT-PCR and western blot analysis, respectively. Next, we adopted both gain- and loss-of-function strategies to explore the significance of HOXA11-AS. TUNEL, flow cytometry, DNA ladder, and tube formation assays were performed to measure cell apoptosis and angiogenesis, respectively. Besides, the potential binding relationship between HOXA11-AS and miR-124-3p, as well as miR-124-3p and TGFßR1 was identified using bioinformatic screening and verified by luciferase reporter assay. Furthermore, we explored the importance of miR-124-3p in HOXA11-AS-induced phenotypes and regulations on TGFß signaling or PI3K/Akt signaling. We found that HOXA11-AS and TGFßR1 were significantly up-regulated, while miR-124-3p was down-regulated both in keloid tissues or fibroblasts than in normal skin tissues or fibroblasts. Functionally, high expression of HOXA11-AS essentially inhibited cell apoptosis and promoted fibroblast-induced angiogenesis. Mechanistically, miR-124-3p was identified as a downstream effector to be involved in HOXA11-AS-mediated phenotypes through directly targeting TGFßR1, thus modulating PI3K/Akt signaling pathway. Taken together, our findings revealed that HOXA11-AS inhibits cell apoptosis and promotes angiogenesis through miR-124-3p/TGFßR1 axis, contributing to the progression of keloid formation, which might provide a novel target for keloid therapy.

[Effect of Tongxinluo on Apoptosis of Rat Cardiac Microvascular Endothelial Cells].

OBJECTIVE: To observe the protective effects of Tongxinluo (TXL) on apoptosis of rat cardiac microvascular endothelial cells (RCMECs) resulting from homocysteine (Hcy) induced endoplasmic reticulum stress (ERS), and to determine the signaling pathway behind its protection. METHODS: Primary cultured RCMECs were isolated from neonatal rats using tissue explant method. The morphology of RCMECs was observed using inverted microscope, identified and differentiated by CD31 immunofluorescence method. Selected were well growing 2nd-4th generations of RCMECs. The optimal action time was determined by detecting the expression of glucose regulated protein 78 (GRP78) using immunofluorescence method. In the next experiment RCMECs were divided into 5 groups, i.e., the blank control group, the Hcy induced group (Hcy 10 mmol/L, 10 h), the Hcy + TXL group (Hcy 10 mmol/L + TXL 400 µg/mL), the Hcy +LY294002 group (Hcy 10 mmol/L + LY294002 5 µmol/L, LY294002 as the inhibitor of PI3K), the Hcy + LY294002 + TXL group (Hcy 10 mmol/L + LY294002 5 µmol/L + TXL 400 µg/mL). The apoptosis rate of RCMECs was detected by flow cytometry. mRNA and protein expressions of GRP78, C/ EBP homologous protein (CHOP), and cysteinyl aspartate specific proteinase-12 (caspase12) were detected by real-time reverse transcription PCR (RT-PCR) and Western blot respectively. Expression levels of phosphorylation of phosphatidylinositol 3-kinase (P-PI3K), total phosphatidylinositol 3-kinase (T- P13K) , phosphorylation of kinase B (P-Akt) , and total kinase B (T-Akt) were detected by Western blot. RESULTS: Ten hours Hcy action time was determined. Compared with the blank control group, the apoptosis rate was increased (22.77%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were increased, protein expressions of P-PI3K and P-Akt,ratios of P-PI3K/T-PI3K and P-Akt/T-Akt were decreased in the Hcy induced group (P < 0.05, P < 0.01). Compared with the Hcy induced group, the apoptosis rate was decreased (10.17%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were decreased, and expression levels of P-PI3K, P-Akt, P-PI3K/T-PI3K, and P-Akt/T-Akt were increased in the Hcy + TXL group (P < 0.05, P < 0.01). Compared with the Hcy + TXL group, the apoptosis rate was increased (17.9%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were increased, expression levels of P-PI3K and P-Akt, ratios of P-PI3K/T-PI3K and P-Akt/T-Akt were decreased in the Hcy + TXL + LY294002 group (P < 0.05, P < 0.01). CONCLUSION: TXL could inhibit the apoptosis of RCMECs resulting from Hcy-induced ERS and its mechanism might be associated with activating PI3K/Akt signaling pathway.

