Zhilong Huoxue Tongyu Capsule regulates the macrophage polarization and inflammatory response via the let-7i/TLR9/MyD88 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Zhilong Huoxue Tongyu Capsule (ZL) is clinically prescribed for acute ischemic stroke (AIS). However, only a few studies have addressed the mechanisms of ZL in treating AIS. AIM OF THE STUDY: To explore the underlying mechanism of macrophage polarization and inflammation mediated by ZL, and to provide a reference for AIS treatment. MATERIALS AND METHODS: Sixteen SD rats were fed with different dose of ZL (0, 0.4, 0.8, and 1.6 g/kg/d) for 4 days to prepare ZL serum. After 500 ng/mL lipopolysaccharide (LPS) stimulation, RAW264.7 cells were administrated with ZL serum. Then, experiments including ELISA, flow cytometry, real-time quantitative PCR and Western blot were performed to verify the effects of ZL on macrophage polarization and inflammation. Next, let-7i inhibitor was transfected in RAW264.7 cells when treated with LPS and ZL serum to verify the regulation of ZL on the let-7i/TLR9/MyD88 signaling pathway. Moreover, the interaction between let-7i and TLR9 was confirmed by the dual-luciferase assay. RESULTS: ZL serum significantly decreased the expression of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α), and increased the expression of IL-10 and transforming growth factor ß1 (TGF-ß1) of LPS stimulated-macrophages. Furthermore, ZL serum polarized macrophages toward M2, decreased the expressions of TLR9, MyD88, and iNOS, as well as increased the expressions of let-7i, CHIL3, and Arginase-1. It is worth mentioning that the effect of ZL serum is dose-dependent. However, let-7i inhibitor restored all the above effects in LPS stimulated-macrophages. In addition, TLR9 was the target of let-7i. CONCLUSIONS: ZL targeted let-7i to inhibit TLR9 expression, thereby inhibiting the activation of the TLR9/MyD88 pathway, promoting the M2 polarization, and inhibiting the development of inflammation in AIS.

Zhilong Huoxue Tongyu Capsule attenuates hemorrhagic transformation through the let-7f/TLR4 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Hemorrhagic transformation after acute ischemic stroke is a life-threatening disease that currently has no effective chemotherapy. Zhilong Huoxue Tongyu Capsule (ZL) is an empirical prescription of traditional Chinese medicine that is used to prevent and treat cardiovascular and cerebrovascular diseases in China. However, only a few studies have addressed the mechanisms of ZL in treating hemorrhagic transformation. AIM OF THE STUDY: To evaluate the anti-inflammatory effects of ZL on hemorrhagic transformation model rats and lipopolysaccharide (LPS)-induced RAW264.7 macrophages and to explore the underlying molecular mechanisms. MATERIALS AND METHODS: Murine RAW264.7 cells were treated with ZL and LPS (1 µg/mL), and cell viability was detected by cell counting kit-8 assay. RT-qPCR was used to detect the expression of inflammatory chemokines, microRNA let-7a/e/i/f, toll like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor kappa-B (NF-κB) p65. The protein expression levels of TLR4, MyD88, NF-κB p65, and apoptosis related molecules were determined by Western blotting. The apoptosis rate of RAW264.7 macrophages was detected by Annexin V-FITC/PI double staining. A hemorrhagic transformation model in rats was established by intraperitoneal injection of high glucose solution combined with thread embolization. Then, the model rats were observed behaviourally, pathologically, and molecularly. The gene expression of TLR4, MyD88, and NF-κB p65 was measured by RT-qPCR and used to evaluate the protective effect of ZL against hemorrhagic transformation in rats. RESULTS: ZL (5, 20, 40 µg/mL) was beneficial in cell proliferation. LPS (1 µg/mL) stimulated the production of inflammatory chemokines and inhibited the production of let-7a/e/i/f, with let-7f being influenced most strongly. Moreover, overexpression of let-7f decreased the gene and protein levels of TLR4, MyD88, and NF-κB p65, downregulated TLR4, and inhibited its transcriptional activity. ZL (5, 20, and 40 µg·mL-1) inhibited the production of TLR4, MyD88, and NF-κB p65 and promoted the production of let-7f in a concentration-dependent manner. Furthermore, the blockade of TLR4 antagonized the promoting effects of TLR4 pathway activation in cell inflammation and apoptosis by downregulating let-7f. Critically, it was confirmed in vivo and in vitro that ZL upregulated the expression of let-7f and inhibited the gene expression of TLR4, MyD88, and NF-κB p65 to reduce inflammatory cell infiltration, which determined the occurrence of hemorrhagic transformation. CONCLUSIONS: ZL can reduce inflammatory response by upregulating let-7f and subsequently inhibiting the TLR4 signaling pathway, thereby decreasing the occurrence of hemorrhagic transformation.

RhoA/Rock2/Limk1/cofilin1 pathway is involved in attenuation of neuronal dendritic spine loss by paeonol in the frontal cortex of D-galactose and aluminum­induced Alzheimer's disease­like rat model.

