Synergistic amelioration between Ligusticum striatum DC and borneol against cerebral ischemia by promoting astrocytes-mediated neurogenesis.

ETHNOPHARMACOLOGICAL RELEVANCE: Ligusticum chuanxiong Hort (LCH), with the accepted name of Ligusticum striatum DC in "The Plant List" database, is a widely used ethnomedicine in treating ischemic stroke, and borneol (BO) is usually prescribed with LCH for better therapy. Our previous study confirmed their synergistic effect on neurogenesis against cerebral ischemia. However, the underlying mechanism is still unclear. AIM OF THE STUDY: More and more evidence indicated that astrocytes (ACs) might be involved in the modulation of neurogenesis via polarization reaction. The study was designed to explore the synergic mechanism between LCH and BO in promoting astrocyte-mediated neurogenesis. MATERIALS AND METHODS: After primary cultures and identifications of ACs and neural stem cells (NSCs), the oxygen-glucose deprivation (OGD) model and the concentrations of LCH and BO were optimized. After the OGD-injured ACs were treated by LCH, BO, and their combination, the conditioned mediums were used to culture the OGD-injured NSCs. The proliferation, migration, and differentiation of NSCs were assessed, and the secretions of BDNF, CNTF, and VEGF from ACs were measured. Then the expressions of C3 and PTX3 were detected. Moreover, the mice were performed a global cerebral ischemia/reperfusion model and treated with LCH and (or) BO. After the assessments of Nissl staining, the expressions of Nestin, DCX, GFAP, C3, PTX3, p65 and p-p65 were probed. RESULTS: The most appropriate duration of OGD for the injury of both NSCs and ACs was 6 h, and the optimized concentrations of LCH and BO were 1.30 µg/mL and 0.03 µg/mL, respectively. The moderate OGD environment induced NSCs proliferation, migration, astrogenesis, and neurogenesis, increased the secretions of CNTF and VEGF from ACs, and upregulated the expressions of C3 and PTX3. For the ACs, LCH further increased the secretions of BDNF and CNTF, enhanced PTX3 expression, and reduced C3 expression. Additionally, the conditioned medium from LCH-treated ACs further enhanced NSC proliferation, migration, and neurogenesis. The in vivo study showed that LCH markedly enhanced the Nissl score and neurogenesis, and decreased astrogenesis which was accompanied by downregulations of C3, p-p65, and p-p65/p65 and upregulation of PTX3. BO not only decreased the expression of C3 in ACs both in vitro and in vivo but also downregulated p-p65 and p-p65/p65 in vivo. Additionally, BO promoted the therapeutic effect of LCH for most indices. CONCLUSION: A certain degree of OGD might induce ACs to stimulate the proliferation, astrogenesis, and neurogenesis of NSCs. LCH and BO exhibited a marked synergy in promoting ACs-mediated neurogenesis and reducing astrogenesis, in which LCH played a dominant role and BO boosted the effect of LCH. The mechanism of LCH might be involved in switching the polarization of ACs from A1 to A2, while BO preferred to inhibit the formation of A1 phenotype via downregulating NF-κB pathway.

Emodin suppresses alkali burn-induced corneal inflammation and neovascularization by the vascular endothelial growth factor receptor 2 signaling pathway.

OBJECTIVE: To investigate the effects of emodin on alkali burn-induced corneal inflammation and neovascularization. METHODS: The ability of emodin to target vascular endothelial growth factor receptor 2 (VEGFR2) was predicted by molecular docking. The effects of emodin on the invasion, migration, and proliferation of human umbilical vein endothelial cells (HUVEC) were determined by cell counting kit-8, Transwell, and tube formation assays. Analysis of apoptosis was performed by flow cytometry. CD31 levels were examined by immunofluorescence. The abundance and phosphorylation state of VEGFR2, protein kinase B (Akt), signal transducer and activator of transcription 3 (STAT3), and P38 were examined by immunoblot analysis. Corneal alkali burn was performed on 40 mice. Animals were divided randomly into two groups, and the alkali-burned eyes were then treated with drops of either 10 µM emodin or phosphate buffered saline (PBS) four times a day. Slit-lamp microscopy was used to evaluate inflammation and corneal neovascularization (CNV) in all eyes on Days 0, 7, 10, and 14. The mice were killed humanely 14 d after the alkali burn, and their corneas were removed and preserved at －80 ℃ until histological study or protein extraction. RESULTS: Molecular docking confirmed that emodin was able to target VEGFR2. The findings revealed that emodin decreased the invasion, migration, angiogenesis, and proliferation of HUVEC in a dose-dependent manner. In mice, emodin suppressed corneal inflammatory cell infiltration and inhibited the development of corneal neovascularization induced by alkali burn. Compared to those of the PBS-treated group, lower VEGFR2 expression and CD31 levels were found in the emodin-treated group. Emodin dramatically decreased the expression of VEGFR2, p-VEGFR2, p-Akt, p-STAT3, and p-P38 in VEGF-treated HUVEC. CONCLUSION: This study provides a new avenue for evaluating the molecular mechanisms underlying corneal inflammation and neovascularization. Emodin might be a promising new therapeutic option for corneal alkali burns.

Targeting mitochondrial bioenergetics by combination treatment with imatinib and dichloroacetate in human erythroleukemic K­562 and colorectal HCT­116 cancer cells.

