Anti-influenza drug screening and inhibition of apigetrin on influenza A virus replication via TLR4 and autophagy pathways.

Activation of Toll-like receptor (TLR) 4 plays important roles in the influenzaA virus (IAV) infection. To explore TLR4 inhibitors, 161 traditional Chinese medicines (TCMs) were screened. Further, we screened out Ixeris sonchifolia Hance, and its active compound, Apigetrin (apigenin-7-O-glucoside). Antiviral activity of Apigetrin was determined by plaque assay. We also further investigated the influence of Apigetrin on immune signaling pathways including TLRs, MAPK, NF-κB and autophagy pathways. The in-vitro results showed that the extract and its several ingredients could significantly inhibit IAV replication. Apigetrin significantly improved IAV-induced oxidative stress, inhibited the IAV-induced cytokine storm by suppressing the excessive activation of TLR3/4/7, JNK/p38 MAPK and NF-κB. Apigetrin decreased autophagosome accumulation and promoted degradation of IAV protein. Interestingly, Apigetrin antiviral activity was reversed by using H2O2 and the agonists of TLR4, JNK/p38, NF-κB and autophagy. Most important, the in-vitro effective concentration is higher than the reported plasma concentration. The in-vivo test showed that Apigetrin significantly increased the average survival time, reduced the lung edema and IAV replication. In conclusion, we have found that Ixeris sonchifolia Hance and its several ingredients can inhibit IAV infection, and the mechanisms of action of Apigetrin against IAV is by regulating TLR4 and autophagy signaling pathways.

Anti-influenza A Virus Effects and Mechanisms of Emodin and Its Analogs via Regulating PPARα/γ-AMPK-SIRT1 Pathway and Fatty Acid Metabolism.

The peroxisome proliferator-activated receptor (PPAR) α/γ-adenosine 5'-monophosphate- (AMP-) activated protein kinase- (AMPK-) sirtuin-1 (SIRT1) pathway and fatty acid metabolism are reported to be involved in influenza A virus (IAV) replication and IAV-pneumonia. Through a cell-based peroxisome proliferator responsive element- (PPRE-) driven luciferase bioassay, we have investigated 145 examples of traditional Chinese medicines (TCMs). Several TCMs, such as Polygonum cuspidatum, Rheum officinale Baillon, and Aloe vera var. Chinensis (Haw.) Berg., were found to possess high activity. We have further detected the anti-IAV activities of emodin (EMO) and its analogs, a group of common important compounds of these TCMs. The results showed that emodin and its several analogs possess excellent anti-IAV activities. The pharmacological tests showed that emodin significantly activated PPARα/γ and AMPK, decreased fatty acid biosynthesis, and increased intracellular ATP levels. Pharmaceutical inhibitors, siRNAs for PPARα/γ and AMPKα1, and exogenous palmitate impaired the inhibition of emodin. The in vivo test also showed that emodin significantly protected mice from IAV infection and pneumonia. Pharmacological inhibitors for PPARα/γ and AMPK signal and exogenous palmitate could partially counteract the effects of emodin in vivo. In conclusion, emodin and its analogs are a group of promising anti-IAV drug precursors, and the pharmacological mechanism of emodin is linked to its ability to regulate the PPARα/γ-AMPK pathway and fatty acid metabolism.

Inhibition of curcumin on influenza A virus infection and influenzal pneumonia via oxidative stress, TLR2/4, p38/JNK MAPK and NF-κB pathways.

Oxidative stress, Nrf2-HO-1 and TLR-MAPK/NF-κB signaling pathways have been proved to be involved in influenza A virus (IAV) replication and influenzal pneumonia. In the previous studies, we have performed several high-throughput drug screenings based on the TLR pathways. In the present study, through plaque inhibition test, luciferase reporter assay, TCID50, qRT-PCR, western blotting, ELISA and siRNA assays, we investigated the effect and mechanism of action of curcumin against IAV infection in vitro and in vivo. The results showed that curcumin could directly inactivate IAV, blocked IAV adsorption and inhibited IAV proliferation. As for the underlying mechanisms, we found that curcumin could significantly inhibit IAV-induced oxidative stress, increased Nrf2, HO-1, NQO1, GSTA3 and IFN-ß production, and suppressed IAV-induced activation of TLR2/4/7, Akt, p38/JNK MAPK and NF-κB pathways. Suppression of Nrf2 via siRNA significantly abolished the stimulatory effect of curcumin on HO-1, NQO1, GSTA3 and IFN-ß production and meanwhile blocked the inhibitory effect of curcumin on IAV M2 production. Oxidant H2O2 and TLR2/4, p38/JNK and NF-κB agonists could significantly antagonize the anti-IAV activity of curcumin in vitro. Additionally, curcumin significantly increased the survival rate of mice, reduced lung index, inflammatory cytokines and lung IAV titer, and finally improved pulmonary histopathological changes after IAV infection. In conclusion, curcumin can directly inactivate IAV, inhibits IAV adsorption and replication; and its inhibition on IAV replication may be via activating Nrf2 signal and inhibiting IAV-induced activation of TLR2/4, p38/JNK MAPK and NF-κB pathways.