Shensongyangxin protects against pressure overload­induced cardiac hypertrophy.

Shensongyangxin (SSYX) is a medicinal herb, which has long been used in traditional Chinese medicine. Various pharmacological activities of SSYX have been identified. However, the role of SSYX in cardiac hypertrophy remains to be fully elucidated. In present study, aortic banding (AB) was performed to induce cardiac hypertrophy in mice. SSYX (520 mg/kg) was administered by daily gavage between 1 and 8 weeks following surgery. The extent of cardiac hypertrophy was then evaluated by pathological and molecular analyses of heart tissue samples. In addition, in vitro experiments were performed to confirm the in vivo results. The data of the present study demonstrated that SSYX prevented the cardiac hypertrophy and fibrosis induced by AB, as assessed by measurements of heart weight and gross heart size, hematoxylin and eosin staining, cross­sectional cardiomyocyte area and the mRNA expression levels of hypertrophic markers. SSYX also inhibited collagen deposition and suppressed the expression of transforming growth factor ß (TGFß), connective tissue growth factor, fibronectin, collagen Ⅰα and collagen Ⅲα, which was mediated by the inhibition of the TGFß/small mothers against decapentaplegic (Smad) signaling pathway. The inhibitory action of SSYX on cardiac hypertrophy was mediated by the inhibition of Akt signaling. In vitro investigations in the rat H9c2 cardiac cells also demonstrated that SSYX attenuated angiotensin II­induced cardiomyocyte hypertrophy. These findings suggested that SSYX attenuated cardiac hypertrophy and fibrosis in the pressure overloaded mouse heart. Therefore, the cardioprotective effect of SSYX is associated with inhibition of the Akt and TGFß/Smad signaling pathways.

The role of AMPKα in high-glucose-induced dysfunction of cultured rat mesangial cells.

AIM: Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) is known as a mammalian cell energy sensor, which could regulate cellular energy metabolism via sensing the alterations of energy balance, such as oversupply or lack of glucose and fatty acid. Recent studies have suggested that AMPK could also regulate many other biological processes, including cell cycling, inflammation, protein synthesis, and so on. In this study, AMPK signaling in high-glucose-induced dysfunction of mesangial cells (MCs) was investigated. METHODS: Established rat glomerular MCs were treated under normal glucose (5.6 mM glucose) or high-glucose conditions (30 mM glucose). mRNA levels of AMPK subunits were detected by reverse transcriptase-polymerase chain reaction. Expressions of AMPKα, phosphorylated AMPKα (p-AMPKα), phosphorylated acetyl-CoA carboxylase (p-ACC), and collagen IV were measured by Western blot. RESULTS: Under high-glucose conditions, AMPKα protein expression and mRNA levels were significantly decreased. High-glucose treatment also induced a notable decrease in p-AMPKα and p-ACC expression. AMPKα activation by 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) effectively ameliorated high-glucose-induced dysfunction of MCs, including cell proliferation, cell-cycle progression, and collagen IV production. CONCLUSION: High glucose impaired AMPKα in its expression and activity; AICAR significantly ameliorated high-glucose-induced proliferation of MCs and collagen IV production, indicating a role of AMPKα in high-glucose-induced dysfunction of MCs.

Exhaustive swimming differentially inhibits P2X1 receptor- and α1-adrenoceptor-mediated vasoconstriction in isolated rat arteries.