Tumor malignant cells are characterized by dysregulation of mitochondrial bioenergetics due to the 'Warburg effect'. In the present study, this metabolic imbalance was explored as a potential target for novel cancer chemotherapy. Imatinib (IM) downregulates the expression levels of SCΟ2 and FRATAXIN (FXN) genes involved in the heme­dependent cytochrome c oxidase biosynthesis and assembly pathway in human erythroleukemic IM­sensitive K­562 chronic myeloid leukemia cells (K­562). In the present study, it was investigated whether the treatment of cancer cells with IM (an inhibitor of oxidative phosphorylation) separately, or together with dichloroacetate (DCA) (an inhibitor of glycolysis), can inhibit cell proliferation or cause death. Human K­562 and IM­chemoresistant K­562 chronic myeloid leukemia cells (K­562R), as well as human colorectal carcinoma cells HCT­116 (+/+p53) and (­/­p53, with double TP53 knock-in disruptions), were employed. Treatments of these cells with either IM (1 or 2 µM) and/or DCA (4 mΜ) were also assessed for the levels of several process biomarkers including SCO2, FXN, lactate dehydrogenase A, glyceraldehyde­3­phosphate dehydrogenase, pyruvate kinase M2, hypoxia inducing factor­1a, heme oxygenase­1, NF­κB, stem cell factor and vascular endothelial growth factor via western blot analysis. Computational network biology models were also applied to reveal the connections between the ten proteins examined. Combination treatment of IM with DCA caused extensive cell death (>75%) in K­562 and considerable (>45%) in HCT­116 (+/+p53) cultures, but less in K­562R and HCT­116 (­/­p53), with the latter deficient in full length p53 protein. Such treatment, markedly reduced reactive oxygen species levels, as measured by flow­cytometry, in K­562 cells and affected the oxidative phosphorylation and glycolytic biomarkers in all lines examined. These findings indicated, that targeting of cancer mitochondrial bioenergetics with such a combination treatment was very effective, although chemoresistance to IM in leukemia and the absence of a full length p53 in colorectal cells affected its impact.

TCM formula for trauma treatment screening and its role of promoting infectious wound coalescence investigating.

In pet clinics, the number of cases using trauma drugs accounts for >10% of the total number of cases, and most wounds are healing by second intention. The prolongation of wound healing time causes inconvenience and burden to pets and pet owners. Therefore, how to reduce wound healing time and achieve maximum recovery of tissue function and aesthetics is one of the focuses of veterinary clinical practice. Wound suppuration caused by Staphylococcus aureus and Pseudomonas aeruginosa is the main cause of delaying wound healing. Clinically, available antimicrobial treatments are almost exhausted due to the production of large numbers of resistant bacteria. At present, there are no bacteria resistant to traditional Chinese medicine (TCM), which makes TCM have the potential to become an effective drug for the treatment of bacterial infections, so the use of TCM in the treatment of traumatic infections has broad prospects. Based on the characteristics of infection syndrome, three different prescriptions were formulated in our laboratory, and the most effective prescription and dosage form was screened and named Lianrong Healing Cream (LRHC). The results showed that LRHC regulated the expression of fibroblast growth factor-2 (FGF-2), epidermal growth factor-1 (EGF-1), transforming growth factor-ß (TGF-ß) and vascular endothelial growth factor-1 (VEGF-1) genes in wound tissues and fibroblasts, thereby accelerating wound healing and repairing wound appearance and function. The results of this study may be help to develop TCM formulation for traumatic infections.

Ethyl cinnamate suppresses tumor growth through anti-angiogenesis by attenuating VEGFR2 signal pathway in colorectal cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Kaempferia galanga Linn. is an aromatic medicinal herb with extensively applied in India, China, Malaysia and other South Asia countries for thousands of years. It has been mentioned to treat abdominal tumors. Ethyl cinnamate (EC), one of the main chemical constituents of the rhizome of K. galanga, exhibited nematocidal, sedative and vasorelaxant activities. However, its anti-angiogenic activity, and anti-tumor effect have not been investigated. AIM OF THE STUDY: To investigate the anti-angiogenic mechanism of EC and its anti-tumor effect by suppressing angiogenesis. MATERIALS AND METHODS: The in vitro anti-angiogenic effect was evaluated using HUVECs model induced by VEGF and zebrafish model in vivo. The influence of the EC on phosphorylation of VEGFR2 and its downstream signaling pathways were evaluated by western blotting assay. Molecule docking technology was conducted to explore the interaction between EC and VEGFR2. SPR assay was used for detecting the binding affinity between EC and VEGFR2. To further investigate the molecular mechanism of EC on anti-angiogenesis, VEGFR2 knockdown in HUVECs and examined the influence of the EC. Anti-tumor activity of EC was evaluated using colony formation assay and apoptosis assay. The inhibitory effect of EC on tumor growth was explored using HT29 colon cancer xenograft model. RESULTS: EC obviously inhibited proliferation, migration, invasion and tube formation of VEGF-induced HUVECs. EC also induced apoptosis of HUVECs. Moreover, it inhibited the development of vessel formation in zebrafish. Further investigations demonstrated that EC could suppress the phosphorylation of VEGFR2, and its downstream signaling pathways were altered in VEGF-induced HUVECs. EC formed a hydrogen bond to bind with the ATP binding site of the VEGFR2, and EC-VEGFR2 interaction was shown in SPR assay. The suppressive effect of EC on angiogenesis was abrogated after VEGFR2 knockdown in HUVECs. EC inhibited the colon cancer cells colony formation and induced apoptosis. In addition, EC suppressed tumor growth in colon cancer xenograft model, and no detectable hepatotoxicity and nephrotoxicity. In addition, it inhibited the phosphorylation of VEGFR2, and its downstream signal pathways in tumor. CONCLUSIONS: EC could inhibit tumor growth in colon cancer by suppressing angiogenesis via VEGFR2 signaling pathway, and suggested EC as a promising candidate for colon cancer treatment.