Panax notoginseng saponins inhibit areca nut extract-induced oral submucous fibrosis in vitro.

BACKGROUND: Oral submucous fibrosis (OSF) is a premalignant and fibrosing disease, which is closely associated with the habit of chewing areca nut. Panax notoginseng Buck F. H. Chen is an often used antifibrotic and antitumor agent. To treat areca nut-induced OSF, we have developed a chewable tablet, in which one of the major medicines is total Panax notoginseng saponins (PNS). In this study, we have investigated the antifibrotic effect and mechanism of PNS on areca nut-induced OSF in vitro. METHODS: Through human procollagen gene promoter luciferase reporter plasmid, hydroxyproline assay, gelatin zymography, qRT-PCR, ELISA, and Western blot, the influences of PNS on areca nut extract (ANE)-induced cell growth, collagen accumulation, procollagen gene transcription, MMP-2/-9 activity, MMP-1/-13 and TIMP-1/-2 expression, cytokine secretion, and the activation of PI3K/AKT, ERK/JNK/p38 MAPK, and TGFß/Smads pathways were detected. RESULTS: Panax notoginseng saponins could inhibit the ANE-induced abnormal growth and collagen accumulation of oral mucosal fibroblasts in a concentration-dependent manner. PNS (25 µg/ml) could significantly inhibit the ANE-induced expression of Col1A1 and Col3A1, augment the ANE-induced decrease of MMP-2/-9 activity, inhibit the ANE-induced increase of TIMP-1/-2 expression, and decrease the ANE-induced transcription and release of CTGF, TGFß1, IL-6, and TNFα. PNS (25 µg/ml) also significantly inhibited the ANE-induced activation of AKT and ERK/JNK/p38 MAPK pathways in oral mucosal fibroblasts and the ANE-induced activation of TGFß/smad pathway in HaCaT cells. CONCLUSION: Panax notoginseng saponins possess excellent anti-OSF activity, and its mechanism may be related to its ability to inhibit the ANE-induced activation of PI3K/AKT, ERK/JNK/p38 MAPK, and TGFß/smad pathways.

Identification of 23-(s)-2-amino-3-phenylpropanoyl-silybin as an antiviral agent for influenza A virus infection in vitro and in vivo.

It has been reported that autophagy is involved in the replication of many viruses. In this study, we screened 89 medicinal plants, using an assay based on the inhibition of the formation of the Atg12-Atg5/Atg16 heterotrimer, an important regulator of autophagy, and selected Silybum marianum L. for further study. An antiviral assay indicated that silybin (S0), the major active compound of S. marianum L., can inhibit influenza A virus (IAV) infection. We later synthesized 5 silybin derivatives (S1 through S5) and found that 23-(S)-2-amino-3-phenylpropanoyl-silybin (S3) had the best activity. When we compared the polarities of the substituent groups, we found that the hydrophobicity of the substituent groups was positively correlated with their activities. We further studied the mechanisms of action of these compounds and determined that S0 and S3 also inhibited both the formation of the Atg12-Atg5/Atg16 heterotrimer and the elevated autophagy induced by IAV infection. In addition, we found that S0 and S3 could inhibit several components induced by IAV infection, including oxidative stress, the activation of extracellular signal-regulated kinase (ERK)/p38 mitogen-activated protein kinase (MAPK) and IκB kinase (IKK) pathways, and the expression of autophagic genes, especially Atg7 and Atg3. All of these components have been reported to be related to the formation of the Atg12-Atg5/Atg16 heterotrimer, which might validate our screening strategy. Finally, we demonstrated that S3 can significantly reduce influenza virus replication and the associated mortality in infected mice. In conclusion, we identified 23-(S)-2-amino-3-phenylpropanoyl-silybin as a promising inhibitor of IAV infection.

