Hyperhubeins A-I, Bioactive Sesquiterpenes with Diverse Skeletons from Hypericum hubeiense.

Nine new sesquiterpenes, hyperhubeins A-I (1-9), and 14 known analogues (10-23) were isolated from the aerial portions of Hypericum hubeiense. Their structures and absolute configurations were determined unambiguously via spectroscopic analysis, single-crystal X-ray diffraction, and electronic circular dichroism calculations. Compounds 1-3 possess an unprecedented sesquiterpene carbon skeleton. Further, a plausible biosynthetic pathway from farnesyl diphosphate (FPP) is proposed. The isolated phytochemicals were evaluated for neuroprotective and anti-neuroinflammatory properties in vitro. Compounds 1, 2, 5-8, 14, and 21 displayed notable neuroprotective activity against hydrogen peroxide (H2O2)-induced lesions in PC-12 cells at 10 µM. Additionally, compounds 1, 2, 12, and 13 exhibited inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) production in BV-2 microglial cells, with their IC50 values ranging from 4.92 to 6.81 µM. Possible interactions between these bioactive compounds and inducible nitric oxide synthase (iNOS) were predicted via molecular docking. Moreover, Western blotting indicated that compound 12 exerted anti-neuroinflammatory activity by suppressing LPS-stimulated expression of toll-like receptor-4 (TLR-4) and inhibiting consequent activation of nuclear factor-kappa-B (NF-κB) signaling.

Network Pharmacology-Based Prediction of Bioactive Compounds and Potential Targets of Wenjing Decoction for Treatment of Endometriosis.

Endometriosis is a chronic estrogen-dependent inflammatory disorder that negatively affects the quality of life in women. The Wenjing decoction (WJD) is a traditional Chinese medicine that has been shown to have a therapeutic effect on endometriosis. Our study systematically explored the mechanism of WJD against endometriosis using a network pharmacology approach. Potentially bioactive compounds of WJD and their possible targets were retrieved from the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform. The protein-protein interaction network and herbs-compounds-genes multinetwork were constructed using Cytoscape for visualization. Subsequently, the signaling pathways of common targets were retrieved from the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, and molecular docking was performed using PyRx software. In total, 48 common targets were screened, such as IL6 and ESR1, which were related to inflammation and the endocrine system. The top five bioactive compounds were quercetin, kaempferol, wogonin, beta-sitosterol, and stigmasterol. KEGG enrichment analysis revealed 65 pathways containing inflammatory- and endocrine-related signaling pathways, such as the "TNF signaling pathway" and the "estrogen signaling pathway." Taken together, the results of our network pharmacology analysis predicted that certain active ingredients of WJD might treat endometriosis by regulating inflammation and/or endocrine, which provided references for further understanding and exploration of WJD on endometriosis.

[Modified Dihuang Decoction improves ovarian reserve in mice by regulating Bcl-2-related mitochondrial apoptosis pathway].

The present study investigated the effect of Modified Dihuang Decoction in improving ovarian reserve in mice through the Bcl-2-related mitochondrial apoptosis pathway. Forty-eight adult female BALB/c mice were randomly divided into the following six groups with eight mice in each group: a blank group, a model group, a femoston group(three cycles of treatment with 0.13 mg·kg~(-1) estradiol tablets for 2 days and 1.43 mg·kg~(-1) estradiol and dydrogesterone tablets for 3 days), and high(64.74 g·kg~(-1))-, medium(43.16 g·kg~(-1))-, and low-dose(21.58 g·kg~(-1)) Modified Dihuang Decoction groups. Mice in other groups except the blank group received a single intraperitoneal injection of 12 mg·kg~(-1) cyclophosphamide and 1.2 mg·kg~(-1) busulfan to induce a model of diminished ovarian reserve(DOR), while those in the blank group received an equal volume of normal saline. Mice were treated with corresponding drugs for 15 d from the 36 th day, once per day, and the mice in the blank group and the model group were treated with an equal volume of normal saline. The general condition and oestrous cycle were observed. The serum hormone levels were detected with the enzyme-linked immunosorbent assay(ELISA). The morphological changes of ovaries were observed by HE staining. Western blot was used to detect the protein expression of cysteinyl aspartate specific proteinase-9(caspase-9), cleaved caspase-3, Bcl-2 associated X protein(Bax), Bcl-2, superoxide dismutase-2(SOD-2), and glutathione peroxidase-1(GPx-1). The mRNA expression of Bax and Bcl-2 was detected by real-time fluorescence-based quantitative polymerase chain reaction(real-time PCR). The results showed that compared with the blank group, the model group showed body weight loss, disordered oestrous cycle, elevated serum levels of follicle-stimulating hormone(FSH) and luteinizing hormone(LH), reduced serum levels of estradiol(E_2), anti-mullerian hormone(AMH), and inhibin B(INHB), the declining number of ovarian follicles and granulosa layers, increased number of atretic follicles, up-regulated protein expression of caspase-9, cleaved caspase-3, and Bax and Bax mRNA expression in ovaries, and down-regulated protein expression of Bcl-2, SOD-2 and GPx-1, and Bcl-2 mRNA expression. Compared with the model group, the Modified Dihuang Decoction groups displayed restored body weight and oestrous cycle, decreased serum levels of FSH and LH, elevated serum levels of E_2, AMH, and INHB, increased number of ovarian follicles, thickened granulosa layers, and declining number of atretic follicles. Additionally, the protein expression of caspase-9, cleaved caspase-3, and Bax, and Bax mRNA expression was down-regulated, and the protein expression of Bcl-2, SOD-2, and GPx-1, and Bcl-2 mRNA expression was up-regulated. The results suggest that Modified Dihuang Decoction can regulate endocrine hormone, promote follicle growth and improve ovarian reserve by enhancing ovarian anti-oxidant capacity, inhibiting the Bcl-2-related mitochondrial apoptosis pathway, and further inhibiting cell apoptosis.

