The mechanism of the anti-inflammatory effect of Oldenlandia diffusa on arthritis model rats: a quantitative proteomic and network pharmacologic study.

Background: In China, Oldenlandia diffusa (OD) has been prescribed as a therapeutic herb for rheumatoid arthritis (RA). We previously conducted a preliminary study of the anti-inflammatory effect of OD, and the purpose of this study is to further investigate its mechanism. Methods: We performed a quantitative proteomic analysis of synovium, identified the differentially expressed proteins, and performed bioinformatics analyses. With the help of network pharmacology, we aimed to find the key synovial proteins which OD or its key compound might influence. To verify the result, liquid chromatography-mass spectrometry (LC-MS) was applied to quantify and qualify the absorbable potential compounds of OD. The anti-inflammatory effect was evaluated by morphological, histopathological, and cytokine analyses. Target proteins were observed by immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA). Results: MMP3 and CAV1 were identified as 2 of the differentially expressed proteins in RA synovium, and might be influenced by quercetin, the active compound of OD. MMP3 might be altered through atherosclerosis signaling, while CAV1 might be altered through caveolar-mediated endocytosis signaling. According to our verification, quercetin was identified as the absorbed and effective compound of OD, and it could exert an anti-inflammatory effect on the collagen-induced arthritis (CIA) model, including serum cytokine expression, synovial hyperplasia and lymphocyte infiltration, articular cartilage lesion. Quercetin could also down-regulate the synovial expression of MMP3 and CAV1, and could exert better effects at a high dose. Conclusions: Quercetin was the main active compound of OD in the treatment of RA. OD might alleviate inflammatory responses in CIA rats by suppressing the expression of MMP3 and CAV1 through quercetin, and at a high dose, quercetin could exert a better anti-inflammatory effect.

Xuefu Zhuyu decoction improves neurological dysfunction by increasing synapsin expression after traumatic brain injury.

Xuefu Zhuyu decoction has been used for treating traumatic brain injury and improving post-traumatic dysfunction, but its mechanism of action needs further investigation. This study established rat models of traumatic brain injury by controlled cortical impact. Rat models were intragastrically administered 9 and 18 g/kg Xuefu Zhuyu decoction once a day for 14 or 21 days. Changes in neurological function were assessed by modified neurological severity scores and the Morris water maze. Immunohistochemistry, western blot assay, and reverse-transcription polymerase chain reaction were used to analyze synapsin protein and mRNA expression at the injury site of rats. Our results showed that Xuefu Zhuyu decoction visibly improved neurological function of rats with traumatic brain injury. These changes were accompanied by increased expression of synaptophysin, synapsin I, and postsynaptic density protein-95 protein and mRNA in a dose-dependent manner. These findings indicate that Xuefu Zhuyu decoction increases synapsin expression and improves neurological deficits after traumatic brain injury.

Anti-Inflammatory Effects of p-Coumaric Acid, a Natural Compound of Oldenlandia diffusa, on Arthritis Model Rats.

OBJECTIVES: In China, Oldenlandia diffusa (OD) is a natural herb that is widely used and has been proven to be effective in the treatment of rheumatoid arthritis (RA). This study aimed to preliminarily reveal the mechanism by which OD exerts its beneficial effect. METHODS: Ultra-performance liquid chromatography photodiode array was applied to identify the absorbable compounds in the plasma of collagen-induced arthritis (CIA) model rats. After 2 weeks, an OD decoction or the identified absorbable compound was administered to CIA rats. Morphology, X-ray images of the joints, pathological images, arthritis index, and cytokine (TNF-α and IL-6) levels were evaluated. RESULTS: p-Coumaric acid (p-CA) was identified as the absorbed compound in plasma. After administration of p-CA solution or the OD decoction, symptoms in the treated rats were alleviated as compared to the untreated model rats, and inflammatory cell infiltration was suppressed. The arthritis index and serum levels of TNF-α and IL-6 were decreased as compared to the control group. CONCLUSIONS: OD may exert its anti-inflammatory effect on RA via its active ingredient, p-CA. This information sheds light on the mechanism by which OD exerts its anti-inflammatory effort in RA and forms the basis for further development of therapeutic agents for RA.

