Investigating the synergy of Shikonin and Valproic acid in inducing apoptosis of osteosarcoma cells via ROS-mediated EGR1 expression.

BACKGROUND: Osteosarcoma is the most prevalent malignant bone tumour with a poor prognosis. Shikonin (SHK) is derived from the traditional Chinese medicine Lithospermum that has been extensively studied for its notable anti-tumour effects, including for osteosarcoma. However, its application has certain limitations. Valproic acid (VPA) is a histone deacetylase inhibitor (HDACI) that has recently been employed as an adjunctive therapeutic agent that allows chromatin to assume a more relaxed state, thereby enhancing anti-tumour efficacy. PURPOSE: This study was aimed to investigate the synergistic anti-tumour efficacy of SHK in combination with VPA and elucidate its underlying mechanism. METHODS/STUDY DESIGN: CCK-8 assays were utilized to calculate the combination index. Additional assays, including colony formation, acridine orange/ethidium bromide double fluorescent staining, and flow cytometry, were employed to evaluate the effects on osteosarcoma cells. Wound healing and transwell assays were utilized to assess cell mobility. RNA sequencing, PCR, and Western blot analyses were conducted to uncover the underlying mechanism. Rescue experiments were performed to validate the mechanism of apoptotic induction. The impact of SHK and VPA combination treatment on primary osteosarcoma cells was also assessed. Finally, in vivo experiments were conducted to validate its anti-tumour effects and mechanism. RESULTS: The combination of SHK and VPA synergistically inhibited the proliferation and migration of osteosarcoma cells in vitro and induced apoptosis in these cells. Through a comprehensive analysis involving RNA sequencing, PCR, Western blot, and rescue experiments, we have substantiated our hypothesis that the combination of SHK and VPA induced apoptosis via the ROS-EGR1-Bax axis. Importantly, our in vivo experiments corroborated these findings, demonstrating the potential of the SHK and VPA combination as a promising therapeutic approach for osteosarcoma. CONCLUSION: The combination of SHK and VPA exerted an anti-tumour effect by inducing apoptosis through the ROS-EGR1-Bax pathway. Repurposing the old drug VPA demonstrated its effectiveness as an adjunctive therapeutic agent for SHK, enhancing its anti-tumour efficacy and revealing its potential value. Furthermore, our study expanded the application of natural compounds in the anti-tumour field and overcame some of their limitations through combination therapy. Finally, we enhanced the understanding of the mechanistic pathways linking reactive oxygen species (ROS) accumulation and apoptosis in osteosarcoma cells. Additionally, we elucidated the role of EGR1 in osteosarcoma cells, offering novel strategies and concepts for the treatment of osteosarcoma.

[Effect mechanism of pricking and cupping therapy combined with Chinese herbal wet compress in treatment of herpes zoster based on Th1/Th2 imbalance and serum substance P].

