Connective tissue disease-related interstitial lung disease is alleviated by tripterine through inhibition of the PI3K/Akt, apoptosis, and TNF-α signalling pathways.

Background: Interstitial lung disease (ILD) is the major cause of morbidity and mortality in patients with various rheumatic diseases. However, more interventions need to be sought. Tripterine, an extract of Tripterygium wilfordii Hook. F, has been widely studied for its powerful anti-inflammatory effect. However, its mechanism of action in treating connective tissue disease-related (CTD)-ILD remains unclear. Purpose: To investigate the mechanism of tripterine in CTD-ILD treatment by combining network pharmacology and an in vivo experiment. Methods: The related targets of tripterine were obtained after searching the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform, Comparative Toxicogenomics Database, GeneCards, Search Tool for Interacting Chemicals database, and SymMap database. Following this, Online Mendelian Inheritance in Man, GeneCards, Genebank, and DrugBank were used to screen the targets of CTD-ILD. A target-signalling pathway network was constructed using Cytoscape. Additionally, topological analysis was performed. Protein interaction analysis was performed using the STRING online analysis platform. Following this, Gene Ontology (GO) and the Kyoto Encyclopaedia of Genes and Genomes (KEGG) signalling pathway enrichment analyses were performed. Subsequently, the molecular docking between tripterine and the core targets was verified. Finally, experimental verification was performed in bleomycin-induced model mice. Results: A total of 134 common targets and 10 core targets of tripterine, including signal transducer and activator of transcription 3, tumour necrosis factor (TNF), v-rel avian reticuloendotheliosis viral oncogene homolog A, protein kinase B (Akt) α (Akt1), mitogen-activated protein kinase (MAPK) 1, Jun transcription factor family, tumour protein 53, MAPK3, nuclear factor kappa B subunit 1, and caspase 8, were obtained. GO enrichment analysis revealed that, while treating CTD-ILD, tripterine was mainly involved in cytokine receptor binding, receptor-ligand activity, signal receptor activation, cytokine activity, protein ubiquitination, deoxyribonucleic acid transcriptase activity, etc. The KEGG pathway enrichment analysis revealed that the most significant signalling pathways were multiple viral infections and the phosphatidylinositol-3-kinase (PI3K)/Akt, TNF, and apoptosis signalling pathways. Molecular docking results revealed that tripterine had good docking activity with the core targets. Experimental studies also demonstrated that tripterine could inhibit the activation of PI3K/Akt, apoptosis, and TNF-α signalling pathways in lung tissue and significantly improve lung pathology and collagen deposition in the model mice. Conclusions: This study preliminarily revealed the potential molecular biological mechanism of tripterine while treating CTD-ILD might be related to inhibiting the PI3K/Akt, apoptosis, and TNF-α signalling pathways. Tripterygium wilfordii Hook. F. and its extract could be used clinically for treating CTD-ILD.

Salvianolic Acid C Attenuates LPS-Induced Inflammation and Apoptosis in Human Periodontal Ligament Stem Cells via Toll-Like Receptors 4 (TLR4)/Nuclear Factor kappa B (NF-κB) Pathway.

BACKGROUND Periodontitis is a chronic inflammatory disease that causes gingival detachment and disintegration of alveolar bone. Salvianolic acid C (SAC) is a polyphenol compound with anti-inflammatory and antioxidant activities that is isolated from Danshen, a traditional Chinese medicine made from the roots of Salvia miltiorrhiza Bunge. The aim of this study was to investigate the mechanisms of underlying its protective effects and its inhibition effect on inflammation and apoptosis in human periodontal ligament stem cells (hPDLSCs). MATERIAL AND METHODS LPS-induced hPDLSCs, as a model mimicking an inflammatory process of periodontitis in vivo, were established to investigate the therapeutic effect of SAC in periodontitis. The inflammatory cytokines secretion and oxidative stress status were measured by use of specific commercial test kits. The hPDLSCs viability was analyzed by Cell Counting Kit-8 assay. The cell apoptosis and cell cycle were assayed with flow cytometry. Expressions levels of proteins involved in apoptosis, osteogenic differentiation, and TLR4/NF-kappaB pathway were evaluated by Western blotting. Alkaline phosphatase (ALP) activity was detected by ALP assay kit and ALP staining. The mineralized nodules formation of hPDLSCs was checked by Alizarin Red S staining. RESULTS Our results showed that LPS induced increased levels of inflammatory cytokines and oxidative stress and mediated the phosphorylation and nuclear translocation of NF­kappaB p65 in hPDLSCs. SAC reversed the abnormal secretion of inflammatory cytokines and inhibited the TLR4/NF­kappaB activation induced by LPS. SAC also upregulated cell viability, ALP activity, and the ability of osteogenic differentiation. The anti-inflammation and TLR4/NF­kappaB inhibition effects of SAC were reversed by TLR4 overexpression. CONCLUSIONS Taken together, our results revealed that SAC effectively attenuates LPS-induced inflammation and apoptosis via the TLR4/NF-kappaB pathway and that SAC is effective in treating periodontitis.

