San Huang Xiao Yan recipe modulates the HMGB1-mediated abnormal inflammatory microenvironment and ameliorates diabetic foot by activating the AMPK/Nrf2 signalling pathway.

BACKGROUND: Diabetic foot (DF) is one of the serious complications of diabetes and lacks of therapeutic drugs. Abnormal and chronic inflammation promoting foot infection and wound healing delay are the main pathogenesis of DF. The traditional prescription San Huang Xiao Yan Recipe (SHXY) has been used in the clinical treatment of DF for several decades as approved hospital experience prescription and showed remarkable therapeutic effect, but the mechanisms by which SHXY treats DF are still unclear. PURPOSE: Objectives of this study were to investigate SHXY anti-inflammatory effect on DF and explore the molecular mechanism for SHXY. METHODS: We detected the effects of SHXY on DF in C57 mouse and SD rat DF models. Animal blood glucose, weight and wound area were detected every week. Serum inflammatory factors were detected by ELISA. H&E and Masson's trichrome were used to observe tissue pathology. Single-cell sequencing data reanalysis revealed the role of M1 macrophages in DF. Venn analysis showed the co-target genes between DF M1 macrophages and compound-disease network pharmacology. Western blotting was used to explored target protein expression. Meanwhile, RAW264.7 cells were treated with drug-containing serum of SHXY to further unravel the roles of target proteins during high glucose-induced inflammation in vitro. The Nrf2 inhibitor ML385 was used on RAW 264.7 cells to further explore the relationship between Nrf2, AMPK and HMGB1. The main components of SHXY were analysed by HPLC. Finally, the treatment effect of SHXY on DF were detected on rat DF model. RESULTS: In vivo, SHXY can ameliorate inflammatory, accelerate wound healing and upregulate expression of Nrf2, AMPK and downregulate of HMGB1. Bioinformatic analysis showed that M1 macrophages were the main inflammatory cell population in DF. Moreover, the Nrf2 downstream proteins HO-1 and HMGB1 were potential DF therapeutic targets for SHXY. In vitro, we also found that SHXY increased AMPK and Nrf2 protein levels and downregulated HMGB1 expression in RAW264.7 cells. Inhibiting the expression of Nrf2 impaired the inhibition effect of SHXY on HMGB1. SHXY promoted Nrf2 translocation into the nucleus and increased the phosphorylation of Nrf2. SHXY also inhibited HMGB1 extracelluar release under high glucose. In rat DF models, SHXY also exhibited significant anti-inflammatory effect. CONCLUSION: The SHXY activated AMPK/Nrf2 pathway to suppress abnormal inflammation on DF via inhibiting HMGB1 expression. These findings provide novel insight into the mechanisms by which SHXY treats DF.

Ruan Jian Qing Mai Recipe Inhibits the Inflammatory Response in Acute Lower Limb Ischemic Mice through the JAK2/STAT3 Pathway.

Ruan jian qing mai recipe (RJQM) is an empirical prescription for treating arteriosclerosis obliterans (ASO). However, the mechanism of RJQM recipe-mediated ASO attenuation has not yet been elucidated. Therefore, this study aimed to explore the mechanism by which the RJQM recipe relieves ASO in a mouse model of lower limb ischemia, which was established by ligating and breaking the femoral artery of the left lower limb. The surgical groups were divided into the ischemic group, beraprost sodium group, low-dose RJQM group, medium-dose RJQM group, and high-dose RJQM group. Normal mice were set as the control group. The blood flow of the lower limb was examined on days 7 and 14. At the end of animal procedures, blood samples were collected, and the rectus femoris of the left lower limb were harvested. Results revealed that mice in the ischemic group demonstrated low blood flow. Additionally, hematoxylin and eosin, and Masson staining results showed that inflammation of the rectus femoris was obvious in the ischemia group, and the level of fibrosis was increased. Blood flow was recovered in all treatment groups compared to the ischemic group, and the inflammatory infiltration and fibrosis of the rectus femoris were relieved after RJQM treatment. The serum levels of interleukin (IL)-17A and IL-21 were decreased, and the expression of JAK2/STAT3 proteins was inhibited in all RJQM treatment groups compared to the ischemia group. Furthermore, the improvement of IL-17A, IL-21, and rectus femoris fibrosis was more obvious with increasing treatment time. In conclusion, RJQM can effectively alleviate ASO and promote the recovery of lower limb blood flow by regulating the JAK2/STAT3 signaling pathway to reduce the inflammatory response.