Hypothalamus-related resting brain network underlying short-term acupuncture treatment in primary hypertension.

The present study attempted to explore modulated hypothalamus-seeded resting brain network underlying the cardiovascular system in primary hypertensive patients after short-term acupuncture treatment. Thirty right-handed patients (14 male) were divided randomly into acupuncture and control groups. The acupuncture group received a continuous five-day acupuncture treatment and undertook three resting-state fMRI scans and 24-hour ambulatory blood pressure monitoring (ABPM) as well as SF-36 questionnaires before, after, and one month after acupuncture treatment. The control group undertook fMRI scans and 24-hour ABPM. For verum acupuncture, average blood pressure (BP) and heart rate (HR) decreased after treatment but showed no statistical differences. There were no significant differences in BP and HR between the acupuncture and control groups. Notably, SF-36 indicated that bodily pain (P = 0.005) decreased and vitality (P = 0.036) increased after acupuncture compared to the baseline. The hypothalamus-related brain network showed increased functional connectivity with the medulla, brainstem, cerebellum, limbic system, thalamus, and frontal lobes. In conclusion, short-term acupuncture did not decrease BP significantly but appeared to improve body pain and vitality. Acupuncture may regulate the cardiovascular system through a complicated brain network from the cortical level, the hypothalamus, and the brainstem.

Drug screening for autophagy inhibitors based on the dissociation of Beclin1-Bcl2 complex using BiFC technique and mechanism of eugenol on anti-influenza A virus activity.

Autophagy is involved in many human diseases, such as cancer, cardiovascular disease and virus infection, including human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza A virus (IAV) and coxsackievirus B3/B4 (CVB3/B4), so a drug screening model targeting autophagy may be very useful for the therapy of these diseases. In our study, we established a drug screening model based on the inhibition of the dissociation of Beclin1-Bcl2 heterodimer, an important negative regulator of autophagy, using bimolecular fluorescence complementation (BiFC) technique for developing novel autophagy inhibitors and anti-IAV agents. From 86 examples of traditional Chinese medicines, we found Syzygium aromaticum L. had the best activity. We then determined the anti-autophagy and anti-IAV activity of eugenol, the major active compound of Syzygium aromaticum L., and explored its mechanism of action. Eugenol could inhibit autophagy and IAV replication, inhibited the activation of ERK, p38MAPK and IKK/NF-κB signal pathways and antagonized the effects of the activators of these pathways. Eugenol also ameliorated the oxidative stress and inhibited the expressions of autophagic genes. We speculated that the mechanism underlying might be that eugenol inhibited the oxidative stress and the activation of ERK1/2, p38MAPK and IKK/NF-κB pathways, subsequently inhibited the dissociation of Beclin1-Bcl2 heterodimer and autophagy, and finally impaired IAV replication. These results might conversely display the reasonableness of the design of our screening model. In conclusion, we have established a drug screening model for developing novel autophagy inhibitor, and find eugenol as a promising inhibitor for autophagy and IAV infection.

A drug screening method based on the autophagy pathway and studies of the mechanism of evodiamine against influenza A virus.

In this research, we have established a drug screening method based on the autophagy signal pathway using the bimolecular fluorescence complementation-fluorescence resonance energy transfer (BiFC-FRET) technique to develop novel anti-influenza A virus (IAV) drugs. We selected Evodia rutaecarpa Benth out of 83 examples of traditional Chinese medicine and explored the mechanisms of evodiamine, the major active component of Evodia rutaecarpa Benth, on anti-IAV activity. Our results showed that evodiamine could significantly inhibit IAV replication, as determined by a plaque inhibition assay, an IAV vRNA promoter luciferase reporter assay and the Sulforhodamine B method using cytopathic effect (CPE) reduction. Additionally, evodiamine could significantly inhibit the accumulation of LC3-II and p62, and the dot-like aggregation of EGFP-LC3. This compound also inhibited the formation of the Atg5-Atg12/Atg16 heterotrimer, the expressions of Atg5, Atg7 and Atg12, and the cytokine release of TNF-α, IL-1ß, IL-6 and IL-8 after IAV infection. Evodiamine inhibited IAV-induced autophagy was also dependent on its action on the AMPK/TSC2/mTOR signal pathway. In conclusion, we have established a new drug screening method, and selected evodiamine as a promising anti-IAV compound.