Global protein expression analysis of molecular markers of DS-1-47, a component of implantation-promoting traditional chinese medicine.

This study investigated the molecular markers of DS-1-47, a component of an implantation- promoting traditional Chinese medicine consisting of Astragalus mongholicus, Atractylodes macrocephala, Scutellaria baicalensis and Dipsacales, in an attempt to clarify the molecular mechanism and action targets of DS-1-47. Controlled ovarian stimulation (COS) method was used to establish the implantation dysfunction models of mice. Animals were divided into normal pregnant group, COS model group and DS-1-47 group. Laser capture microdissection-double dimensional electrophoresis-mass spectrum (LCM-DE-MS) was used to analyze the uterine protein molecules that were possibly involved in the promotion of implantation. Twenty-three proteins in DS-1-47 group were significantly changed as compared to those in COS model group, with 7 proteins down-regulated and 16 proteins up-regulated. Except for some constituent proteins, the down-regulated proteins included collagen α-1 (VI) chain, keratin 7, keratin 14, myosin regulatory light chain 12B, myosin light polypeptide 9, heat shock protein ß-7, and C-U-editing enzyme APOBEC-2; the up-regulated proteins included apolipoprotein A-I, calcium regulated protein-3, proliferating cell nuclear antigen, L-xylulose reductase, and calcium binding protein. These 23 proteins that were regulated by DS-1-47 represented a broad diversity of molecule functions. The down-regulated proteins were associated with stress and immune response, and those up-regulated proteins were related to proliferation. It was suggested that these proteins were important in regulating the uterine environment for the blastocyst implantation. By identification of DS-1-47 markers, proteomic analysis coupled with functional assays is demonstrated to be a promising approach to better understand the molecular mechanism of traditional Chinese medicine.

Anti-arthritic activity of Fu-Fang-Lu-Jiao-Shuang on collagen-induced arthritis in Balb/c mice and its underlying mechanisms.

BACKGROUND: Rheumatoid arthritis (RA) is a common, autoimmune disorder characterized by progressive multiple joint destruction, deformity, disability and premature death in most patients. Fu-Fang-Lu-Jiao-Shuang (FFLJS) is an effective traditional Chinese medicine, which has long been used clinically to treat RA patients. OBJECTIVE: The objective of this study is aimed to evaluate the anti-rheumatic effects of FFLJS on collagen induced arthritis (CIA) model, as well as the underlying mechanisms, which have not previously been explored. MATERIALS AND METHODS: CIA was induced by immunization with type II collagen (CII) in male Balb/c mice. The mice in the onset of arthritis were treated daily with FFLJS (125 or 500 mg/kg) or 1% carboxymethyl cellulose-Na for 28 days. Paw thickness and arthritic score were evaluated to confirm the anti-arthritic effect of FFLJS on CIA in mice. Levels of anti-CII antibody, proinflammatory cytokines interleukin-1 (IL-1) ß, IL-17, and tumor necrosis factor-α (TNF-α) as well as prostaglandin E-2 (PGE-2) in serum and histological changes in the ankle joint were also analyzed. In addition, expressions of matrix metalloproteinases-1 (MMP-1), MMP-3 and tissue inhibitors of matrix metalloproteases-1 (TIMP-1) in synovial tissue were also detected to further study the molecular mechanism of the anti-arthritic effects of FFLJS. RESULTS: During therapeutic treatment, FFLJS significantly reduced paw thickness and arthritic score in CIA mice, decreased the amounts of TNF-α, IL-1 ß, IL-17, PGE-2 and anti-CII antibody in serum. In addition, FFLJS treatment could prevent the bone destruction by reducing the expression of MMP-1 and MMP-3, increasing the expression of TIMP-1 in synovial tissue of CIA mice. CONCLUSION: These findings offer the convincing evidence for the first time that the anti-rheumatic effects of FFLJS might be related to down-regulation of TNF-α, IL-1 ß, IL-17 and PGE-2 levels for acute arthritis, and regulation of MMP-1, MMP-3 and TIMP-1 protein expression for chronic arthritis.