OBJECTIVE: To observe the effects of pricking and cupping therapy combined with Chinese herbal wet compress on Th1/Th2 balance and the concentration of substance P (SP) in treatment of patients with herpes zoster at acute stage and explore the mechanism of clinical efficacy of this combined therapeutic method. METHODS: The patients of herpes zoster at acute stage were randomly divided into a treatment group (58 cases), a control group No.1 (57 cases), a control group No.2 (58 cases) and a control group No.3 (59 cases). In the treatment group, the therapeutic regimen was pricking and cupping+Chinese herbal wet compress+basic treatment. In the control group No.1, pricking and cupping + basic treatment were administered. In the control group No.2, Chinese herbal wet compress+basic treatment were provided. In the control group No.3, only basic treatment was delivered. The treatment duration was 9 days in each group. The score of pain degree, the score of sleep quality and the rehabilitation conditions of pain and herpes were observed in 4 groups. The concentration of serum interleukin 4 (IL-4), interferon gamma (IFN-γ) and SP were detected by ELISA. The clinical efficacy was evaluated. The occurrence of adverse reaction during treatment was observed. RESULTS: After treatment, the score of pain degree and the concentrations of serum IL-4 and SP were all lower than those before treatment in 4 groups (P<0.05); and the score of subjective sleep quality, the concentrations of serum IFN-γ and IFN-γ/IL-4 were all increased in comparison with those before treatment (P<0.05). In the treatment group and the control groups No.1 and No.2, the score of pain degree and the concentrations of serum IL-4 and SP were all lower than those in the control group No.3 (P<0.05); and the score of subjective sleep quality, the concentrations of serum IFN-γ and IFN-γ/IL-4 were higher than those in the control group No.3, respectively (P<0.05). Besides, every indicator in the treatment group was improved more significantly than that either in the control group No.1 or the control group No.2 (P<0.05). After treatment, compared with the control group No.3, the time of pain relief and disappearance, and the time of blister termination, incrustation and decrustation were all shorter in the treatment group, the control group No.1 and the control group No.2 separately (P<0.05); and every indicator in the treatment group was reduced more significantly in comparison with either the control group No.1 or the control group No.2 (P<0.05). The total effective rate in the treatment group (96.55%, 56/58), the control group No.1 (92.98%, 53/57) and the control group No.2 (91.38%, 53/58) was higher than that in the control group No.3 (74.58%, 44/59, P<0.05). No any adverse reaction occurred in the 4 groups. CONCLUSION: The pricking and cupping therapy combined with Chinese herbal wet compress effectively promotes the recovery of Th1/Th2 balance and reduces the concentration of serum SP in patients with herpes zoster at acute stage. This combined therapeutic regimen is conductive to the recovery of patients, the improvement of therapeutic effects and the decrease of the risk of pos-therpetic neuralgia.

Investigation of the effective components inhibited macrophage foam cell formation in Ophiopogonis Radix.

ETHNOPHARMACOLOGICAL RELEVANCE: Ophiopogonis Radix, the commonly used traditional Chinese medicine in clinic for treating cardiovascular diseases, is returned to the stomach, lung and heart meridian. It is reported to nourish yin, moisten lung and is used to treat heart yin deficiency syndromes and asthenia of heart and lung, which indicated that Ophiopogonis Radix may have a protective effect on heart disorders. Atherosclerosisis is an important process in the development of cardiovascular diseases and abnormal lipid deposition induced macrophage foam cells is its crucial foundation. Our previous study showed the extract of Ophiopogonis Radix (EOR) ameliorates atherosclerosis in vitro. However, it may protect against cardiovascular diseases through inhibiting macrophage foam cell formation and its potential effective components and mechanisms are still unclear. AIM OF THE STUDY: Our study aimed to investigate the effect of Ophiopogonis Radix on macrophage foam cell formation and its potential active constituents and mechanisms. MATERIALS AND METHODS: Ox-LDL induced macrophage cells were employed to evaluate the effect of Ophiopogonis Radix on macrophage foam cell formation. Then the potential active constituents inhibited formation of macrophage foam cells were screened by biospecific cell extraction and its underlying mechanisms were also explored by Western blot. RESULTS: The extract of Ophiopogonis Radix was found to significantly inhibit macrophage foam cell formation, evidenced by the decrease of TG and TC and Oil Red O staining analysis in macrophage cells, which indicated that EOR reduced the formation of macrophage foam cells. At the same time, EOR was showed to increase antioxidant capacity in macrophage cells. After treatment with EOR, two potential active components interacted with macrophage foam cells specifically were identified to inhibit macrophage foam cell formation including methylophiopogonanone A and methylophiopogonanone B. Methylophiopogonanone A was then proved to decrease the expression of CD36, Lox-1 and SREBP2, increase the expression of ABCA1 obviously, while the expression of ABCG1 and SREBP1 had no changes. CONCLUSIONS: In our study, Ophiopogonis Radix was found to protect against atherosclerosis through suppressing ox-LDL induced macrophage foam cell formation and two potential compounds were identified by biospecific cell extraction including methylophiopogonanone A and methylophiopogonanone B. Moreover, methylophiopogonanone A was proved to inhibit foam cells through reducing uptake, synthesis and increasing efflux, which may provide guidance and reference for application of Ophiopogonis Radix and investigation of the effective components of TCMs.