[Meta-analysis of Kuntai Capsules combined with GnRH-a in treatment of endometriosis].

To systemically evaluate the efficacy and safety of Kuntai Capsules combined with GnRH-a in the treatment of endome-triosis. The databases of CNKI, WanFang, VIP, PubMed, EMbase and Cochrane Library were searched from their establishment to May 2019 to collect the randomized controlled trials of Kuntai Capsules combined with GnRH-a in the treatment of endometriosis. The data were searched, screened and extracted by two researchers according to the inclusion and exclusion criteria, and the data were analyzed by using RevMan 5.3 software. A total of 58 articles were collected and 13 studies were included. The total sample size was 1 041 cases, including 523 cases in the experimental group and 518 cases in the control group. The results of Meta-analysis showed that Kuntai Capsules combined with GnRH-a can reduce the level of follicle stimulating hormone(FSH), luteinizing hormone(LH) and estradiol(E_2) in patients with endometriosis as compared with GnRH-a alone. With a low incidence of adverse events of peri-meno-pausal symptoms during treatment(RR=0.46, 95%CI[0.35, 0.60], P<0.000 01), it can reduce the VAS score of dysmenorrhea(MD=-1.85,95%CI[-1.92,-1.78],P<0.000 01). The recurrence rate in the combined treatment group was lower than that in the control group(RR=0.27, 95%CI[0.09,0.77], P=0.01). This study showed that Kuntai Capsules combined with GnRH-a can reduce the level of FSH, LH and E_2 in patients with endometriosis, reduce the VAS score of dysmenorrhea, with lower incidence of adverse events and recurrence rate, but it still needs large-scale, multicenter, randomized, double-blind and high-quality clinical trials for support and evidence.

Esculetin Inhibits Proliferation, Invasion, and Migration of Laryngeal Cancer In Vitro and In Vivo by Inhibiting Janus Kinas (JAK)-Signal Transducer and Activator of Transcription-3 (STAT3) Activation.

BACKGROUND Laryngeal cancer is one of the most common malignant tumors of the head and neck. Natural compounds in traditional Chinese medicine provide many valuable potential compounds for tumor chemotherapy. Esculetin, a coumarin derivative from several herbs, inhibits proliferation of many types of cancer cells, but its anticancer effect in laryngeal cancer is still not clear. MATERIAL AND METHODS We performed in vitro proliferation assay, invasion assay, and migration assay to assess the effect of esculetin against LC, and in vivo nude mouse xenograft animal model was used as well. Flow cytometry was conducted to analyze the effect of esculetin on cell cycle of LC cells, and Western blot analysis was used to assess the effect esculetin on the JAK-STAT signaling pathway. RESULTS Esculetin remarkably inhibits proliferation, migration, and invasion of LC cells, and reduces in vivo xenograft tumor growth and tumor weight in a dose-dependent manner. Our molecular mechanism study demonstrated that esculetin significantly inhibits STAT3 phosphorylation and blocks translocation of STAT3 into the nucleus, and esculetin also blocks the cell cycle in G1/S phase. CONCLUSIONS In a summary, by inhibiting the STAT3 activation, esculetin shows potential anticancer effects against the laryngeal cancer.