DS147 improves pregnancy in mice with embryo implantation dysfunction induced by controlled ovarian stimulation.

The study examined the effect of DS147, the bioactive component of the traditional herbal recipe Bangdeyun, on pregnancy in mice with embryo implantation dysfunction induced by controlled ovarian stimulation (COS), and the underlying mechanisms. Female mice were superovulated by intraperitoneal injection of 7.5 IU of pregnant mare serum gonadotropin (PMSG) followed by an additional injection of 7.5 IU hCG 48 h later to establish embryo implantation dysfunction (EID) model. Pregnant mice were randomly divided into normal control group, COS group and DS147-treated groups. The pregnancy rate and the average implantation site were obtained on pregnancy day 8 (PD8). The side effect of 200 mg/kg of DS147 on naturally pregnant mice was also observed. Further, the uterine and ovarian tissue samples were collected on PD5 for measuring their weights, observing the development of the endometrium and ovary, and detecting the endometrial expression of MMP-2, TIMP-2, CD34 and angiogenin (ANG). The female mice treated with DS147 at doses of 100 to 800 mg/kg showed a higher pregnancy rate than those in COS group, and the highest pregnancy rate of 83.3% occurred in the 200 mg/kg DS147-treated group. Moreover, no obvious side effect was found in mice treated with 200 mg/kg DS147 on PD8 and PD16. The ovarian and uterine weights, and the expression levels of MMP-2, ANG and CD34 were significantly increased in DS147-treated groups when compared with COS group. The TIMP-2 expression level was much lower in DS147-treated mice than in COS mice and the ratio of MMP-2/TIMP-2 was much higher in DS147-treated group than in COS group, and even higher than normal control group. In all, these findings suggest that DS147 may improve pregnancy in mice with COS-induced EID by promoting matrix degradation and angiogenesis, and improving the development of corpus luteum and endometrial decidualization around the implantation window.

Topical anti-inflammatory and analgesic activity of kirenol isolated from Siegesbeckia orientalis.

ETHNOPHARMACOLOGICAL RELEVANCE: Siegesbeckia orientalis has been traditionally used as a topical anti-inflammatory and analgesic agent. AIMS OF THE STUDY: Current study was designed to explore the topical anti-inflammatory and analgesic effects of a constituent isolated from Siegesbeckia orientalis (Compositae), in order to validate its folk use. MATERIALS AND METHODS: Kirenol was isolated from ethanolic extract of Siegesbeckia orientalis. Several topical formulations containing kirenol were investigated for anti-inflammatory and analgesic activities in rat. The effects were studied using carrageenan-induced rat acute inflammation model, complete Freund's adjuvant (CFA)-induced chronic inflammation and formalin test in rats. Piroxicam gel and methyl salicylate ointment were studied as positive control for anti-inflammatory and analgesic activity, respectively. RESULTS: The anti-inflammatory effect of kirenol 0.4-0.5% (w/w) was similar to the effect of piroxicam gel 4h after carrageenan injection. The analgesic activity of topical preparation with more than 0.4% (w/w) was observed in the late phase. These effects may be due, at least in part, to the pro-inflammatory cytokine production of IL-1ß and TNF-α. The administration of kirenol cream at the dose of 0.3, 0.4 and 0.5% (w/w) significantly inhibited the development of joint swelling induced by CFA, which was auxiliary supported by histopathological studies. CONCLUSION: Kirenol has demonstrated its significant potential to be further investigated for its discovery as a new lead compound for management of topical pain and inflammation, although further pharmacological research is necessary to fully understand its mechanism of action. It also supports the potential beneficial effect of topically administered Siegesbeckia orientalis in inflammatory diseases.

In vitro and in vivo evaluation of the wound healing properties of Siegesbeckia pubescens.