Co-treatment with bone marrow-derived mesenchymal stem cells and curcumin improved angiogenesis in myocardium in a rat model of MI.

BACKGROUND: The cardioprotective properties of mesenchymal stem cells and the therapeutic potential of curcumin (CUR) have been explored. Combining these approaches may enhance stem cell effectiveness and expedite healing. This study aimed to investigate the synergistic effects of co-treating bone marrow mesenchymal stem cells (BMSCs) with curcumin on vascular endothelial growth factor (VEGF) levels, in a rat model of myocardial ischemia (MI). METHODS AND RESULTS: Sixty-five male rats were divided into four groups: G1 (healthy control), G2 (MI induced by isoproterenol hydrochloride), G3 (treated with BMSCs), and G4 (co-treated with curcumin and BMSCs). Blood and tissue samples were collected at specific time points (day 1, 7, 15 and 21) after MI induction. Serum levels of lactate dehydrogenase (LDH), creatine kinase (CK), cardiac troponin I (cTnI), aspartate aminotransferase (AST), CK-MB and VEGF were measured. VEGF mRNA and protein expression were evaluated using RT-qPCR and Western blot techniques. Histopathological assessments were performed using H&E staining and CD31 immunofluorescence staining. VEGF expression significantly increased on days 7 and 15 in the CUR-BMSCs group, peaking on day 7. Western blot analysis confirmed elevated VEGF protein expression on days 7 and 15 post-MI. ELISA results demonstrated increased serum VEGF levels on days 7 and 15, reaching the highest level on day 7 in CUR-BMSCs-treated animals. Treated groups showed lower levels of LDH, AST, CK, CK-MB and cTnI compared to the untreated MI group. H&E staining revealed improved myocardial structure, increased formation of new capillaries, in both treatment groups compared to the MI group. CONCLUSION: Combining curcumin with BMSCs promotes angiogenesis in the infarcted myocardium after 15 days of MI induction. These findings suggest the potential of this combined therapy approach for enhancing cardiac healing and recovery.

Ononin promotes radiosensitivity in lung cancer by inhibiting HIF-1α/VEGF pathway.

BACKGROUND: In our previous study, we provided evidence that Astragalus mongholicus Bunge(AM) and its extracts possess a protective capability against radiation-induced damage, potentially mediated through the reduction of reactive oxygen species (ROS) and nitric oxide (NO). However, we were pleasantly surprised to discover during our experimentation that AM not only offers protection against radiation damage but also exhibits a radiation sensitization effect. This effect may be attributed to a specific small molecule present in AM known as ononin. Currently, radiation sensitizers are predominantly found in nitrazole drugs and nanomaterials, with no existing reports on the radiation sensitization properties of ononin, nor its underlying mechanism. PURPOSE: This study aims to investigate the sensitization effect of the small molecule ononin derived from AM on lung cancer radiotherapy, elucidating its specific molecular mechanism of action. Additionally, the safety profile of combining astragalus small molecule ononin with radiation therapy will be evaluated. METHODS: The effective concentration of ononin was determined through cell survival experiments, and the impact of ononin combined with varying doses of radiation on lung cancer cells was observed using CCK-8 and cell cloning experiments. The apoptotic effect of ononin combined with radiation on lung cancer cells was assessed using Hochester staining, flow cytometry, and WB assay. Additionally, WB and immunofluorescence analysis were conducted to investigate the influence of ononin on HIF-1α/VEGF pathway. Furthermore, Molecular Dynamics Simulation was employed to validate the targeted binding ability of ononin and HIF-1α. A lung cancer cell line was established to investigate the effects of knockdown and overexpression of HIF-1α. Subsequently, the experiment was repeated using tumor bearing nude mice and C57BL/6 mouse models in an in vivo study. Tumor volume was measured using a vernier caliper, while HE, immunohistochemistry, and immunofluorescence techniques were employed to observe the effects of ononin combined with radiation on tumor morphology, proliferation, and apoptosis. Additionally, Immunofluorescence was employed to examine the impact of ononin on HIF-1α/VEGF pathway in vivo, and its effect on liver function in mice was assessed through biochemistry analysis. RESULTS: At a concentration of 25 µM, ononin did not affect the proliferation of lung epithelial cells but inhibited the survival of lung cancer cells. In vitro experiments demonstrated that the combination of ononin and radiation could effectively inhibit the growth of lung cancer cells, induce apoptosis, and suppress the excessive activation of the Hypoxia inducible factor 1 alpha/Vascular endothelial growth factor pathway. In vivo experiments showed that the combination of ononin and radiation reduced the size and proliferation of lung cancer tumors, promoted cancer cell apoptosis, mitigated abnormal activation of the Hypoxia inducible factor 1 alpha pathway, and protected against liver function damage. CONCLUSION: This study provides evidence that the combination of AM and its small molecule ononin can enhance the sensitivity of lung cancer to radiation. Additionally, it has been observed that this combination can specifically target HIF-1α and exert its effects. Notably, ononin exhibits the unique ability to protect liver function from damage while simultaneously enhancing the tumor-killing effects of radiation, thereby demonstrating a synergistic and detoxifying role in tumor radiotherapy. These findings contribute to the establishment of a solid basis for the development of novel radiation sensitizers derived from traditional Chinese medicine.

[Tetramethylpyrazine regulates angiogenesis of endothelial cells in cerebral ischemic stroke injury via SIRT1/VEGFA signaling pathway].