Fumagillin prodrug nanotherapy suppresses macrophage inflammatory response via endothelial nitric oxide.

Antiangiogenesis has been extensively explored for the treatment of a variety of cancers and certain inflammatory processes. Fumagillin, a mycotoxin produced by Aspergillus fumigatus that binds methionine aminopeptidase 2 (MetAP-2), is a potent antiangiogenic agent. Native fumagillin, however, is poorly soluble and extremely unstable. We have developed a lipase-labile fumagillin prodrug (Fum-PD) that eliminated the photoinstability of the compound. Using αvß3-integrin-targeted perfluorocarbon nanocarriers to deliver Fum-PD specifically to angiogenic vessels, we effectively suppressed clinical disease in an experimental model of rheumatoid arthritis (RA). The exact mechanism by which Fum-PD-loaded targeted nanoparticles suppressed inflammation in experimental RA, however, remained unexplained. We herein present evidence that Fum-PD nanotherapy indirectly suppresses inflammation in experimental RA through the local production of endothelial nitric oxide (NO). Fum-PD-induced NO activates AMP-activated protein kinase (AMPK), which subsequently modulates macrophage inflammatory response. In vivo, NO-induced AMPK activation inhibits mammalian target of rapamycin (mTOR) activity and enhances autophagic flux, as evidenced by p62 depletion and increased autolysosome formation. Autophagy in turn mediates the degradation of IkappaB kinase (IKK), suppressing the NF-κB p65 signaling pathway and inflammatory cytokine release. Inhibition of NO production by N(G)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, reverses the suppression of NF-κB-mediated inflammatory response induced by Fum-PD nanotherapy. These unexpected results uncover an activity of Fum-PD nanotherapy that may be further explored in the treatment of angiogenesis-dependent diseases.

Suppression of inflammation in a mouse model of rheumatoid arthritis using targeted lipase-labile fumagillin prodrug nanoparticles.

Nanoparticle-based therapeutics are emerging technologies that have the potential to greatly impact the treatment of many human diseases. However, drug instability and premature release from the nanoparticles during circulation currently preclude clinical translation. Herein, we use a lipase-labile (Sn 2) fumagillin prodrug platform coupled with a unique lipid surface-to-surface targeted delivery mechanism, termed contact-facilitated drug delivery, to counter the premature drug release and overcome the inherent photo-instability of fumagillin, an established anti-angiogenic agent. We show that α(v)ß(3)-integrin targeted fumagillin prodrug nanoparticles, administered at 0.3 mg of fumagillin prodrug/kg of body weight suppress the clinical disease indices of KRN serum-mediated arthritis in a dose-dependent manner when compared to treatment with the control nanoparticles with no drug. This study demonstrates the effectiveness of this lipase-labile prodrug nanocarrier in a relevant preclinical model that approximates human rheumatoid arthritis. The lipase-labile prodrug paradigm offers a translatable approach that is broadly applicable to many targeted nanosystems and increases the translational potential of this platform for many diseases.

[Effects of bushen zhuyun recipe on protein expressions of estrogen receptor, progesterone receptor and integrin alpha5 and beta3 in endometrium of rats at the implantation stage].

OBJECTIVE: To observe the effects of Bushen Zhuyun Recipe (BZR) on protein expressions of estrogen receptor (ER), progesterone receptor (PR) and integrin alpha5 and beta3 in endometrium of rats at the implantation stage, for exploring the possible mechanism of the recipe in treating luteal phase defect (LPD) infertility. METHODS: Female SD rats were randomly divided into 6 groups, the blank group, the model group, the WM group treated by Western medicine, and the three BZR groups treated by low-, middle- and high-dose BZR respectively. Rats were made to pregnancy and sacrificed at the implantation stage, their middle segment of uterus, about 1 cm in length was gotten for detecting the protein expressions by Western blot. Results The protein expressions of endometrial ER and PR were significantly higher, while those of integrin alpha5 and beta3 were significantly lower than those of the control group (P < 0.05). The protein expressions of endometrial ER and PR were significantly lower, but those of integrin alpha5 and integrin beta3 were higher in rats treated by middle- and high- dose BZR than those in model rats (P < 0.05). CONCLUSION: BZR can raise the receptivity of rats' endometrium through down-regulating the expressions of ER, PR and increasing the protein expression of integrin alpha5 and beta3 in endometrium and thus to enhance the pregnant rate.

Alphavbeta3-targeted nanotherapy suppresses inflammatory arthritis in mice.