ETHNOPHARMACOLOGICAL RELEVANCE: Siegesbeckia pubescens (SP) has been traditionally used as a wound healing agent. AIM OF THE STUDY: Investigate in vitro and in vivo healing properties of SP extract. MATERIALS AND METHODS: The methanolic extract of SP was tested for the ability to stimulate the growth of mouse fibroblast NIH3T3 in vitro. The viability and proliferation of fibroblasts were evaluated at 72 h after cell seeding by MTT assay at 570 nm. To study wound healing properties in vivo, excision and incision wound models were used on rats and SP (3, 4, 5%, w/w) was topically administered. After treatment, wound contraction, epithelialization period and hexosamine content were evaluated in the excision wound model. In the incision wound model, wound sites were removed for histopathological analysis and skin-breaking strength determination. RESULTS: The methanol extract showed significant stimulation of the growth of mouse fibroblast NIH3T3 at 0.5-100 µg/mL. In excision wound, animals treated with 4, 5% (w/w) SP exhibited significant increases in the rate of wound contraction, period of epithelialization and content of hydroxyproline. In incision wound, the animals treated with both the 4 and 5% (w/w) SP extracts showed an increase in breaking strength when compared with the control, which was additionally supported by histopathological studies. CONCLUSION: The experimental data revealed that the methanolic extract of SP displayed remarkable wound healing activity, corroborating its traditional use.

Local immune regulatory effects of Bangdeyun on the endometrium of mice with embryo implantation dysfunction during the implantation time.

This study examined the effects of Bangdeyun on the expressions of nuclear factor-kappaB (NF-kappaB), interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) in the endometrium of mice with embryo implantation dysfunction (EID) during the implantation time (namely on pregnancy day 5, 6, 7 and 8) and explored the local immune regulatory effects of Bangdeyun. The gestational mice were randomly divided into normal group, model group and Bangdeyun-treated group. EID models of mice were established by using indomethacin. The endometrial expression of NF-kappaB was detected by immunohistochemistry and Western blotting. IFN-gamma and IL-10 were measured by enzyme-linked immunosorbent assay (ELISA). The results showed that in the normal group, NF-kappaB and IFN-gamma were weakly expressed and IL-10 was strongly expressed in the endometrium during the whole implantation period. In the model group, the expressions of NF-kappaB and IFN-gamma were increased on pregnancy day 5, 6 and 7, and IL-10 expression decreased during the whole implantation time when compared with those in the normal group (P<0.01 for all). In the Bangdeyun-treated group, little amount of NF-kappaB and IFN-gamma was expressed and IL-10 expression was strong, much the way they were expressed in the normal group (P>0.05). The expressions of NF-kappaB and IFN-gamma were much lower in the Bangdeyun-treated group than those in the model group on pregnancy day 5, 6 and 7 (P<0.01 for all), while the expression of IL-10 was much higher than in the model group during the whole implantation time (P<0.01). It was suggested Bangderun may favor a shift from Th1- to Th2-type immune response, therefore inhibiting the maternal immune rejection, inducing the immune tolerance and improving the fetal implantation.

Antiinflammatory and analgesic activity of topical administration of Siegesbeckia pubescens.

Several topical formulations containing methanolic extract of Siegesbeckia pubescens was investigated for antiinflammatory and analgesic activities in rat. The effects were studied using carrageenan-induced edema and formalin testing. Piroxicam gel and methyl salicylate ointment were studied as positive control for antiinflammatory and analgesic activity, respectively. The edema inhibition of the preparations containing extract at the doses of 1-5% w/w were significantly different from the control group. The antiinflammatory effect of Siegesbeckia pubescens 4-5% w/w was similar to the effect of piroxicam gel 3 h after carrageenan injection. The analgesic activity of topical preparation with more than 4% w/w was observed in the late phase. The topical analgesic activity of the extract was less than the analgesic activity of methyl salicylate ointment. The results of the present study further confirm the use of Siegesbeckia pubescens traditionally for the treatment of painful inflammatory conditions and can be useful for the treatment of local inflammation.

[Effect of Xingding Injection on expression of heat shock protein 72 in vascular endothelial cells].