Baicalin relaxes vascular smooth muscle and lowers blood pressure in spontaneously hypertensive rats.

Scutellaria baicalensis Georgi is an extensively used medicinal herb for the treatment of hypertension in traditional Chinese medicine. Baicalin is the most abundant flavone compound present in Scutellaria baicalensis Georgi and endothelium-dependent vascular activities of baicalin have been suggested. However, the pharmacological implications and mechanisms of baicalin under hypertensive conditions remain to be investigated. The current study examined the blood pressure-lowering effect of baicalin in a spontaneously hypertensive rat (SHR) model. Moreover, vascular activities and mechanisms of baicalin were investigated under hypertensive conditions. The results demonstrate that baicalin treatment lowers the blood pressure in SHRs in vivo. Ex vivo vascular reactivity assay reveals that baicalin relaxes phenylephrine (PE)-constricted SHR aortas in an endothelium-independent manner. Baicalin attenuates Angiotensin II (Ang II) or potassium chloride (KCl)-induced vasoconstriction in SHR aortas as well. Baicalin also relaxes SHR aortas in the presence of different Ca2+ channel blockers such as nifedipine and SKF96365 in response to PE-induced constriction. Most importantly, ATP-sensitive potassium channel (KATP) blockade partially abrogated the vasorelaxant effect of baicalin. In summary, the current study demonstrates for the first time that intracellular Ca2+ regulation in vascular smooth muscle is mechanistically implicated in the vasorelaxant effect of baicalin under hypertensive conditions. Furthermore, activated KATP channels are in part required for the vasorelaxant effect of baicalin under hypertensive conditions. Thus, the work here sheds novel pharmacological and mechanistic insights into the blood pressure-lowering effect of baicalin, which may help better understand the therapeutic application of Scutellaria baicalensis Georgi in the treatment of hypertension.

Two new phenols from Lysimachia patungensis.

Two new phenols, methyl 3-(2-O-ß-d-glucopyranosyl-3-hydroxy-5-methoxyphenyl) propionate (1) and myricetin-3,3',5'-tri-O-α-l-rhamnopyranoside (2), together with six known phenols compounds (3-8), were isolated from the whole plant of Lysimachia patungensis Hand.-Mazz. Their structures were elucidated on the basis of the interpretation of spectroscopic data, viz., ESI-MS, HR-TOF-MS, UV, IR, and NMR. All the known phenols were isolated from the genus Lysimachia for the first time. A preliminary bioassay revealed that compounds 3 and 7 exhibited significant protective effects against hydrogen peroxide-induced damage in human retinal endothelial cells (HRECs) with the concentration of 10 µM, respectively. Compound 1 showed moderate activity against the HRECs damage at 100 µM.

[Anti-atherosclerotic Effects of Bear Bile Powder in Shexiang Tongxin Dripping Pill: a Mechanism Study].