The purpose of this study was to assess whether an alternative treatment approach that targets angiogenesis, delivered through ligand-targeted nanotherapy, would ameliorate inflammatory arthritis. Arthritis was induced using the K/BxN mouse model of inflammatory arthritis. After arthritis was clearly established, mice received three consecutive daily doses of alpha(v)beta(3)-targeted fumagillin nanoparticles. Control groups received no treatment or alpha(v)beta(3)-targeted nanoparticles without drugs. Disease score and paw thickness were measured daily. Mice that received alpha(v)beta(3)-targeted fumagillin nanoparticles showed a significantly lower disease activity score (mean score of 1.4+/-0.4; P<0.001) and change in ankle thickness (mean increase of 0.17+/-0.05 mm; P<0.001) 7 d after arthritis induction, whereas the group that received alpha(v)beta(3)-targeted nanoparticles without drugs exhibited a mean arthritic score of 9.0 +/- 0.3 and mean change in ankle thickness of 1.01 +/- 0.09 mm. Meanwhile, the group that received no treatment showed a mean arthritic score of 9.8 +/- 0.5 and mean change in ankle thickness of 1.05 +/- 0.10 mm. Synovial tissues from animals treated with targeted fumagillin nanoparticles also showed significant decrease in inflammation and angiogenesis and preserved proteoglycan integrity. Ligand-targeted nanotherapy to deliver antiangiogenic agents may represent an effective way to treat inflammatory arthritis.

Therapeutic strategies for Parkinson's disease: the ancient meets the future--traditional Chinese herbal medicine, electroacupuncture, gene therapy and stem cells.

In China, it has been estimated that there are more than 2.0 million people suffering from Parkinson's disease, which is currently becoming one of the most common chronic neurodegenerative disorders during recent years. For many years, scientists have struggled to find new therapeutic approaches for this disease. Since 1994, our research group led by Drs. Ji-Sheng Han and Xiao-Min Wang of Neuroscience Research Institute, Peking University has developed several prospective treatment strategies for the disease. These studies cover the traditional Chinese medicine-herbal formula or acupuncture, and modern technologies such as gene therapy or stem cell replacement therapy, and have achieved some original results. It hopes that these data may be beneficial for the research development and for the future clinical utility for treatment of Parkinson's disease.

Triptolide protects dopaminergic neurons from inflammation-mediated damage induced by lipopolysaccharide intranigral injection.

Converging lines of evidence suggest that neuroinflammatory processes may account for the progressive death of dopaminergic neurons in Parkinson's disease (PD). Therefore, anti-inflammatory strategies have attracted much interest for their potential to prevent further deterioration of PD. Our previous study showed that triptolide, a traditional Chinese herbal compound with anti-inflammatory and immunosuppressive properties, protected dopaminergic neurons from lipopolysaccharide (LPS)-induced damage in primary embryonic midbrain cell cultures. To examine further if triptolide can protect dopaminergic neurons from inflammation-mediated damage in vivo, microglial activation and injury of dopaminergic neurons were induced by LPS intranigral injection, and the effects of triptolide treatment on microglial activation and survival ratio and function of dopaminergic neurons were investigated. Our results demonstrated that microglial activation induced by a single intranigral dose of 10 mug of LPS reduced the survival ratio of tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the substantia nigra pars compacta (SNpc) to 29% and the content of dopamine (DA) in striatum to 37% of the non-injected side. Intriguingly, treatment with triptolide of 5 mug/kg for 24 days once per day dramatically improved the survival rate of TH-ir neurons in the SNpc to 79% of the non-injected side. Meanwhile, treatment with triptolide of 1 or 5 mug/kg for 24 days once per day significantly improved DA level in striatum to 70% and 68% of the non-injected side, respectively. Complement receptor 3 (CR3) immunohistochemical staining revealed that triptolide treatment potently inhibited LPS-elicited deleterious activation of microglia in SNpc. The excessive production of cytokines, such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta, was significantly abolished by triptolide administration. These results, together with our previous data in vitro, highly suggest the effectiveness of triptolide in protecting dopaminergic neurons against inflammatory challenge.

Electro-acupuncture stimulation protects dopaminergic neurons from inflammation-mediated damage in medial forebrain bundle-transected rats.