AIM: To investigate the effects of exhaustive swimming exercise on P2X1 receptor- and α1-adrenoceptor-mediated vasoconstriction of different types of arteries in rats. METHODS: Male Wistar rats were divided into 2 groups: the sedentary control group (SCG) and the exhaustive swimming exercise group (ESEG). The rats in the ESEG were subjected to a swim to exhaustion once a day for 2 weeks. Internal carotid, caudal, pulmonary, mesenteric arteries and aorta were dissected out. Isometric vasoconstrictive responses of the arteries to α,ß-methylene ATP (α,ß-MeATP) or noradrenaline (NA) were recorded using a polygraph. RESULTS: The exhaustive swimming exercise did not produce significant change in the EC(50) values of α,ß-MeATP or NA in vasoconstrictive response of most of the arteries studied. The exhaustive swimming exercise inhibited the vasoconstrictive responses to P2X1 receptor activation in the internal carotid artery, whereas it reduced the maximal vasoconstrictive responses to α1-adrenoceptor stimulation in the caudal, pulmonary, mesenteric arteries and aorta. The rank order of the reduction of the maximal vasoconstriction was as follows: mesenteric, pulmonary, caudal, aorta. CONCLUSION: Exhaustive swimming exercise differentially affects the P2X1 receptor- and α1-adrenoceptor-regulated vasoconstriction in internal carotid artery and peripheral arteries. The ability to preserve purinergic vasoconstriction in the peripheral arteries would be useful to help in maintenance of the basal vascular tone during exhaustive swimming exercise.

Physiological significance of P2X receptor-mediated vasoconstriction in five different types of arteries in rats.

P2X(1) receptors, the major subtype of P2X receptors in the vascular smooth muscle, are essential for α,ß-methylene adenosine 5'-triphosphate (α,ß-MeATP)-induced vasoconstriction. However, relative physiological significance of P2X(1) receptor-regulated vasoconstriction in the different types of arteries in the rat is not clear as compared with α(1)-adrenoceptor-regulated vasoconstriction. In the present study, we found that vasoconstrictive responses to noncumulative administration of α,ß-MeATP in the rat isolated mesenteric arteries were significantly smaller than those to single concentration administration of α,ß-MeATP. Therefore, we firstly reported the characteristic of α,ß-MeATP-regulated vasoconstrictions in rat tail, internal carotid, pulmonary, mesenteric arteries, and aorta using single concentration administration of α,ß-MeATP. The rank order of maximal vasoconstrictions for α,ß-MeATP (E (max·α,ß-MeATP)) was the same as that of maximal vasoconstrictions for noradrenaline (E (max·NA)) in the internal carotid, pulmonary, mesenteric arteries, and aorta. Moreover, the value of (E (max·α,ß-MeATP)/E (max·KCl))/(E (max·NA)/E (max·KCl)) was 0.4 in each of the four arteries, but it was 0.8 in the tail artery. In conclusion, P2X(1) receptor-mediated vasoconstrictions are equally important in rat internal carotid, pulmonary, mesenteric arteries, and aorta, but much greater in the tail artery, suggesting its special role in physiological function.

Chinese medicine Tongxinluo modulates vascular endothelial function by inducing eNOS expression via the PI-3K/Akt/HIF-dependent signaling pathway.

AIM OF THE STUDY: To investigate the molecular mechanisms whereby the Chinese medicinal compound Tongxinluo improves vascular endothelial function through studying the induction of endothelial nitric oxide synthase (eNOS) and its upstream signaling pathway. MATERIALS AND METHODS: Hyperhomocysteinemia was induced in Wistar rats by a methionine-rich diet followed by Tongxinluo treatment. The aorta ring was isolated for measuring vascular dilation of aorta and eNOS expression. Human umbilical vein endothelial cells (HUVECs) were transfected with AP-1, NF-κB, HRE or eNOS reporter plasmid followed by Tongxinluo exposure. Expression of the reporter genes was measured by luciferase assay. The level of eNOS was studied by western blot and the nitric oxide content was measured using the nitrate reductase method. HUVECs were also transiently transfected with the dominant negative mutant of HIF-1, PI-3K or Akt to explore the role of HIF and PI-3K/Akt pathway in eNOS induction by Tongxinluo. RESULTS: Tongxinluo could significantly up-regulate the expression of eNOS in the aortic tissue and improve the endothelium-dependent vasodilation of the aorta ring. Additionally, Tongxinluo at various doses could significantly enhance the expression of HRE and eNOS reporter gene as well as up-regulate the protein level of eNOS. Meanwhile, Tongxinluo caused a dose-dependent increase in the NO content in the supernatant of HUVECs. Suppression of HIF-1 activation by DN-HIF or inhibition of PI-3K/Akt pathway by ΔP85 or DN-Akt both attenuated HRE reporter gene activation and eNOS induction by Tongxinluo. CONCLUSION: Tongxinluo, a compound Chinese traditional medicine, up-regulates the expression of eNOS via the PI-3K/Akt/HIF-dependent signaling pathway, thus improving the endothelium-dependent vasodilation.