UNLABELLED: OBJECTIVE : To study the anti-atherosclerotic mechanism of bear bile powder (BBP) in Shexiang Tongxin Dripping Pill (STDP) , and to provide scientific evidence for treating atherosclerosis (AS) by its therapeutic characteristics of cool resuscitation. METHODS: AS model was duplicated using ApoE-/- gene knocked mice fed with high-fat diet. Thirty ApoE-/- deficient male mice were divided into four groups according to body weight using random digit table, i.e., the model group (A, n =9), the STDP group (B, n=E7), the STDP without BBP group (C, n =7), and the BBP group (D, n =9). Besides, another 9 C57BL/6J male mice of the same age were recruited as a normal control group (E). All mice in Group B, C, and D were respectively administered with corresponding drugs (30, 30, and 0. 33 mg/kg) by gastrogavage. Equal volume of normal saline was administered to mice in Group A and E. All medication lasted for 8 successive weeks. Serum levels of inflammatory cytokines such as interleukin 2 (IL-2), interleukin 6 (IL-6), tumor necrosis factor a (TNF-α), interferon y (IFNγ), and oxidized low-density lipoprotein (ox-LDL) were measured by ELISA. Serum levels of malondialdehyde (MDA), activities of glutathione (GSH) and superoxide dismutase (SOD) were determined using biochemical assay. Contents of reactive oxygen species (ROS) in the aortic root was detected by dihydroethidum (DHE) fluorescent probe. Expression levels of microRNAs (such as miR-20, miR-21, miR-126, and miR-155) were detected by real-time PCR. RESULTS: The fluorescence intensity of the aorta was obviously enhanced in Group A. But it was obviously attenuated in Group B, C, and D, and the attenuation was the most in Group B. Compared with Group E, serum levels of IL-2, IL-6, TNF-α, IFN-γ, oxLDL, and MDA all increased (P <0. 01), GSH contents and SOD activities decreased (P <0. 01), expression levels of miR-126, miR-21, and miR-155 in aorta increased (P <0. 01), and the expression level of miR-20 decreased in Group A (P<0. 01). Compared with Group A, serum levels of IL-2, IL-6, TNF-α, IFN-γ, oxLDL, and MDA were all down-regulated (P <0. 01), GSH contents and SOD activities were up-regulated (P <0. 01), expression levels of miR-126, miR-21, and miR-155 in aorta were down-regulated in Group B, C, and D (P <0. 01). The expression level of miR20 was up-regulated in Group B and D (P <0. 01). Compared with Group B, serum levels of IL-2, IL-6, TNF-α, IFN-γ increased (P <0.01); GSH contents and SOD activities decreased, levels of MDA and oxLDL increased (P <0. 01) in Group C and D. Expression levels of miR-20 and miR-155 were down-regulated in Group C and D (P <0. 01). CONCLUSIONS: STDP played roles in significantly regulating inflammatory factors and oxidative stress factors. Its mechanism might be possibly associated with regulating expressions of miR-126, miR-21, miR-155, and miR-20 in aorta. BBP played significant roles in STDP.

Shexiang Tongxin dropping pill attenuates atherosclerotic lesions in ApoE deficient mouse model.

ETHNOPHARMACOLOGICAL RELEVANCE: Shexiang Tongxin dropping pill (STDP) is a formulation of Traditional Chinese Medicine mainly used for clinical treatment of stable angina pectoris in China. AIM OF THE STUDY: To investigate the effects and mechanisms of STDP treatment on atherosclerosis. MATERIALS AND METHODS: ApoE deficient (ApoE(-/-)) mice were utilized to evaluate the effect of STDP treatment (30 mg/kg/day) on atherosclerotic lesions. Histopathological features of atherosclerotic lesions, serum levels of lipid proteins, parameters of oxidative stress and pro-inflammatory cytokines were measured by H&E staining, Masson's trichrome staining and ELISA, respectively. Real-time PCR analyses were performed to examine the aortic expression of atherosclerosis-associated microRNAs. RESULTS: The STDP treatment resulted in attenuated atherosclerotic lesion manifested by reduced lipid deposition, fibrosis and oxidative stress. It also led to increase in serum levels of GSH and SOD, decrease in MDA, decrease in CHO, TG, LDL, ox-LDL and increase in HDL, respectively. Additionally, the levels of pro-inflammatory cytokines including IL-2, IL-6, TNF-α and γ-IFN were markedly reduced by STDP treatment. Furthermore, STDP treatment was associated with a significant reduction in the aortic expression of miR-21a, miR-132, miR-126a, miR-155 and increased expression of miR-20a. CONCLUSION: Our results demonstrated for the first time that STDP attenuated atherosclerotic lesions in ApoE(-/-) mouse model. Moreover, STDP treatment exhibited multi-targeting effects on pathological, biochemical and molecular aspects of atherosclerosis implicating lipid regulation, fibrosis, inflammation and oxidative stress. Findings from the current study warrant further evaluation of the clinical application of STDP in atherosclerosis treatment.