Through producing a variety of cytotoxic factors upon activation, microglia are believed to participate in the mediation of neurodegeneration. Intervention against microglial activation may therefore exert a neuroprotective effect. Our previous study has shown that the electro-acupuncture (EA) stimulation at 100 Hz can protect axotomized dopaminergic neurons from degeneration. To explore the underlying mechanism, the effects of 100 Hz EA stimulation on medial forebrain bundle (MFB) axotomy-induced microglial activation were investigated. Complement receptor 3 (CR3) immunohistochemical staining revealed that 24 sessions of 100 Hz EA stimulation (28 days after MFB transection) significantly inhibited the activation of microglia in the substantia nigra pars compacta (SNpc) induced by MFB transection. Moreover, 100 Hz EA stimulation obviously inhibited the upregulation of the levels of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta mRNA in the ventral midbrains in MFB-transected rats, as revealed by reverse transcriptase polymerase chain reaction (RT-PCR). ED1 immunohistochemical staining showed that a large number of macrophages appeared in the substantia nigra (SN) 14 days after MFB transection. The number of macrophages decreased by 47% in the rats that received 12 sessions of EA simulation after MFB transection. These data indicate that the neuroprotective role of 100 Hz EA stimulation on dopaminergic neurons in MFB-transected rats is likely to be mediated by suppressing axotomy-induced inflammatory responses. Taken together with our previous results, this study suggests that the neuroprotective effect of EA on the dopaminergic neurons may stem from the collaboration of its anti-inflammatory and neurotrophic actions.

[Effect of Chinese herb Tripterygium wilfordii Hook F monomer triptolide on apoptosis of PC12 cells induced by Abeta1-42].

Recent studies indicate that beta-amyloid (Abeta) is the key factor to cause neuronal degeneration in Alzheimer's disease (AD). In the present study, we set up an Abeta induced PC12 cell damage modle and studied the protective effect and related mechanisms of T(10), monomer extracted from Chinese herb Tripterygium wilfordii Hook F. PC12 cells were treated with different concentrations of Abeta (5x10(-4), 5x10(-3), 5x10(-2), 5x10(-1), 5, 50 micromol/L) for 48 h, cell viability was detected by MTT conversion. The apoptotic rate of PC12 cells was quantitatively determined using FACS assay. After PC12 cells were treated with 1x10(-11) mol/L T(10) for 48 h and then co-treated with 50 micromol/LAbetafor 48 h, the apoptotic rate and the change in intracellular Ca(2+) concentration of PC12 cells were analyzed by FACS assay and confocal, respectively. It was found that 5 micromol/L Abeta decreased the cell viability to 66.3% and 50 micromol/L Abeta decreased it to 55.1%, significantly different from that of the control group. After treatment with 50 micromol/L Abeta for 48 h, the apoptotic rate of PC12 cells increased obviously. The apoptotic rate was 5.37% in the control group, while after treatment with 0.5, 5 and 50 micromol/L Abeta for 48 h, the apoptotic rate of PC12 cells went up to 10.19%, 8.02% and 16.63%, respectively. At the same time, the concentration of intracellular Ca(2+) increased greatly after treatment with 50 micromol/L Abeta for 48 h. At the concentration of 1x10(-11) mol/L T(10) remarkably inhibited the apoptosis induced by 50 micromol/L Abeta. In the naive group, the apoptotic rate was 4.83%. The apoptotic rate went up to 17.24% after treatment with 50 micromol/L Abeta for 48 h. After co-treatment with 1x10(-11) mol/L T(10) and 50 micromol/L Abeta, the apoptotic rate decreased to 8.91%, significantly different from that of the control group. At the same time, at the concentration of 1x10(-11 )mol/L T(10) remarkably inhibited the increase of intracellular Ca(2+) concentration induced by Abeta. The results indicate that T(10) has obvious protective effect on PC12 cells, which may be related to the inhibition of the cell apoptosis and increment of intracellular Ca(2+) concentration induced by Abeta.

Triptolide, a Chinese herbal extract, protects dopaminergic neurons from inflammation-mediated damage through inhibition of microglial activation.

Mounting lines of evidence have suggested that brain inflammation participates in the pathogenesis of Parkinson's disease. Triptolide is one of the major active components of Chinese herb Tripterygium wilfordii Hook F, which possesses potent anti-inflammatory and immunosuppressive properties. We found that triptolide concentration-dependently attenuated the lipopolysaccharide (LPS)-induced decrease in [3H]dopamine uptake and loss of tyrosine hydroxylase-immunoreactive neurons in primary mesencephalic neuron/glia mixed culture. Triptolide also blocked LPS-induced activation of microglia and excessive production of TNFalpha and NO. Our data suggests that triptolide may protect dopaminergic neurons from LPS-induced injury and its efficiency in inhibiting microglia activation may underlie the mechanism.