OBJECTIVE: To study the effect of Xingding Injection on the expression of heat shock protein 72 (HSP 72) in vascular endothelial cells after ultraviolet radiation. METHODS: Porcine aortic endothelial cells were cultured for 72 hours in culture mediums with different concentrations (0.1, 0.5, 1.0, 5.0, 10 mg/ml) of Xingding Injection. Ultraviolet radiation was administered to the cultured cells for 30 minutes. Western-blot assay was used to measure the expression of HSP 72 in the vascular endothelial cells. RESULTS: There was no expression of HSP 72 in the cultured vascular endothelial cells without ultraviolet radiation, but there was some expression of HSP 72 after ultraviolet radiation. Xingding Injection of different concentrations could significantly improve the expression of HSP 72. The expression of HSP 72 in the vascular endothelial cells cultured in culture medium with 1.0 mg/ml Xingding Injection was the highest, and there was no more increase of expression when the concentration was higher, instead the expression decreased. CONCLUSION: Xingding Injection can protect the vascular endothelial cells from injury during stress. It may be one of its mechanisms in preventing and treating cardio-cerebrovascular disorders.

Measuring dynamics of caspase-3 activity in living cells using FRET technique during apoptosis induced by high fluence low-power laser irradiation.

BACKGROUND AND OBJECTIVES: Low-power laser irradiation (LPLI) has been used for therapies such as curing spinal cord injury, healing wound etc. Yet, the mechanism of LPLI remains unclear. In order to determine the effects of high fluence LPLI on cell growth and caspase-3 activity, we have measured the dynamics of caspase-3 activity during cell apoptosis induced by high fluence LPLI treatment. STUDY DESIGN/MATERIALS AND METHODS: He-Ne laser was used to irradiate human lung adenocarcinoma cells (ASTC-a-1). Cell Counting Kit-8 was used for cytotoxicity assay. A fluorescent microscope was used to perform fluorescence resonance energy transfer (FRET) imaging. A luminescence spectrometer was used to acquire the fluorescent emission spectrum. Statistical analysis was performed with Student's paired t-test. RESULTS: Cytotoxicity assay showed that when light irradiation fluence exceeded 60 J/cm2, LPLI treatment induced ASTC-a-1 cell apoptosis in a fluence-dependent manner. FRET imaging and spectrofluorometric analysis demonstrated that caspase-3 was activated during high fluence LPLI-induced cell apoptosis. CONCLUSIONS: Using FRET technique, we have reported that high fluence LPLI can induce human lung adenocarcinoma cells (ASTC-a-1) apoptosis. The activation of caspase-3 plays an important role in the apoptotic process.

[Effect of traditional Chinese medicine on expression of extracellular signal-regulating kinase-1 and mitogen activated protein kinase phosphatase-1 in brain and liver of fetal rats with fetal growth restriction].

OBJECTIVE: To investigate the effect of reinforcing kidney, replenishing Qi and blood activating prescription (RKRQBAP) on extracellular signal regulating kinase-1 (ERK-1) and mitogen activated protein kinase phosphatase-1 (MKP-1) of fetal rats with fetal growth restriction (FGR). METHODS: Passive smoking was used to establish animal model of FGR. All pregnant rats were divided into four groups: FGR group, L-arg group: rats with FGR treated with L-arginine, Chinese medicine group (CHM group): rats with FGR treated by traditional Chinese medicine and normal control group. The expression of ERK-1 and MKP-1 in brain and liver were measured by western-blot. RESULTS: (1) According to FGR group, L-arg group, CHM group and normal group, the expression of ERK-1 in brain was 7.63 +/- 0.22, 10.03 +/- 0.41, 11.03 +/- 0.30, 11.44 +/- 0.09 and MKP-1 was 7.41 +/- 0.38, 10.35 +/- 0.60, 10.60 +/- 0.14, 11.60 +/- 0.62. (2) The expression of ERK-1 in liver was 4.73 +/- 0.54, 5.83 +/- 0.17, 11.37 +/- 0.12, 12.34 +/- 0.14 accordingly and MKP-1 was 9.07 +/- 0.61, 10.66 +/- 0.08, 14.27 +/- 0.73, 14.92 +/- 0.17. (3) The expression of ERK-1 and MKP-1 in brain and liver was much lower in FGR group than in normal group (P < 0.01), and that of CHM group was much higher than in FGR group (P < 0.01) and closed to normal group (P > 0.05). The expression of ERK-1 in brain was much higher in CHM group than in L-arg group (P < 0.05), the expression of MKP-1 was almost the same (P > 0.05); Both of them in liver were higher in CHM group than in L-arg group (P < 0.01). CONCLUSION: The possible mechanism which the RKRQBAP cure FGR is to affect the expression of ERK-1 and MKP-1 and to promote growth- related genes expression and restrain apoptosis. In addition, RKRQBAP is superior to L-arginine in treatment of FGR.