Alzheimer's disease (AD) has become the most prevalent neurodegenerative disorder. Given the pathogenesis of AD is unclear, there is currently no drug approved to halt or delay the progression of AD. Therefore, it is pressing to explore new targets and drugs for AD. In China, polyphenolic Chinese herbal medicine has been used for thousands of years in clinical application, and no toxic effects have been reported. In the present study, using D­galactose and aluminum­induced rat model, the effects of paeonol on AD were validated via the Morris water maze test, open field test, and elevated plus maze test. Neuronal morphology in frontal cortex was assessed using ImageJ's Sholl plugin and RESCONSTRUCT software. RhoA/Rock2/Limk1/cofilin1 signaling pathway­related molecules were determined by Western blotting. Cofilin1 and p­cofilin1 were analyzed by immunofluorescence. Results showed that pre­treatment with paeonol attenuated D­galactose and aluminum­induced behavioral dysfunction and AD­like pathological alterations in the frontal cortex. Accompanied by these changes were the alterations in the dendrite and dendritic spine densities, especially the mushroom­type and filopodia­type spines in the apical dendrites, as well as actin filaments. In addition, the activity and intracellular distribution of cofilin1 and the molecules RhoA/Rock2/Limk1 that regulate the signaling pathway for cofilin1 phosphorylation have also changed. Our data suggests that paeonol may be through reducing Aß levels to alleviate the loss of fibrillar actin and dendrites and dendritic spines via the Rho/Rock2/Limk1/cofilin1 signaling pathway in the frontal cortex, and ultimately improving AD­like behavior.

Weichang'an Formula Inhibits Tumor Growth in Combination with Bevacizumab in a Murine Model of Colon Cancer-Making up for the Deficiency of Bevacizumab by inhibiting VEGFR-1.

Aim: Angiogenesis plays an important role in the initiation, development, and metastasis of malignant tumors. Antiangiogenic drugs combined with immune therapy are considered to have a synergistic effect on anti-tumor strategy. Weichang'an formula (WCAF) is a prescription of traditional Chinese medicine (TCM) based on pharmaceutical screening and clinical experience. The aim of this study is to examine the effect of WCAF and its combined action with Bevacizumab (BEV) in colorectal cancer, and to identify the possible mechanism of action. Methods: A human colon cancer cell (HCT 116) subcutaneous xenograft model was established in BALB/c-nu/nu mice. Tumor-bearing mice were randomized into each of four groups: control, WCAF treated, BEV treated, and WCAF plus BEV treated. Apoptosis was detected by TUNEL assay. Western blot was used to assess the protein levels of Leptin-R, STAT3, p-STAT3, BCL-2, and VEGFR-1. Immunohistochemistry was used to detect the micro-vessel density (MVD) and AKT1. Leptin and Vascular endothelial growth factor A (VEGF-A) mRNA expression were detected by Real-time PCR (RT-PCR). A network pharmacology study and validation assay were carried out to find the underlying molecular targets of WCAF related to immune regulation. Results: Compared with the control group, WCAF reduced tumor weight and volume, as well as promoted tumor cell apoptosis. WCAF treatment decreased the mRNA expression of Leptin and VEGF-A, while the protein levels of CD31, LEP-R, VEGFR-1, STAT3, and p-STAT3 were decreased in tumor tissues. In addition, VEGFR-1 protein expression was decreased in the WCAF group and the WCAF plus BEV group but not in the BEV group. The combination of WCAF and BEV demonstrated a partial additive anti-tumor effect in vivo. The pharmacological network also found there are 26 WCAF target proteins related to cancer immune and 12 cancer immune related pathways. The AKT1 protein expression in the WCAF and WCAF + BEV groups were significantly lower than the that in the control group (p < 0.01). Conclusion: WCAF can inhibit tumor growth and promote apoptosis and inhibit tumor angiogenesis in subcutaneous xenografts of human colon cancer HCT-116 in nude mice. WCAF also makes up for the deficiency of BEV by inhibiting VEGFR-1. The VEGFR-1 expression between the combination group and BEV alone achieved statistically significant difference (p < 0.01). Combined with BEV, WCAF showed a partial additive anti-tumor effect. The mechanism may be related to Leptin/STAT3 signal transduction, VEGF-A, VEGFR-1 and WCAF target proteins related to cancer immune such as leptin and AKT1.

Ligustrum lucidum Ait. fruit extract induces apoptosis and cell senescence in human hepatocellular carcinoma cells through upregulation of p21.

Nü-zhen-zi, the fruit of Ligustrum lucidum Ait., is one of the most frequently used liver Yin tonifying Chinese herbs for the treatment of liver cancer. However, the effect of Ligustrum lucidum fruit on hepatocarcinoma cells remains unknown. In the present study, we evaluated the effects of a Ligustrum lucidum fruit extract (LLFE) on human hepatocellular carcinoma Bel-7402 cells. The results showed that LLFE inhibited the proliferation of the Bel-7402 cells in a dose- and time-dependent manner. LLFE induced apoptosis in Bel-7402 cells accompanied by activation of caspase-3, -8 and -9. LLFE-induced apoptosis was completely abrogated by a pan caspase inhibitor, Z-VAD-FMK. LLFE treatment also caused a large and flat morphologic cellular change, positive SA-ß-gal staining, and G0/G1 phase cell cycle arrest in the Bel-7402 cells, accompanied by upregulation of p21 and downregulation of RB phosphorylation. Specific knockdown of p21 expression by RNA interference partially abrogated LLFE-induced apoptosis, and significantly abrogated LLFE-induced cell senescence. These observations suggest that Nü-zhen-zi is a potential anticancer herb and support the traditional use of Nü-zhen-zi for hepatocarcinoma treatment.