This study aims to investigate whether tetramethylpyrazine(TMP) can stimulate angiogenesis in cerebral microvascular endothelial cells and alleviate cerebral ischemic stroke(CIS) and to explore the underlying mechanisms. In the animal study, adult Sprague-Dawley rats(n=15) were assigned into sham surgery(sham), middle cerebral artery occlusion/reperfusion(MCAO/R), and MCAO/R+TMP(intraperitoneal injection of 20 mg·kg~(-1)) groups. The neurological function was evaluated by the Z-Longa method. The cerebral infarction volume was detected by TTC staining. Enzyme-linked immunosorbent assay(ELISA) was employed to detect the expression of vascular endothelial growth factor(VEGF), angiopoietin(Ang), and platelet-derived growth factor(PDGF). Immunofluorescence staining was employed to detect Ki67 and the expression of vascular endothelial growth factor A(VEGFA) and slient information regulator 1(SIRT1). Western blot was employed to determine the expression levels of VEGFA, SIRT1, angiopoietin-2(Ang-2), and platelet-derived growth factor B(PDGFB). In the cell study, mouse brain-derived endothelial cells(Bend.3) were cultured, and the optimal concentration of TMP was determined. Then, VEGF, Ang, and PDGF were detected by ELISA after the addition of cabozantinib. Western blot was employed to measure the expression of VEGFA, Ang-2, and PDGFB. Immunofluorescence staining was used to detect CD31, CD34, and Ki67, and the proliferation, migration, and tube formation ability of Bend.3 cells were observed in vitro. Western blot and immunofluorescence staining were performed to measure the expression of SIRT1 and VEGFA after addition of the SIRT1-specific inhibitor selisistat(EX-527). The results showed that compared with the sham group, the MCAO/R group had severe neurological function damage, increased infarction volume, up-regulated expression of VEGF, VEGFA, Ang, Ang-2, PDGF, and PDGFB, and down-regulated expression of Ki67 and SIRT1(P<0.01). Compared with the MCAO/R group, the MCAO/R+TMP group presented alleviated neurological function damage, reduced infarction volume, and activated expression of VEGF, VEGFA, Ang, Ang-2, PDGF, PDGFB, Ki67, and SIRT1(P<0.01). The cell experiments showed that compared with the normal group, Bend.3 cells were activated by oxygen glucose deprivation/reoxygenation(OGD/R) treatment(P<0.05, P<0.01). Compared with the OGD/R group, the OGD/R+TMP group upregulated the expression levels of VEGF, VEGFA, Ang, Ang-2, PDGF, PDGFB, SIRT1, Ki67, CD31, and CD34, enhanced the angiogenic ability of Bend.3 cells without being inhibited by BMS or EX-527(P<0.05, P<0.01, P<0.001). The results suggest that TMP can activate the SIRT1/VEGFA signaling pathway to stimulate angiogenesis and alleviate CIS injury.

Hu-Qi-Zheng-Xiao Decoction Inhibits the Metastasis of Hepatocellular Carcinoma Cells by Suppressing the HIF-1α Signaling Pathway to Inhibit EMT, LCSC, and Angiogenic Process.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common clinical malignant tumor of the digestive system. Hu-Qi-Zheng-Xiao (HQZX) decoction has been clinically found to prolong the survival of patients with hepatocellular carcinoma and improve the quality of patients' survival, but its antitumor biological mechanism is still unclear. METHODS: A nude mouse hollow fiber hepatocellular carcinoma model was constructed to analyze the in vivo efficacy of HQZX decoction against 7 different hepatocellular carcinoma cells. The subcutaneous graft tumor model was again validated. In vitro, the effect of HQZX decoction on the growth and metastasis of the cell line with the highest growth inhibition was evaluated. The cell line with the best efficacy response screened was again used to construct a hollow fiber hepatocellular carcinoma model and hollow fiber conduit cells were extracted to detect the expression of HIF-1α, VEGF, EMT-related molecules, LCSCs-related molecules, and to observe the density of the subcutaneous vascular network of hollow fiber conduits. The liver metastasis model of splenic injection was constructed to observe the effect of HQZX decoction on tumor metastasis. RESULTS: The hollow fiber hepatocellular carcinoma model was evaluated for the efficacy of HQZX decoction, and it was found to have the highest growth inhibition of LM3-luc cells. In vitro, the CCK8 assay revealed that HQZX decoction could inhibit tumor migration and invasion and promote apoptosis. In addition, the mechanism study of extracting cells from hollow fiber tubes found that HQZX decoction could inhibit metastasis-associated HIF-1α, VEGF, EMT-related molecules, and LCSCs-related molecules expression. capillary network around subcutaneous fiber tubes was reduced in the HQZX decoction gavage group of mice. It inhibited tumor metastasis in nude mice. CONCLUSIONS: HQZX decoction inhibited the growth of a variety of hepatocellular carcinoma cells. HQZX decoction suppressed the expression of metastasis-associated VEGF, EMT-related molecules, and LCSCs-related molecules and inhibited tumor angiogenesis and growth and metastasis, which may be related to the inhibition of the HIF-1α signaling pathway. It reveals that HQZX decoction may be a promising herbal compound for anti-HCC therapy, and also reveals the accurate feasibility of the hollow fiber hepatocellular carcinoma model for in vivo pharmacodynamic evaluation and mechanism study.