Serum Proteome Alterations in Patients with Cognitive Impairment after Traumatic Brain Injury Revealed by iTRAQ-Based Quantitative Proteomics.

Background. Cognitive impairment is the leading cause of traumatic brain injury- (TBI-) related disability; however, the underlying pathogenesis of this dysfunction is not completely understood. Methods. Using an isobaric tagging for relative and absolute quantitation- (iTRAQ-) based quantitative proteomic approach, serum samples from healthy control subjects, TBI patients with cognitive impairment, and TBI patients without cognitive impairment were analysed to identify differentially expressed proteins (DEPs) related to post-TBI cognitive impairment. In addition, DEPs were further analysed using bioinformatic platforms and validated using enzyme-linked immunosorbent assays (ELISA). Results. A total of 56 DEPs were identified that were specifically related to TBI-induced cognitive impairment. Bioinformatic analysis revealed that a wide variety of cellular and metabolic processes and some signaling pathways were involved in the pathophysiology of cognitive deficits following TBI. Five randomly selected DEPs were validated using ELISA in an additional 105 cases, and the results also supported the experimental findings. Conclusions. Despite limitations, our findings will facilitate further studies of the pathological mechanisms underlying TBI-induced cognitive impairment and provide new methods for the research and development of neuroprotective agents. However, further investigation on a large cohort is warranted.

Evidence-based review of oral traditional Chinese medicine compound recipe administration for treating weight drop-induced experimental traumatic brain injury.

BACKGROUND: Recently, a number of studies conducted and published in China have suggested that traditional Chinese medicine compound recipe (TCMCR) may be beneficial in the treatment of experimental traumatic brain injury (TBI). In this study, we conducted a systematic review and meta-analysis of the efficacy of TCMCR in TBI model with weight drop method to provide robust evidence on the effects of TCMCR and to determine whether TCMCR can be recommended for routine treatment or considered as a standard treatment for TBI. METHODS: We identified eligible studies by searching five electronic databases on April 1, 2014, and pooled the data using the random-effects model. Results were reported in terms of standardized mean difference (SMD). We also calculated statistical heterogeneity, evaluated the studies' methodological quality and investigated the presence of publication bias. RESULTS: Totally, 187 relevant publications were searched from databases, 25 of which met our inclusion criteria. The overall methodological quality of the most studies was poor, and there was evidence of statistical heterogeneity among studies along with small-study effects. Meta-analysis showed statistically significant effects indicating that TCMCR has a beneficial effect on TBI. CONCLUSIONS: Despite the limitations, we concluded that TCMCR may reduce brain water content, improve BBB permeability, and decrease TNF-α/NO expression after experimental TBI in terms of overall efficacy. However, our review also indicates that more well-designed and well-reported animal studies are needed.

Impact of statins on cognitive deficits in adult male rodents after traumatic brain injury: a systematic review.

The efficacy of statin treatment on cognitive decline is controversial, and the effect of statins on cognitive deficits in individuals with traumatic brain injury (TBI) has yet to be investigated. Therefore, we systematically reviewed the effect of statins on cognitive deficits in adult male rodents after TBI. After identifying eligible studies by searching four electronic databases on February 28, 2014, we assessed study quality, evaluated the efficacy of statin treatment, and performed stratified metaregression and metaregression to assess the influence of study design on statin efficacy. Eleven studies fulfilled our inclusion criteria from a total of 183 publications. The overall methodological quality of these studies was poor. Meta-analysis showed that statins exert statistically significant positive effects on cognitive performance after TBI. Stratified analysis showed that atorvastatin has the greatest effect on acquisition memory, simvastatin has the greatest effect on retention memory, and statin effects on acquisition memory are higher in closed head injury models. Metaregression analysis further showed that that animal species, study quality, and anesthetic agent impact statin effects on retention memory. We conclude that statins might reduce cognitive deficits after TBI. However, additional well-designed and well-reported animal studies are needed to inform further clinical study.

Anti-Inflammatory Effects of the Bioactive Compound Ferulic Acid Contained in Oldenlandia diffusa on Collagen-Induced Arthritis in Rats.