Regional homogeneity analysis on acupoint specificity with resting-state functional magnetic resonance imaging.

BACKGROUND: The mechanism of acupuncture analgesia in craniotomy has been widely studied. However, the theoretical basis for selection of acupoints has not been examined. In this study, we used the regional homogeneity method blood oxygen level-dependent (BOLD) signals to determine changes in brain activity in response to transcutaneous electrical stimulation on acupoints and non-acupoints in resting state functional magnetic resonance imaging (fMRI). METHODS: Twelve healthy volunteers were enrolled in this study. BOLD fMRI scanning of the brain was performed for 306 seconds before and 30 minutes after transcutaneous electrical stimulation on acupoints UB63 (Jinmen), LV3 (Tai chong), ST36 (Zusanli), and GB40 (Qiuxu). The procedure was repeated after one week with stimulation on non-acupoints (one was 9 above BL67, the second was 12 above BL67 (Kunlun), the third was 7 above KI3, and the fourth was 10 above KI3 (Taixi)). RESULTS: The regional homogeneity in the acupoint group was increased in the left thalamus, caudate, putamen, lentiform nucleus (BA19, 30, 39), postcentral gyrus, precentral gyrus (BA3, 4, 30, 32), calcarine fissure, middle temporal gyrus (BA30), right superior temporal gyrus, inferior temporal gyrus (BA38), cuneus, and precuneus (BA7, 19) when compared to the non-acupoint group. The regional homogeneity of the acupoint group was decreased in the left cerebellum posterior lobe, middle frontal gyrus (BA10), double-side precuneus (BA7), and the postcentral gyrus (BA40). CONCLUSIONS: The brain region activated following acupoint stimulation is the ipsilateral pain-related brain region, which may relate to the therapeutic effect of acupuncture on pain relief. Further acupoint stimulation causes different central nervous responses compared to non-acupoint stimulation.

High-throughput screening for anti-influenza A virus drugs and study of the mechanism of procyanidin on influenza A virus-induced autophagy.

In this research, we have established a high-throughput screening (HTS) platform based on the influenza A virus (IAV) vRNA promoter. Using this HTS platform, we selected 35 medicinal plants out of 83 examples of traditional Chinese medicine and found that 7 examples had not been reported. After examining many previous reports, we found that Vaccinium angustifolium Ait., Vitis vinifera L, and Cinnamomum cassia Presl had a common active compound, procyanidin, and then determined the anti-IAV effect of procyanidin and explored its mechanism of action. With a plaque inhibition assay and a time-of-addition experiment, we found that procyanidin could inhibit the IAV replication at several stages of the life cycle. In the Western blot and EGFP-LC3 localization assays, we found that procyanidin could inhibit the accumulation of LC3II and the dot-like aggregation of EGFP-LC3. In the RT-PCR and Western blot assays, we found procyanidin could inhibit the expression of Atg7, Atg5, and Atg12. Finally, by the bimolecular fluorescence complementation-fluorescence resonance energy transfer and co-immunoprecipitation assays, we found that procyanidin could inhibit the formation of the Atg5-Atg12/Atg16 heterotrimer and the dissociation of the beclin1/bcl2 heterodimer. In conclusion, we have established an HTS platform and identified procyanidin as a novel and promising anti-IAV agent.

Investigation of acupoint specificity by multivariate granger causality analysis from functional MRI data.

PURPOSE: To investigate the acupoint specificity by exploring the effective connectivity patterns of the poststimulus resting brain networks modulated by acupuncture at the PC6, with the same meridian acupoint PC7 and different meridian acupoint GB37. MATERIALS AND METHODS: The functional MRI (fMRI) study was performed in 36 healthy right-handed subjects receiving acupuncture at three acupoints, respectively. Due to the sustained effects of acupuncture, a novel experimental paradigm using the nonrepeated event-related (NRER) design was adopted. Psychophysical responses (deqi sensations) were also assessed. Finally, a newly multivariate Granger causality analysis (mGCA) was used to analyze effective connectivity patterns of the resting fMRI data taken following acupuncture at three acupoints. RESULTS: Following acupuncture at PC6, the red nucleus and substantia nigra emerged as central hubs, in comparison with the fusiform gyrus following acupuncture at GB37. Red nucleus was also a target following acupuncture at PC7, but with fewer inputs than those of PC6. In addition, the most important target following acupuncture at PC7 was located at the parahippocampus. CONCLUSION: Our findings demonstrated that acupuncture at different acupoints may exert heterogeneous modulatory effects on the causal interactions of brain areas during the poststimulus resting state. These preliminary findings provided a clue to elucidate the relatively function-oriented specificity of acupuncture effects.