Bidirectional regulation of angiogenesis and miR-18a expression by PNS in the mouse model of tumor complicated by myocardial ischemia.

BACKGROUND: Panax Notoginseng Saponins (PNS) is the major class of active constituents of notoginseng, a natural product extensively used as a therapeutic agent in China. Tumor when accompanied by cardiovascular disorders poses a greater challenge for clinical management given the paradoxical involvement of angiogenesis, therefore gaining increased research attention. This study aim to investigate effects of PNS and its activity components in the mouse model of tumor complicated with myocardial ischemia. METHODS: Tumor complexed with myocardial ischemia mouse model was first established, which was followed by histological and immunohistochemistry examination to assess the effect of indicated treatments on tumor, myocardial ischemia and tissue specific angiogenesis. MicroRNA (miRNA) profiling was further carried out to identify potential miRNA regulators that might mechanistically underline the therapeutic effects of PNS in this complex model. RESULTS: PNS and its major activity components Rg1, Rb1 and R1 suppressed tumor growth and simultaneously attenuated myocardial ischemia. PNS treatment led to decreased expression of CD34 and vWF in tumor and increased expression of these vascular markers in heart. PNS treatment resulted in reduced expression of miR-18a in tumor and upregulated expression of miR-18a in heart. CONCLUSIONS: Our data demonstrated for the first time that PNS exerts tissue specific regulatory effects on angiogenesis in part through modulating the expression of miR-18a, which could be responsible for its bidirectional effect on complex disease conditions where paradoxical angiogenesis is implicated. Therefore, our study provides experimental evidence warranting evaluation of PNS and related bioactive component as a rational therapy for complex disease conditions including co-manifestation of cancer and ischemic cardiovascular disease.

Panax notoginseng saponins (PNS) inhibits breast cancer metastasis.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax notoginseng (Burkill) F.H. Chen (Araliaceae) has been extensively used as a therapeutic agent to treat a variety of diseases. Panax notoginseng saponins (PNS) consist of major therapeutically active components of Panax notoginseng. PNS inhibit the growth of a variety of tumor cells in vitro and in vivo. The aim of the study is to investigate the effects and underlying mechanisms of PNS on breast cancer metastasis. MATERIALS AND METHODS: 4T1 cell, a highly metastatic mouse breast carcinoma cell line, was utilized for in vitro and in vivo assays. In vitro assays were first performed to examine the effects of PNS on 4T1 cell viability, migration and invasion, respectively. Real-time PCR analyses were also performed to examine the effects of PNS on the expression of genes associated with tumor metastasis. The effect of PNS on 4T1 tumor cell metastasis was further assessed in spontaneous and experimental metastasis models in vivo. RESULTS: PNS treatment exhibited a dose-dependent effect on impairing 4T1 cell viability in vitro. However, when examined at a lower dose that did not affect cell viability, the migration and invasion of 4T1 cell was remarkably inhibited in vitro. Meanwhile, PNS treatment led to upregulated expression of genes known to inhibit metastasis and downregulated expression of genes promoting metastasis in cultured 4T1 cells. These results suggested a selective effect of PNS on 4T1 migration and invasion. This hypothesis was further addressed in 4T1 metastasis models in vivo. The results showed that the lung metastasis was significantly inhibited by PNS treatment in both spontaneous and experimental metastasis models. CONCLUSION: Taken together, our results demonstrated an inhibitory effect of PNS on 4T1 tumor metastasis, warranting further evaluation of PNS as a therapeutic agent for treating breast cancer metastasis.