Objectives. This study aimed to identify the active compounds in Oldenlandia diffusa (OD) decoction and the compounds absorbed into plasma, and to determine whether the absorbed compounds derived from OD exerted any anti-inflammatory effects in rats with collagen induced arthritis (CIA). Methods. The UPLC-PDA (Ultra Performance Liquid Chromatography Photo-Diode Array) method was applied to identify the active compounds both in the decoction and rat plasma. The absorbable compound was administered to the CIA rats, and the effects were dynamically observed. X-ray films of the joints and HE stain of synovial tissues were analyzed. The levels of IL-1 ß and TNF- α in the rats from each group were measured by means of ELISA. The absorbed compound in the plasma of CIA rats was identified as ferulic acid (FA), following OD decoction administration. Two weeks after the administration of FA solution or OD decoction, the general conditions improved compared to the model group. The anti-inflammatory effect of FA was inferior to that of the OD decoction (P < 0.05), based on a comparison of IL-1 ß TNF- α levels. FA from the OD decoction was absorbed into the body of CIA rats, where it elicited anti-inflammatory responses in rats with CIA. Conclusions. These results suggest that FA is the bioactive compound in OD decoction, and FA exerts its effects through anti-inflammatory pathways.

UPLC-PDA determination of paeoniflorin in rat plasma following the oral administration of Radix Paeoniae Alba and its effects on rats with collagen-induced arthritis.

Rheumatoid arthritis (RA) is a chronic disabling autoimmune disease with characteristics of chronic, progressive inflammatory joint synovial damage, which mainly encroaches upon the synovium of the joint. The use of traditional medicine to treat RA slows the development of RA to a certain extent; however, it often has numerous side-effects. Therefore, the focus of RA research is the identification of a new, safe and effective medicine. The aim of the present study was to use an ultra performance liquid chromatography and photo diode array (UPLC-PDA) method to detect the paeoniflorin component in a Radix Paeoniae Alba decoction and in rat plasma following the oral administration of Radix Paeoniae Alba decoction. In addition, the effects of paeoniflorin on collagen-induced arthritis (CIA) in rats were investigated. The results indicate that a UPLC-PDA method for determining the presence of paeoniflorin in the Radix Paeoniae Alba decoction was successfully established. The method was fast, simple, sensitive, precise and valid. Paeoniflorin was shown to be a bioactive component of the Radix Paeoniae Alba decoction that was absorbed into rat plasma. Paeoniflorin significantly improved the disease resistant ability of RA rats and reduced the levels of the inflammatory cytokines, IL-1ß and TNF-α, thereby inhibiting inflammation and bone erosion in the rats with CIA. The observations are likely to lay the foundation for further study of the mechanism of paeoniflorin in the treatment of RA.

Effect of Bizhongxiao decoction and its dismantled formulae on IL-1 and TNF levels in collagen-induced arthritis in rat synovial joints.

BACKGROUND: Rheumatoid arthritis (RA), a chronic autoimmune disease, affects sufferers in many different ways. Treatment of this chronic condition is particularly challenging. Traditional Chinese Medicine (TCM) provides alternatives. Bizhongxiao decoction (BZX) is a TCM complex, which has been used clinically for many years to treat RA. The purpose of this study is to compare the effects of BZX decoction and its dismantled formulae on IL-1 and TNF-1 levels in rats with RA, and to elucidate its mechanism of action. METHODS: Ninety healthy normal female SD rats were randomly divided into six groups: normal (control), model, BZX decoction, and the three dismantled formulae (I: heat-clearing and detoxication, II: dissipating dampness, and III: blood circulation promotion). Apart from the normal (control) group, the rats in each group were injected subcutaneously with bovine type II collagen and complete Freund adjuvant to establish a collagen-induced arthritis model, so that inhibition of foot swelling in the rats by BZX decoction and its dismantled formulae could be observed. Immunohistochemistry was used to assess the levels of the inflammatory cytokines IL-1 and TNF in synovial joints at various time points. RESULTS: Twenty-one days after the model was established, the levels of TNF and IL-1 were significantly higher in the model group, BZX decoction group and dismantled formula groups I, II and III than in the normal controls (P < 0.05). The levels of these cytokines were significantly higher in the model group than the BZX decoction or the three dismantled formula groups (P <0.01). At longer times, the TNF and IL-1 levels in model group rose gradually; those in the BZX decoction and dismantled formula groups were gradually reduced. The cytokine levels in the BZX decoction group were lower than in the three dismantled formula groups and continued to decline. CONCLUSIONS: BZX decoction and the three dismantled formulae examined down-regulated the inflammatory factors IL-1 and TNF in collagen-induced arthritis rat models, but BZX exerted the strongest effect.