[Fire needling stripping after local anesthesia for verruca vulgaris: a multi-center randomized controlled trial].

OBJECTIVE: To compare the clinical therapeutic effect of fire needling stripping after local anesthesia, simple fire needling and liquid nitrogen cryotherapy on verruca vulgaris. METHODS: A total of 900 patients with verruca vulgaris were randomized into a fire needling stripping group (300 cases, 2 cases dropped off), a fire needling group (300 cases, 4 cases dropped off) and a liquid nitrogen cryotherapy group (300 cases, 5 cases dropped off). After local anesthesia of compound lidocaine cream, fire needling therapy was adopted, and the necrotic tissue of verruca was stripped in the fire needling stripping group. Simple fire needling therapy was adopted in the fire needling group, without local anesthesia and stripping. Liquid nitrogen cryotherapy was adopted in the liquid nitrogen cryotherapy group. The treatment was given once a week, and totally 3 weeks were required in the 3 groups. The skin lesion scores of number, area, thickness, color, pruritus, isomorphism and the level of T lymphocyte (CD+3、CD+4、CD+8、CD+4/ CD+8) in peripheral blood were observed before and after treatment, and the adverse reaction was recorded in the 3 groups. Five weeks after treatment, the therapeutic effect was evaluated. RESULTS: Compared before treatment, the skin lesion scores were decreased (P<0.05), the levels of T lymphocyte in peripheral blood were increased in the 3 groups (P<0.05). After treatment, all the items of the skin lesion score in the fire needling stripping group were lower than those in the fire needling group and the liquid nitrogen cryotherapy group (P<0.05), the levels of T lymphocyte in peripheral blood were higher than those in the fire needling group and the liquid nitrogen cryotherapy group (P<0.05); all the items of the skin lesion score in the liquid nitrogen cryotherapy group were lower than those in the fire needling group (P<0.05). At the follow-up, the total effective rate was 88.6% (264/298) in the fire needling stripping group, which was superior to 81.4% (241/296) in the fire needling group and 81.4% (240/295) in the liquid nitrogen cryotherapy group (P<0.05). The cases of infection, causalgia and cicatrix in the liquid nitrogen cryotherapy group were more than those in the fire needling stripping group and the fire needling group (P<0.05). CONCLUSION: Fire needling stripping after local anesthesia can effectively treat the verruca vulgaris, improve the skin lesion and immunity, its therapeutic effect is superior to simple fire needling and liquid nitrogen cryotherapy.

Coating of Epimedii Folium Water Extract onto TiO2 Nanotube Surfaces Promotes Differentiation and Proliferation of Osteoblasts.

The surface modification of titanium is effective in promoting osseointegration and is widely used in the treatment of bone diseases. Epimedii Folium (EF) plays an important role in the treatment of metabolic bone diseases. However, few studies have so far been reported on their combined use in such treatments. In the present study, EF water extract was coated onto the surface of TiO2 nanotubes (TNT) by electrochemical anodization to obtain EF-TNT. Through analysis of surface morphology characteristics, it was demonstrated that EF was successfully coated on the surface of TiO2 nanotubes. In vitro drug release data suggested that the quantity of EF water extract released was a significant quantity over 4 days, reaching a total of 80%, the release continuing in total for approximately 2 weeks. By using scanning electron microscopy and immunofluorescent staining, it was found that, EF-TNT more strongly promoted adhesion, proliferation, and differentiation of MC3T3-E1 osteoblasts compared with Ti and TNT. Quantitative reverse transcript polymerase chain reaction (qRT-PCR) analysis indicated that the expression of key genes for proliferation and differentiation of osteoblasts, such as COL1a1, ALP, OPN, and Runx2, were up-regulated by EF-TNT. Network pharmacology analysis suggested that EF water extract not only regulated the proliferation and differentiation of osteoblasts but also caused a regulatory effect on osteoclasts via multiple signaling pathways, such as RANKL-RANK-induced signaling and TGF-ß signaling. These findings indicate that the EF-TNT promotes differentiation and proliferation of osteoblasts, and represents considerable potential for use in clinical applications.