Comparative evaluation of four Lycium barbarum cultivars on NaIO3-induced retinal degeneration mice via multivariate statistical analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruit of Lycium barbarum L. (goji berry) is a traditional Chinese medicine and is often used to improve vision. While various goji cultivars may differentially treat retinal degeneration, however their comparative effectiveness remains unclear. AIM OF THE STUDY: To evaluate the protective effects of four goji cultivars on NaIO3-induced retinal degeneration mouse model and identify the most therapeutically potent cultivar. MATERIALS AND METHODS: The principal compounds in the extracts of four goji cultivars were characterized by UPLC-Q-TOF/MS. A retinal degeneration mouse model was established via NaIO3 injection. Dark-light transition and TUNEL assays were used to assess visual function and retinal apoptosis. The levels of antioxidative, inflammatory, and angiogenic markers in serums and eyeballs were measured. Hierarchical cluster analysis, principal component analysis and partial least squares-discriminant analysis were used to objectively compare the treatment responses. RESULTS: Sixteen compounds were identified in goji berry extracts. All goji berry extracts could reverse NaIO3-induced visual impairment, retinal damage and apoptosis. The samples from the cultivar of Ningqi No.1 significantly modulated oxidative stress, inflammation, and vascular endothelial growth factor levels, which are more effectively than the other cultivars based on integrated multivariate profiling. CONCLUSION: Ningqi No.1 demonstrated a stronger protective effect on mouse retina than other goji cultivars, and is a potential variety for further research on the treatment of retinal degeneration.

Tetrahydropalmatine promotes random skin flap survival in rats via the PI3K/AKT signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Flap necrosis is the most common complication after flap transplantation, but its prevention remains challenging. Tetrahydropalmatine (THP) is the main bioactive component of the traditional Chinese medicine Corydalis yanhusuo, with effects that include the activation of blood circulation, the promotion of qi, and pain relief. Although THP is widely used to treat various pain conditions, its impact on flap survival is unknown. AIM OF THE STUDY: To explore the effect and mechanism of THP on skin flap survival. MATERIALS AND METHODS: In this study, we established a modified McFarlane flap model, and the flap survival rate was calculated after 7 days of THP treatment. Angiogenesis and blood perfusion were evaluated using lead oxide/gelatin angiography and laser Doppler, respectively. Flap tissue obtained from zone II was evaluated histopathologically, by hematoxylin and eosin staining, and in assays for malondialdehyde content and superoxide dismutase activity. Immunofluorescence was performed to detect interleukin (IL)-6, tumor necrosis factor (TNF)-α, hypoxia-inducible factor (HIF)-1α, Bcl-2, Bax, caspase-3, caspase-9, SQSTM1/P62, Beclin-1, and LC3 expression, and Western blot to assess PI3K/AKT signaling pathway activation and Vascular endothelial growth factor (VEGF) expression. The role played by the autophagy pathway in flap necrosis was examined using rapamycin, a specific inhibitor of mTOR. RESULTS: Experimentally, THP improved the survival rate of skin flaps, promoted angiogenesis, and improved blood perfusion. THP administration reduced the inflammatory response, oxidative stress, and apoptosis in addition to inhibiting autophagy via the PI3K/AKT/mTOR pathway. Rapamycin partially reversed these effects. CONCLUSION: THP promotes skin flap survival via the PI3K/AKT signaling pathway.

Potent anti-angiogenic component in Kaempferia galanga L. and its mechanism of action.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditionally, the roots of Kaempferia galanga has been used to treat high blood pressure, chest pain, headache, toothache, rheumatism, indigestion, cough, inflammation and cancer in Asia. Nevertheless, most of its pharmacological studies were focused on ethanolic extracts and volatile oils. The exact active chemical constituents and their underlying mechanisms are still poorly understood, especially towards its anti-cancer treatment. Inhibition of angiogenesis is an important atrategy to inhibit tumor growth. It has been reported that the low polar component of the plant possessed anti-angiogenic activity. Yet, the potent compound which is responsible for the effect and its molecular mechanism has not been reported. AIM OF THE STUDY: To determine the potent anti-angiogenic component in K.galanga and its mechanism of action. MATERIAL AND METHODS: The low polar components of the plant were concentrated using the methods of supercritical fluid extraction (SFE), subcritical extraction (SCE) and steam distillation (SD). The anti-angiogenic activity of the three extracts was evaluated using a zebrafish model. The content of the active compound in those extracts was determined with HPLC analysis. The in-vitro and in-vivo activity of the isolated compound was evaluated using human umbilical vein endothelial cells (HUVECs) model, the aortic ring assay and the matrigel plug assay, respectively. Its molecular mechanism was further studied by the western blotting assay and computer-docking experiments. Besides, its cytotoxicity on cancer and normal cell lines was evaluated using the cell-counting kit. RESULTS: HPLC results showed that trans-ethyl p-methoxycinnamate (TEM) was the major component of the extracts. The extract of SFE showed the best effect as it has the highest content of TEM. TEM could inhibit vascular endothelial growth factor (VEGF)-induced viability, migration, invasion and tube formation in human umbilical vein endothelial cells (HUVECs) in vitro. Moreover, it inhibited VEGF-induced sprout formation ex vivo and vessel formation in vivo. Mechanistic study showed that it could suppress tyrosine kinase activity of the receptor of VEGF (VEGFR2) and alter its downstream signaling pathways. In addition, the molecular docking showed that the binding of TEM and VEGFR2 is stable, which mainly attributed to the non-covalent binding interaction. Beside, TEM possessed little toxicity to both cancer and normal cells. CONCLUSION: TEM is the major anti-angiogenic component present in K. galanga and its anti-angiogenic property rather than toxicity provides scientific basis for the traditional use of K. galanga in cancer treatment.