The temporal-spatial encoding of acupuncture effects in the brain.

BACKGROUND: Functional acupoint specificity is crucial to the clinical efficacy of acupuncture treatment, such as pain relief. Whether acupuncture needling at a peripheral acupoint produces distinct patterns of brain responses remains controversial. RESULTS: This fMRI study employed the complex network analysis (CNA) to test the hypothesis that acupuncture stimulation at an acupoint correspondingly induced activity changes in one or more intrinsic or resting-state brain networks. Built upon the sustained effect of acupuncture and its time-varying characteristics, we constructed a dynamic encoding system with the hub anchored at the posterior cingulate cortex and precuneus (PCC/pC). We found that needling at two visual acupoints (GB37 and BL60) and a non-visual acupoint (KI8) induced a spatially converging brain response, which overlapped at the PCC/pC. We also found distinct neural modulations during and after acupoint stimulation. During this period, the PCC/pC interacted with a visual resting-state network in different patterns. Furthermore, there was a delayed functional correspondence between the intrinsic visual network and manipulation over the visual acupoints (i.e., GB37 or BL60), but not the non-visual acupoint (KI8) via the PCC/pC, implicating a specific temporal-spatial encoding/decoding mechanism underlying the post-effect of acupuncture. CONCLUSIONS: This study provided an integrated view exploring the functional specificity of acupuncture in which both the needling sensation and the following neural cascades may contribute to the overall effect of acupuncture through dynamic reconfiguration of complex neural networks.fMRI, acupoints, posterior cingulate cortex, precuneus, temporal-spatial encoding, resting-state networks.

Acupuncture modulates temporal neural responses in wide brain networks: evidence from fMRI study.

BACKGROUND: Accumulating neuroimaging studies in humans have shown that acupuncture can modulate a widely distributed brain network, large portions of which are overlapped with the pain-related areas. Recently, a striking feature of acupuncture-induced analgesia is found to be associated with its long-last effect, which has a delayed onset and gradually reaches a peak even after acupuncture needling being terminated. Identifying temporal neural responses in these areas that occur at particular time--both acute and sustained effects during acupuncture processes--may therefore shed lights on how such peripheral inputs are conducted and mediated through the CNS. In the present study, we adopted a non-repeated event-related (NRER) fMRI paradigm and control theory based approach namely change-point analysis in order to capture the detailed temporal profile of neural responses induced by acupuncture. RESULTS: Our findings demonstrated that neural activities at the different stages of acupuncture presented distinct temporal patterns, in which consistently positive neural responses were found during the period of acupuncture needling while much more complex and dynamic activities found during a post-acupuncture period. These brain responses had a significant time-dependent effect which showed different onset time and duration of neural activities. The amygdala and perigenual anterior cingulate cortex (pACC), exhibited increased activities during the needling phase while decreased gradually to reach a peak below the baseline. The periaqueductal gray (PAG) and hypothalamus presented saliently intermittent activations across the whole fMRI session. Apart from the time-dependent responses, relatively persistent activities were also identified in the anterior insula and prefrontal cortices. The overall findings indicate that acupuncture may engage differential temporal neural responses as a function of time in a wide range of brain networks. CONCLUSIONS: Our study has provided evidence supporting a view that acupuncture intervention involves complex modulations of temporal neural response, and its effect can gradually resolve as a function of time. The functional specificity of acupuncture at ST36 may involve multiple levels of differential activities of a wide range of brain networks, which are gradually enhanced even after acupuncture needle being terminated.

Regulatory action of charred Gossamer urocteae on the functions of mouse oral fibroblasts.