[Effects of Tongxinluo on repeated hypoxic tolerance in mice].

OBJECTIVE: To observe the effects of Tongxinluo (TXL) on repeated hypoxic tolerance in mice and explore the underlying mechanisms. METHODS: Mice were randomly divided into groups of repeated hypoxia (control) and TXL according to body weights. The mice in each group were exposed to acute repeated hypoxia for 0 run (H0), 1 run (H1), 3 runs (H3) and 5 runs (H5). The animal's tolerance time of each hypoxic exposure was recorded. Western blot was used to measure the protein levels of hypoxia inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in cortex tissue. RESULTS: The hypoxic tolerance time in control and TXL groups significantly increased run by run. Both HIF-1alpha and VEGF proteins in two groups increased gradually. Compared with control group, the tolerance time in H1 of TXL group [(18.0 +/- 2.4) minvs (15.6 +/- 2.0) min], H3 [(68.3 +/- 13.2) min vs (41.7 +/- 9.0) min) and H5 (85.9 +/- 7.0) min vs (51.4 +/- 14.4) min] increased (P < 0.05 or P < 0.01); the HIF-1alpha protein expression in H1 of TXL group (0.95 +/- 0.04 vs 0.79 +/- 0.02), H3 (1.01 +/- 0.03 vs 0.85 +/- 0.02), H5 (1.16 +/- 0.02 vs 0.92 +/- 0.03) increased (P < 0.05 or P < 0.01); the VEGF protein expression in H3 of TXL group (1.14 +/- 0.02 vs 0.89 +/- 0.03), H5 (1.34 +/- 0.05 vs 0.99 +/- 0.07) increased (P < 0.05 or P < 0.01). CONCLUSIONS: Under repeated hypoxia, an organism has a strong adaptive ability. The rises of HIF-1alpha and VEGF may be an adaptive mechanism. TXL can increase obviously the adaptive ability of mice to hypoxia.

Comfortable lifestyle-induced imbalance of neuro-endocrine-immunity network: a possible mechanism of vascular endothelial dysfunction.

OBJECTIVE: To observe the changes of vascular endothelial functions and general neuro-endocrine-immunity (NEI) network under the state of qi-deficiency syndrome induced by excessive idleness and to approach their internal relevance and illuminate initially the pathophysiological mechanism of vascular lesion induced by excessive idleness. METHODS: A total of 100 male Wistar rats were randomly divided into the control group and the qi-deficiency syndrome model group, 50 rats in each group. The qi-deficiency syndrome model was established by feeding the animals with hyper-alimentation diet in combination with restricting movement for 10 weeks. Changes of common chemical signal molecules related to NEI and vascular endothelial functions were measured by the end of the experiment. Furthermore, their internal relevance was analyzed by the method of canonical correlation analysis. RESULTS: The vascular endothelial structure and function were obviously injured in the model group. Compared with the control group, the chemical signal molecules, such as 5-hydroxytryptamine (5-HT), corticosterone (CORT), triiodothyronine (T3), tetraiodothyronine (T4), angiotensin II (Ang II), interleukin-1 (IL-1), and tumor necrosis factor-alpha (TNF-alpha) in peripheral blood of the model group (n=43) were changed significantly (P<0.05 or P<0.01). Canonical correlation analysis showed that vascular endothelial dysfunction was correlated to the changes of these signal molecules in the NEI network. CONCLUSIONS: Comfort-based lifestyle induced not only vascular endothelial dysfunction but also an imbalance of the NEI network. Vascular endothelial dysfunction and the imbalanced NEI network interacted with each other, and an imbalance of the NEI network may be the pathophysiologic basis for the genesis and development of vascular endothelial dysfunction, even diseases of the blood vessel.