Brucea javanica derived exosome-like nanovesicles deliver miRNAs for cancer therapy.

Triple-negative breast cancer (TNBC) is characterized by complex heterogeneity, high recurrence and metastasis rates, and short overall survival, owing to the lack of endocrine and targeted receptors, which necessitates chemotherapy as the major treatment regimen. Exosome-like nanovesicles derived from medicinal plants have shown great potential as novel biotherapeutics for cancer therapy by delivering their incorporated nucleic acids, especially microRNAs (miRNAs), to mammalian cells. In this study, we isolated exosome-like nanovesicles derived from B. javanica (BF-Exos) and investigated their influence and underlying molecular mechanisms in TNBC. We found that BF-Exos delivered 10 functional miRNAs to 4T1 cells, significantly retarding the growth and metastasis of 4T1 cells by regulating the PI3K/Akt/mTOR signaling pathway and promoting ROS/caspase-mediated apoptosis. Moreover, BF-Exos were shown to inhibit the secretion of vascular endothelial growth factor, contributing to anti-angiogenesis in the tumor microenvironment. In vivo, BF-Exos inhibited tumor growth, metastasis, and angiogenesis in breast tumor mouse models, while maintaining high biosafety. Overall, BF-Exos are considered promising nanoplatforms for the delivery of medicinal plant-derived nucleic acids, with great potential to be developed into novel biotherapeutics for the treatment of TNBC.

Qi-Ju-Di-Huang-Pill delays the progression of diabetic retinopathy.

ETHNOPHARMACOLOGICAL RELEVANCE: Qi-Ju-Di-Huang-Pill (QJDH pill) is a Chinese decoction. Although it is commonly used to treat eye conditions, such as diabetic retinopathy (DR), its exact mechanism of action is unknown. AIM OF THE STUDY: To investigate the specific mechanism by which QJDH pill slows the progression of diabetic retinopathy (DR) based on animal and cellular experiments. MATERIAL AND METHODS: The major components of QJDH pill were characterized by ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLCMS/MS). C57BL/6J mice were randomly divided into five groups as follows: normal group (control group), model group (STZ group), low-dosage QJDH pill group (QJDH-L group), medium-dosage QJDH pill group (QJDH-M group) and high-dosage QJDH pill group (QJDH-H group). Changes in water intake, urination, food intake, and body mass were monitored weekly, while changes in blood glucose were monitored monthly. Fluorescein fundus angiography (FFA), optical coherence tomography angiography (OCTA), and optical coherence tomography (OCT) were utilized to analyze the changes in fundus imaging indications. Hematoxylin & eosin (H&E) and transmission electron microscopy (TEM) were employed to examine histopathologic and ultrastructural changes in retina. The levels of interleukin-6 (IL-6), interleukin-17 (IL-17), tumor necrosis factor-α (TNF-α), and vascular endothelial growth factor (VEGF) in peripheral blood were detected using Enzyme-linked immunosorbent assay (ELISA). The mouse retina apoptotic cells were labeled with green fluorescence via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (Tunel). The protein levels of Bcl-2-Associated X (Bax), B cell lymphoma 2 (Bcl-2), Caspase-3, PI3K, phosphorylated PI3K (p-PI3K), protein kinase B (AKT) and phosphorylated AKT (p-AKT) were quantified by Western blot (WB). The retinal pigment epithelium (RPE) cells were cultured and classified into five groups as follows: normal glucose group (NG group), high glucose group (HG group), high glucose + QJDH pill group (HG + QJDH group), high glucose + inhibitor group (HG + LY294002 group), and high glucose + inhibitor + QJDH pill group (HG + LY294002 + QJDH group). Cell viability and apoptosis were detected via Cell Counting Kit-8 (CCK8) and then analyzed by flow cytometry. RESULTS: In vivo experiments revealed that the QJDH pill effectively reduced blood glucose, symptoms of increased water intake, elevated urination, increased food intake and decreased body mass in DR mice. QJDH pill also slowed the development of a series of fundus imaging signs, such as retinal microangiomas, tortuous dilatation of blood vessels, decreased vascular density, and thinning of retinal thickness, downregulated IL-6, IL-17, TNF-α, and VEGF levels in peripheral blood, and inhibited retinal cell apoptosis by activating the PI3K/AKT signaling pathway. Moreover, in vitro experiments showed that high glucose environment inhibited RPE cell viability and activated RPE cell apoptosis pathway. In contrast, lyophilized powder of QJDH pill increased RPE cell viability, protected RPE cells from high glucose-induced damage, and decreased apoptosis of RPE cells by activating the pi3k pathway. CONCLUSION: QJDH pill induces hypoglycemic, anti-inflammatory effects, anti-VEGF and anti-retinal cell apoptosis by activating PI3K/AKT signaling pathway, and thus can protect the retina and slow the DR progression.

VEGF and EGFR signaling pathways are involved in the baicalein attenuation of OVA-induced airway inflammation and airway remodeling in mice.