Ursolic acid inhibits T-cell activation through modulating nuclear factor-κ B signaling.

OBJECTIVE: To investigate the effects of ursolic acid (UA) on T-cell proliferation and activation, as well as to examine its effect on nuclear factor-κB (NF-κB) signaling pathway in T cells. METHODS: T-cells isolated from BALB/c mice were incubated with UA at concentrations ranging from 5-30 µmol/L in the presence of phorbol 12-myristate 13-acetate (PMA) or PMA plus ionomycin. The proliferation of T cells was measured by the MTT assay. The expressions of CD69, CD25, and CD71 on T-cell surface were analyzed using flow cytometry. The level of interleukin-2 (IL-2) in the culture supernatant of activated T cells was quantified by enzyme-linked immunosorbent assay (ELISA). The level of phosphorylated IκB-α (p-IκB-α) in total protein and p65, a subunit of NF-κB, nuclear translocation were measured by Western blot analysis. RESULTS: UA in a dose-dependent manner significantly decreased the proliferation and inhibited the surface expressions of CD69, CD25, and CD71 in murine T lymphocytes upon in vitro activation (P<0.01). Significant reduction of IL-2 production was found in activated T cells treated with UA (P<0.01). The PMA-induced increase in p-IκB-α protein was inhibited, and nuclear translocation of p65 from the cytoplasm was blocked by UA. CONCLUSION: UA is a potent inhibitor for T cell activation and proliferation; these effects are associated with the inhibition of NF-κB signaling pathway.

[Proteomics study on the essence of wind syndrome caused by gan-yang hyperactivity in Chinese medicine].

OBJECTIVE: To preliminarily study the essence of wind syndrome caused by Gan-yang hyperactivity (WSGH) in Chinese medicine at the protein expression level. METHODS: WSGH was strictly differentiated from wind stirring due to yin deficiency syndrome in patients with intracerebral hemorrhage (ICH) and those with cerebral infarction (CI); from Gan-yang hyperactivity syndrome in patients with cervical spondylosis (CS); from wind syndrome induced by blood deficiency in patients with Parkinson's disease (PD) according to Chinese medicine syndrome typing standard. Control studies were performed. Peripheral blood mononuclear cells (PBMCs) were isolated from all patients of the aforesaid syndromes and healthy subjects. The total proteins were extracted, two-dimensional gel electrophoresis (2-DE) conducted and analyzed by PDQuest software. The peptide mass fingerprint (PMF) was determined using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). The SwissProt database was inquired using Mascot reference system. Proteins of different and same expressions in PBMCs of patients suffering from the same disease of different syndromes, different diseases of the same syndrome, and syndromes of the same kind were compared. RESULTS: The 2-DE map of PBMCs' total proteins in the aforesaid syndrome groups and healthy subjects was established. Through comparison, analysis, and appraisement, there was 1 protein dot of the same expression and 22 protein dots of different expressions between ICH patients of WSGH and ICH patients of wind stirring due to yin deficiency syndrome. There were 6 protein dots of the same expression and 21 protein dots of different expressions between CI patients of WSGH and CI patients of wind stirring due to yin deficiency syndrome. There were 3 protein dots of the same expression and 12 protein dots of different expressions between CS patients of WSGH and CS patients of Gan-yang hyperactivity syndrome. There was no protein dot of the same expression and 12 protein dots of different expressions between PD patients of WSGH and PD patients of wind syndrome induced by blood deficiency. There were 13 protein dots of the same expression in different diseases of the same syndrome. There was 1 protein dot (Thioredoxin-dependent peroxide reductase, TPx) of the same expression in the four diseases of the same kind syndrome. CONCLUSIONS: Different connotations of the essence existed (having multiple different protein expressions) in patients with the same disease of different syndromes. Syndromes of the same kind share the same material bases (having the same protein expression). These suggested that Chinese medicine syndrome has its own material bases and essence findable.

[Effect of Bizhongxiao decoction on proteomics of peripheral blood mononuclear cells in patients with rheumatoid arthritis].