Effect of serum from overfatigue rats on JNK/c-Jun/HO-1 pathway in human umbilical vein endothelial cells and the intervening effect of Tongxinluo superfine powder.

OBJECTIVE: To cultivate human umbilical vein endothelial cells (HUVECs) in the serum of overfatigue rats with the intervention of Tongxinluo superfine powder (TXLSP). By examining the variation of the activity of JNK/c-Jun/HO-1 pathway, the possible mechanisms of vascular endothelial dysfunction under overfatigue conditions and the intervening effect of TXLSP were explored. METHODS: The HUVECs were randomly divided into the normal control group, the model group, the SP600125 (a specific antagonist of JNK) group, the TXLSP group and the TXLSP + SP600125 group. The content of carboyhemoglobin (COHb) and the leak rate of lactic dehydrogenase (LDH) in different groups were measured. The mRNA and protein expression of JNK, c-Jun, HO-1 and the phosphorylation level of c-Jun (P-c-Jun) were detected using Western blot and PCR methods. RESULTS: Compared with the normal control group, the COHb level in supernatant was increased significantly in the model group, and the expression of HO-1, JNK, c-Jun mRNA and corresponding proteins and P-c-Jun were also increased remarkably. The increases in these parameters were significantly decreased by SP600125. TXLSP showed remarkable up-regulation on the expression of JNK, c-Jun, P-c-Jun and HO-1 mRNA and their protein expression. Compared with the SP600125 group, the expressions of JNK, c-Jun, P-c-Jun and HO-1 mRNA and its protein in the TXLSP+SP600125 group were significantly increased at different time points (P<0.05, P<0.01). CONCLUSIONS: The vascular endothelial dysfunction under overfatigue conditions is related to the activity of the JNK/c-Jun/HO-1 pathway. One of the mechanisms of TXLSP in improving the vascular endothelial function is to adjust the activity of the JNK/c-Jun/HO-1 pathway at gene and protein levels.

[Experimental study on effect of Tongxinluo on nerve cell apoptosis after cerebral ischemia in middle cerebral arterial obstructive model rats].

OBJECTIVE: To explore the action mechanism of Tongxinluo Capsule (TXL) in protecting brain from ischemic damage. METHODS: SD rats were divided into five groups randomly, the sham operation group, the model group, the MK-801 group, the large and low dosage TXL groups (TXLL and TXLS). After the middle cerebral arterial obstructive (MCAO) model was established, peritoneal injection of MK-801 0.5 mg/kg per day was given to the MK-801 group, and 1.0 g/(kg x d) and 0.5 g/(kg x d) of TXL powder was administered in twice via gastrogavage to the two TXL groups respectively. The nerve cell apoptosis rate, protein and mRNA expressions of Caspase-3, p53 and heat shock protein (HSP70) were observed using flow cytometry, Western blot and RT-PCR technique. RESULTS: Both TXL and MK-801 could obviously lower the apoptosis rate in model rat (P < 0.05, P < 0.01), TXLL showed the optimal effect. Caspase-3, p53 protein and mRNA expression in the model group were obviously higher than those in the sham operated group. As compared with the model group, the expressions of Caspase-3 and p53 were lower and those of HSP70 and mRNA were higher in the two TXL and MK-801 groups (P < 0.05 or P < 0.01). CONCLUSION: TXL displays it brain protective effect through reducing nerve cell apoptosis rate in MCAO model rats, the mechanism may be related to its actions in inhibiting apoptosis related factors Caspase-3 and p53, and promoting stress protecting factor HSP70.