OBJECTIVE: To explore the influence of charred Gossamer urocteae (CGU) on the functions of primary cultured mouse oral fibroblasts and reveal its mechanism in wound healing. METHODS: CGU was extracted with different solvents and ethanol extract (EE), ethyl acetate fraction (EF), n-butanol fraction (BF) and aqueous fraction (AF) were obtained. The effects of different fractions on the proliferation, matrix metalloproteinase-2,9 (MMP-2,9) activities, synthesis of collagen and tissue inhibitor of metalloproteinase 1 (TIMP-1) in the mouse oral fibroblasts were determined by MTT, gelatin zymography, chloramine-T method, and enzyme-linked immunosorbent assay (ELISA) respectively. RESULTS: EE, EF and BF at high concentrations could significantly inhibit proliferation of fibroblasts (P<0.05 or P<0.01), and at low concentrations EF and BF could promote proliferation of fibroblasts, and BF and AF could significantly inhibit collagen synthesis (P<0.05 or P<0.01). EE, EF and AF at high concentrations could significantly increase the MMP-9 activity, and BF and AF could significantly inhibit synthesis of TIMP-1. CONCLUSION: CGU at high concentrations can inhibit the proliferations of fibroblasts and synthesis of collagen, and in healing of wound, CGU at high concentrations possibly has the functions of anti-fibrosis and anti-scar, and the mechanism to promote degradation of collagen is possibly related to the increase in MMP-9 activity and the inhibition of TIMP-1 synthesis.

Preoperative 3T high field blood oxygen level dependent functional magnetic resonance imaging for glioma involving sensory cortical areas.

BACKGROUND: Localization of sensory cortical areas during the operation is essential to preserve the sensory function. Intraoperative direct electrostimulation under awake anesthesia is the golden standard but time-consuming. We applied 3T high field blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI) to identify the relationship between glioma and cortical sensory areas preoperatively and to guide intraoperative direct electrostimulation for quick and precise localization. METHODS: Five glioma patients with sensory cortex involvement by or next to the lesion had preoperative BOLD fMRI to determine the spatial relationship of cortical sensory areas to the tumours. Bilateral hand opposite movement was performed by these patients for fMRI. Precentral and postcentral gyri were identified by electrical stimulation during the operation. Karnofsky Performance Status scores of the patients' pre- and postoperative and the role of BOLD fMRI were evaluated. RESULTS: The cortical sensory areas were all activated in five glioma patients involving postcentral gyrus areas by BOLD fMRI with bilateral hand opposite movement. The detected activation areas corresponded with the results from cortical electrical stimulation. CONCLUSIONS: The relationship between cortical sensory areas and tumour can be accurately shown by BOLD fMRI before operation. And the information used to make the tumour resection could obtain good clinical results.

Partial correlation investigation on the default mode network involved in acupuncture: an fMRI study.

Certain clinical reports and researches have shown that acupuncture effects can sustain a period during the post-stimulation state, and modulate the default mode network (DMN). In this study, partial correlation approach was utilized to investigate whether or not electro-acupuncture stimulation (EAS) at the three acupoints (GB37 (Guangming), BL60 (Kunlun) and KI8 (Jiaoxin)) and one sham point on the left leg modulated the DMN and how to change the intrinsic connectivity of the DMN. The results indicated that DMN could be modulated after EAS, and there existed different modulation patterns of the four points. Meanwhile, we found that the posterior cingulate cortex and precuneus (PCC/pC) strongly interacted with other nodes during the pre- and post-stimulation states. The correlation was interrupted between the PCC/pC and anterior cingulate cortex (ACC). The orbital prefrontal cortex (OFC) negatively interacted with the left medial temporal cortex (lMTC) at the acupoints. We suggested that the distinct modulation patterns to the DMN attributed to the different effects evoked by the three acupoints and one sham point.

[Regulation of calculus bovis on the function of mice oral fibroblasts].

To explore the influence of calculus bovis on the function of primary cultured mice oral fibroblasts, we determined the effects of calculus bovis on the fibroblast proliferation, collagen production, matrix metalloproteinases-2, -9 activities and tissue inhibitor of metalloproteinase-1 production by MTT assay, chloramine T method, gelatin zymography and enzyme-linked immunosorbent assays respectively. The results showed that calculus bovis could significantly inhibit the proliferation of fibroblasts and collagen synthesis in a concentration dependent manner, could significantly (P<0.05) suppress matrix metalloproteinases-2 activity and very significantly (P<0.01) inhibit the production of tissue inhibitor of metalloproteinase-1. In conclusion, the major function of calculus bovis in the process of ulcer healing is not to promote tissue regeneration, the mechanism that calculus bovis inhibits collagen synthesis may be partly due to its ability to very significantly (P<0.01) suppress the production of tissue inhibitor of metalloproteinase-1.