OBJECTIVE: To probe in the possible acting mechanism of Bizhongxiao Decoction (BZXD) for treatment of early active rheumatoid arthritis (RA) by way of observing the two-dimensional gel electrophoresis map of proteins in peripheral blood mononuclear cells (PBMCs) of healthy persons and RA patients (intervened or un-intervened with BZXD), analyzing the differential proteins and seeking out the RA associated proteins. METHODS: Eighteen patients with early active RA were randomized into the BZXD group and the methotrexate (MTX) group, nine in each group, they were treated with BZXD (contained 15 Chinese herbs, as Herba Hedyotis diffusae, Herba Sarcandrae glabrae, Radix Salviae miltiorrhizae, Caulis Trachelosperi, Rhizoma Drynariae, Semen Coicis, etc.) and MTX combined with nimesulide Tablets respectively, three months as a treatment course, and their blood samples were collected for observation. Besides, blood samples from 9 healthy persons were taken as normal controls. PBMCs were isolated from blood using lymphozytes separation medium, and total protein in the cells was extracted through immobilized pH gradient two-dimensional gel electrophoresis. After Coomassie brilliant blue G250 staining, gel-image analysis was performed using PDQuest software. The differentially expressed proteins were identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). Then partial proteins were validated by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The 2-DE protein profile of PBMCs from healthy persons and RA patients before and 3 months after treatment were obtained, and 23 differential protein spots were found, 14 from 18 differential protein spots were successfully identified, of which 8 proteins were up-regulated and 6 proteins were down-regulated in RA patients as compared with control. After 3-month treatment, 5 differentially expressed proteins showed more obvious in the BZXD group than in the MTX group. RT-PCR verified that the expression of ApoA-I in all the three groups was consistent with the outcomes of 2-DE. CONCLUSIONS: Some differentially expressed proteins exist in the PBMCs of RA patients, which may play a potential role in the pathogenesis of RA; BZXD may treat RA by way of regulating the expression of some differential proteins in patients.

[Effect of bizhongxiao decoction on the expression of 2-dimensional polyacrylamide gel electrophoresis protein in the synovial tissue with collagen-induced arthritis in rats].

OBJECTIVE: To explore the effect of bizhongxiao decoction (BZXD) on the protein maps of BZXD-treated synovitis of collagen-induced arthritis (CIA) in rats in 2-dimensional gel electrophoresis (2-DE), and to provide new clues for illuminating the active mechanism of BZXD in treating the rheumatoid arthritis (RA). METHODS: Seventy SD rats were randomly divided into nor- mal group, model group and BZXD group. The experimental arthritis rat model was established by subcutaneouly injecting Type II collagen and complete Freunds adjuvant. The total proteins of synovial tissue of rat joints in the normal group, model group and BZXD group were seperated by 2-DE respectively. The gels of the 3 groups were stained by Coomassie brilliant blue. Electron pictures were obtained by scanning the gels, and then the differential proteins among the normal group, model group and BZXD group were examined by comparing the spots density volume in the gels. The electrophoregrams of the gels were analyzed in Pdquest software. RESULTS: The incidence of arthritis in the rats was approximately 88%. The 2-DE maps of rat synovial tissue in the normal group, model group and BZXD group were well duplicated. The average protein spots in the normal group, model group and BZXD group were 947 +/- 39, 994 +/- 41, and 1031 +/- 52, and the match rates were 92%, 91%, and 94.2% respectively. The average deviations of spot position were (0.896 +/- 0.217) mm in isoelectric focusing (IEF) and (1.102 +/- 0.104) mm in sodiumdo-decylsufate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Three hundred twenty-eight differential proteins were observed between the model group and BZXD group, of which 174 were up-regulated, 147 were down-regulated in the BZXD group, and 7 proteins were expressed only in the model group. One hundred ninty-three differential proteins were displayed between the model group and the normal group, of which 123 proteins were up-regulated and 70 were down-regulated in the model group. CONCLUSION: 2-DE protein expression profiles of synovial tissue in CIA rats are established, and many differential proteins are discovered. Further analysis on the differential proteins may serve as a new method to study the moleculer mechanism of BZXD in treating the rheumatoid arthritis.