Screening and identification of potential active components in Ophiopogonis Radix against atherosclerosis by biospecific cell extraction.

Atherosclerosis is a chronic disease and an important pathological process associated with cardiovascular disease. Endothelial dysfunction, vascular smooth muscle cells (VSMCs) proliferation and neutrophil activation are involved in the development of atherosclerosis. Ophiopogonis Radix is a common traditional Chinese medicine use to treat cardiovascular diseases, however, its active constituents remain to be elucidated. In this study, primary vascular endothelial cells, primary VSMCs and neutrophils were prepared, and extract of Ophiopogonis Radix (EOR) was investigated to ameliorate H2O2 induced reactive oxygen species (ROS) and nitric oxide (NO) production. The results showed that EOR decreased levels of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, its protective effects against oxidative damage of endothelia and endothelial dysfunction. Additionally, EOR treatment inhibited oxidized low-density lipoprotein-induced VSMC proliferation, phorbol-12-myristate-13-acetate-mediated ROS production and neutrophil activation, malondialdehyde production, and decreased superoxide dismutase activity and myeloperoxidase release. By HPLC-Q-TOF-MS/MS analysis, 51 compounds in EOR were identified including 22 saponins and 24 homoisoflavonoids. Then biospecific cell extraction and LC-MS technique were employed to screening the antiatherosclerosis active components in Ophiopogonis Radix. After co-cultured with EOR, the multi-effective active constituents including four saponins and two homoisoflavonoids were acquired and subsequently verified to restore properties including endothelial injury, VSMC proliferation and neutrophil activation, indicating that these compounds may be multi-effective active constituents that were responsible for atherosclerosis and the cardiovascular protection of Ophiopogonis Radix.

Resolvin D1 attenuates imiquimod-induced mice psoriasiform dermatitis through MAPKs and NF-κB pathways.

BACKGROUND: Resolvin D1 (RvD1), a pro-resolution lipid mediator derived from docosahexaenoic acid (DHA), has been described to promote several kinds of inflammatory resolution. However, the effects and anti-inflammatory mechanisms of RvD1 on psoriasis have not been previously reported. OBJECTIVE: The present study aimed to determine the protective effects and the underlying mechanisms of RvD1 on imiquimod (IMQ)-induced psoriasiform dermatitis. METHODS: Mice were topically treated with IMQ to develop psoriasiform dermatitis on their shaved back, pretreated intraperitoneally (i.p.) with or without RvD1 or tert-butoxycarbonyl Met-Leu-Phe peptide (Boc), a lipoxin A4 (ALX) receptor antagonist. The severity was monitored and graded using a modified human scoring system, the Psoriasis Area and Severity Index (PASI), histopathology, and the signature cytokines of psoriasis (IL-23, IL-17, IL-22 and TNF-α). The mRNA and protein levels of inflammatory cytokines were quantified by quantitative real-time PCR (QRT-PCR) and ELISA. The expressions of signaling proteins MAPKs and NF-κB p65 were analyzed using western blotting. Electrophoretic mobility shift assay (EMSA) was used to check NF-κB p65 DNA binding activity. RESULTS: Our study showed that RvD1 alleviated IMQ-induced psoriasiform dermatitis and improved skin pathological changes. RvD1 markedly inhibited IMQ-induced activation of ERK1/2, p38, JNK (c-Jun N-terminal protein kinase, a subfamily of MAPKs), and NF-κB. Furthermore, pretreatment with Boc, would not exacerbate skin inflammation of IMQ-induced mice, but significantly reversed the beneficial effects of RvD1 on IMQ-induced psoriasiform inflammation. CONCLUSION: RvD1 can obviously improve skin inflammation in IMQ-induced mice psoriasiform dermatitis. The protective mechanisms might be related to its selective reaction with lipoxin A4 receptor/Formyl-peptide receptor 2 (ALX/FPR2), by downregulating relevant cytokines of the IL-23/IL-17 axis expression, the inhibition of MAPKs and NF-κB signaling transduction pathways. Thus, these results show that RvD1 could be a possible candidate for psoriasis therapy.