BACKGROUND: Although Traditional Chinese Medicine (TCM) has been used for treating asthma for centuries, the understanding of its mechanism of action is still limited. Thus, the purpose of this study was to explore the possible therapeutic effects, and underlying mechanism of baicalein in the treatment of asthma. METHODS: Freely availabled atabases (e.g. OMIM, TTD, Genecards, BATMAN-TCM, STITCH 5.0, SEA, SwissTargetPrediction) and software (e.g. Ligplot 2.2.5 and PyMoL) were used for disease drug target prediction and molecular docking by network pharmacology. The efficacy and mechanism of action of baicalein in the treatment of asthma were validated using an ovalbumin (OVA)-induced asthma mouse model and molecular biology techniques. RESULTS: A total of 1655 asthma-related genes and 161 baicalein-related targets were identified from public databases. Utilizing common databases and software for network pharmacology and molecular docking analysis, seven potential target proteins for the therapeutic effects of baicalein on asthma were selected, including v-akt murine thymoma viral oncogene homolog 1 (AKT1), vascular endothelial growth factor A (VEGFA), epidermal growth factor receptor (EGFR), proto-oncogene tyrosine-protein kinase Src (SRC), mitogen-activated protein kinase 3 (MAPK3), matrix metallopeptidase 9 (MMP9), and MAPK1. In vivo, baicalein treatment via intraperitoneal injection at a dose of 50 mg/kg significantly reduced airway inflammation, collagen deposition, smooth muscle thickness, lung interleukin (IL)-4 and IL-13 levels, peripheral blood immunoglobulin (Ig)E levels, as well as the count and ratio of eosinophils in bronchoalveolar lavage fluid (BALF) in an OVA-induced asthma mouse model. Further validation by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting analysis revealed that the VEGF and EGFR signaling pathways involving VEGFA, MAPK1, MAPK3, and EGFR were inhibited by baicalein in the asthma mouse model. CONCLUSION: Baicalein attenuates airway inflammation and airway remodeling through inhibition of VEGF and EGFR signaling pathways in an OVA-induced asthma mouse model. This will provide a new basis for the development of baicalein as a treatment for asthma and highlights the potential of network pharmacology and molecular docking in drug discovery and development.

Rat hair-follicle-associated pluripotent (HAP) stem cells can differentiate into atrial or ventricular cardiomyocytes in culture controlled by specific supplementation.

There has been only limited success to differentiate adult stem cells into cardiomyocyte subtypes. In the present study, we have successfully induced beating atrial and ventricular cardiomyocytes from rat hair-follicle-associated pluripotent (HAP) stem cells, which are adult stem cells located in the bulge area. HAP stem cells differentiated into atrial cardiomyocytes in culture with the combination of isoproterenol, activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF), and cyclosporine A (CSA). HAP stem cells differentiated into ventricular cardiomyocytes in culture with the combination of activin A, BMP4, bFGF, inhibitor of Wnt production-4 (IWP4), and vascular endothelial growth factor (VEGF). Differentiated atrial cardiomyocytes were specifically stained for anti-myosin light chain 2a (MLC2a) antibody. Ventricular cardiomyocytes were specially stained for anti-myosin light chain 2v (MLC2v) antibody. Quantitative Polymerase Chain Reaction (qPCR) showed significant expression of MLC2a in atrial cardiomyocytes and MLC2v in ventricular cardiomyocytes. Both differentiated atrial and ventricular cardiomyocytes showed characteristic waveforms in Ca2+ imaging. Differentiated atrial and ventricular cardiomyocytes formed long myocardial fibers and beat as a functional syncytium, having a structure similar to adult cardiomyocytes. The present results demonstrated that it is possible to induce cardiomyocyte subtypes, atrial and ventricular cardiomyocytes, from HAP stem cells.

A new lymphedema treatment using pyro-drive jet injection.

Lymphedema, resulting from impaired lymphatic drainage, causes inflammation, fibrosis and tissue damage leading to symptoms such as limb swelling and restricted mobility. Despite various treatments under exploration, no standard effective therapy exists. Here a novel technique using the pyro-drive jet injection (PJI) was used to create artificial clefts between collagen fibers, which facilitated the removal of excess interstitial fluid. The PJI was used to deliver a mixture of lactated Ringer's solution and air into the tail of animals with secondary skin edema. Edema levels were assessed using micro-CT scanning. Histopathological changes and neovascularization were evaluated on the injury-induced regenerative tissue. Regarding tissue remodeling, we focused on connective tissue growth factor (CTGF) and vascular endothelial growth factor (VEGF)-C. PJI markedly diminished soft tissue volume in the experimental lymphedema animals compared to the non-injected counterparts. The PJI groups exhibited a significantly reduced proportion of inflammatory granulation tissue and an enhanced density of lymphatic vessels and α-smooth muscle actin (αSMA)-positive small vessels in the fibrous granulation tissue compared to the controls. In addition, PJI curtailed the prevalence of CTGF- and VEGF-C-positive cells in regenerative tissue. In a lymphedema animal model, PJI notably ameliorated interstitial edema, promoted lymphatic vessel growth, and bolstered αSMA-positive capillaries in fibrous granulation tissue. PJI's minimal tissue impact post-lymph node dissection indicates significant potential as an early, standard preventative measure. Easily applied in general clinics without requiring specialized training, it offers a cost-effective and highly versatile solution to the management of lymphedema.

Chanling Gao suppresses colorectal cancer via PI3K/Akt/mTOR pathway modulation and enhances quality of survival.