Combining spatial and temporal information to explore function-guide action of acupuncture using fMRI.

PURPOSE: To investigate the brain response patterns of modulation of GB37 (Guangming) and KI8 (Jiaoxin). MATERIALS AND METHODS: An experiment using nonrepeated event-related fMRI design was carried out on 28 subjects with electroacupuncture stimulation (EAS) at GB37 or KI8 on the left leg. The discrete cosine transform and functional connectivity methods were adopted to detect the differences related with these two acupoints before and after the EAS. RESULTS: Spatial patterns were distinct for EAS at the two acupoints, and the overlapping brain regions were mainly located in the posterior cingulate cortex (PCC) and precuneus (pC). Two opposite patterns of modulation in the default mode network were detected from the temporal patterns with the overlapping PCC/pC as the region of interest. Furthermore, the specific responses of sustained effects at these acupoints were also identified. CONCLUSION: Spatial and temporal patterns of the sustained effect modulation of GB37 and KI8 were distinct. We suggest these findings may attribute to the functional specificity of a certain acupoint. Moreover, our current results reflect a significant methodological contribution to future acupuncture studies.

Acupuncture modulates spontaneous activities in the anticorrelated resting brain networks.

Neuroimaging studies of acupuncture have demonstrated extensive signal attenuations in the core regions of a "default mode" network as well as signal potentiations in the regions of a "central-executive" network. We proposed that this acupuncture-related dichotomy may represent the anticorrelation in these intrinsic brain networks showing spontaneous fluctuations during rest. According to a plentiful clinical report, acupuncture can provide pain relief beyond the time it is being administrated; therefore, imaging its sustained effect (rather than acute effect) on the brain networks may further help elucidate the mechanisms by which acupuncture achieves its therapeutic effects. As an interface, the anterior insula (AI) has recently been shown to be a network hub, which initiates dynamic switching between these intrinsic networks. Here, we attempt to explore how acupuncture can modulate spontaneous coherences of these resting networks anchored by the AI. Using a spontaneous activity detection approach, we identified an AI-related dichotomy showing spontaneous activations in the CEN along with wide spontaneous deactivations located exclusively in the DMN. Following verum acupuncture, but not sham control, there was a prominently enhanced dichotomy in the CEN and DMN networks. More importantly, a long-lasting effect of acupuncture could further modulate intrinsic coherences of the wide interoceptive-autonomic areas, including the paralimbic regions and brainstem nuclei. These findings suggested that acupuncture may not only enhance the dichotomy of the anticorrelated resting networks, but also modulate a larger spatio-temporal extent of spontaneous activities in the salient interoceptive-autonomic network, contributing to potential actions in the endogenous pain-modulation circuits and homeostatic control mechanisms.

Time-varied characteristics of acupuncture effects in fMRI studies.

When studying the neural responses to acupuncture with a block-designed paradigm, its temporal dynamics predicted by the general linear model (GLM) conforms to typical "on-off" variations during a limited period of the experiment manipulation. Despite a lack of direct evidence associating its psychophysiological response, numerous clinical reports suggest that acupuncture can provide pain relief beyond a needling session. Therefore, a typical GLM analysis may be insensitive or inappropriate for identifying altered neural responses resulting from acupuncture. We developed a new approach to investigate the dynamics underlying sustained effects of acupuncture. Specifically, we designed two separate models to evaluate the baseline activities (prior to stimulation) and neural activities in sequential epochs, using three block-designed functional runs: acupuncture at acupoint ST36, nonmeridian point (NMP) stimulation, and a visual task. We found that the activity patterns during rest were associated with the stimulus types and that the resting activities might be even higher than that of stimulation phases. Such effects of the elevated activity during rest may reduce or eliminate the activity during stimulus conditions or even reverse the sign of brain activation using conventional GLM analysis. Moreover, such sustained responses, followed by acupuncture at ST36 and NMP, exhibited distinct patterns in wide brain structures, particularly in the limbic system and brainstem. These findings may pose great implications for the design and interpretation of a range of acupuncture neuroimaging studies.