The Chinese medicine formula Chanling Gao (CLG) exhibits significant tumor inhibitory effects in colorectal cancer (CRC) nude mice. However, the detailed mechanisms remain elusive. CRC in situ nude mouse models were treated with CLG. Small animal magnetic resonance imaging (MRI) tracked tumor progression, and overall health metrics such as food and water intake, body weight, and survival were monitored. Posttreatment, tissues and blood were analyzed for indicators of tumor inhibition and systemic effects. Changes in vital organs were observed via stereoscope and hematoxylin-eosin staining. Immunohistochemistry quantified HIF-1α and P70S6K1 protein expression in xenografts. Double labeling was used to statistically analyze vascular endothelial growth factor (VEGF) and CD31 neovascularization. Enzyme-linked immunosorbent assay was used to determine the levels of VEGF, MMP-2, MMP-9, IL-6, and IL-10 in serum, tumors, and liver. Western blotting was used to assess the expression of the PI3K/Akt/mTOR signaling pathway-related factors TGF-ß1 and smad4 in liver tissues. CLG inhibited tumor growth, improved overall health metrics, and ameliorated abnormal blood cell counts in CRC nude mice. CLG significantly reduced tumor neovascularization and VEGF expression in tumors and blood. It also suppressed HIF-1α, EGFR, p-PI3K, Akt, p-Akt, and p-mTOR expression in tumors while enhancing PTEN oncogene expression. Systemic improvements were noted, with CLG limiting liver metastasis, reducing pro-inflammatory cytokines IL-6 and IL-10 in liver tissues, decreasing MMP-2 in blood and MMP-2 and MMP-9 in tumors, and inhibiting TGF-ß1 expression in liver tissues. CLG can enhance survival quality and inhibit tumor growth in CRC nude mice, likely through the regulation of the PI3K/Akt/mTOR signaling pathway.

L-Borneol promotes skin flap survival by regulating HIF-1α/NF-κB pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: The clinical application of skin flaps in surgical reconstruction is frequently impeded by the occurrence of distant necrosis. L-Borneol exhibits myogenic properties in traditional Chinese medicine and is used in clinical settings to promote wound healing and conditions such as stroke. Nevertheless, the precise mechanism by which borneol exerts its protective effects on skin flap survival remains unclear. AIM OF THE STUDY: To explore the potential of L-borneol to promote skin flap survival and elucidate the underlying mechanisms. MATERIALS AND METHODS: Thirty-six male Sprague-Dawley rats were randomly divided into three groups: a high-dose (200 mg/kg L-borneol per day), a low-dose (50 mg/kg/day), and control group (same volume of solvent). In each rat, a modified rectangular McFarlane flap model measuring 3 × 9 cm was constructed. Daily intragastric administration of L-borneol or solvent was performed. The flap was divided into three square sections of equal size, namely Zone I (the proximal zone), Zone II (the intermediate zone), and Zone III (the distal zone). The survival rate was quantified, and the histological state of each flap was evaluated on the seventh day following the surgical procedure. The assessment of angiogenesis was conducted using lead oxide/gelatin angiography, whereas the evaluation of blood flow in the free flap was performed using laser Doppler flow imaging. Superoxide dismutase activity was detected using the water-soluble tetrazolium salt-8 method. The quantities of vascular endothelial growth factor, interleukin (IL)-1ß, IL-6, and tumour necrosis factor-α were determined using immunohistochemistry. The levels of nuclear transcription factor-κB, hypoxia-inducible factor-1, B-cell lymphoma-2 (BCL-2), and BCL-2-associated X (BAX) were determined by Western blotting technique. RESULTS: Flap survival rate significantly improved and neutrophil recruitment and release were enhanced after treatment with the compound. Angiogenesis was promoted. L-borneol protected against oxidative stress by increasing superoxide dismutase activity and decreasing malondialdehyde content. It downregulated the hypoxia-inducible factor nuclear transcription factor-κB pathway, leading to the inhibition of several inflammatory factors. Simultaneously, it facilitated the expression of vascular endothelial growth factor and BCL-2. CONCLUSION: The study shows that L-borneol may promote skin flap survival by inhibiting HIF-1α/NF-κB pathway.

Protective Effects of Danmu Extract Syrup on Acute Lung Injury Induced by Lipopolysaccharide in Mice through Endothelial Barrier Repair.

OBJECTIVE: To investigate the effects of Danmu Extract Syrup (DMS) on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice and explore the mechanism. METHODS: Seventy-two male Balb/C mice were randomly divided into 6 groups according to a random number table (n=12), including control (normal saline), LPS (5 mg/kg), LPS+DMS 2.5 mL/kg, LPS+DMS 5 mL/kg, LPS+DMS 10 mL/kg, and LPS+Dexamethasone (DXM, 5 mg/kg) groups. After pretreatment with DMS and DXM, the ALI mice model was induced by LPS, and the bronchoalveolar lavage fluid (BALF) were collected to determine protein concentration, cell counts and inflammatory cytokines. The lung tissues of mice were stained with hematoxylin-eosin, and the wet/dry weight ratio (W/D) of lung tissue was calculated. The levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1 ß in BALF of mice were detected by enzyme linked immunosorbent assay. The expression levels of Claudin-5, vascular endothelial (VE)-cadherin, vascular endothelial growth factor (VEGF), phospho-protein kinase B (p-Akt) and Akt were detected by Western blot analysis. RESULTS: DMS pre-treatment significantly ameliorated lung histopathological changes. Compared with the LPS group, the W/D ratio and protein contents in BALF were obviously reduced after DMS pretreatment (P<0.05 or P<0.01). The number of cells in BALF and myeloperoxidase (MPO) activity decreased significantly after DMS pretreatment (P<0.05 or P<0.01). DMS pre-treatment decreased the levels of TNF-α, IL-6 and IL-1 ß (P<0.01). Meanwhile, DMS activated the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway and reversed the expressions of Claudin-5, VE-cadherin and VEGF (P<0.01). CONCLUSIONS: DMS attenuated LPS-induced ALI in mice through repairing endothelial barrier. It might be a potential therapeutic drug for LPS-induced lung injury.