Induction of ferroptosis and apoptosis in endometrial cancer cells by dihydroisotanshinone I.

Danshen, also known as Salvia miltiorrhiza, is a medicinal herb used in traditional Chinese medicine. Its potential impact on endometrial cancer has not been thoroughly investigated. This study aimed to examine the effect of dihydroisotanshinone I (DT), a compound found in Danshen, on the viability of ARK1 and ARK2 endometrial cancer cells and its mechanisms. The results showed that 10 µM DT inhibited cell viability of ARK1 and ARK2 cells by inducing apoptosis and ferroptosis, which was achieved by blocking the expression of GPX4. In vivo experiments using a xenograft nude mouse model indicated that DT treatment significantly reduced tumor volume without causing any adverse effects. These findings suggest that DT may be a potential therapeutic agent for inhibiting endometrial cancer cell viability, but further research is needed to confirm these results.

The anti-SARS-CoV-2 effect and mechanism of Chiehyuan herbal oral protection solution.

The Chiehyuan herbal oral protection solution (GB-2) is a herbal mixture commonly utilized in Taiwan for combating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as per traditional Chinese medicine practices. This study assessed the clinical impact of GB-2 through prospective clinical trials. With twice-daily use for a week, GB-2 was shown to diminish the expression of angiotensin-converting enzyme 2 (ACE2) in oral mucosal cells. Moreover, after two weeks of use, it could reduce transmembrane protease, serine 2 (TMRPSS2) expression in these cells. Additionally, in vitro experiments demonstrated that GB-2 lessened the entry efficiency of the Omicron, L452R-D614G, T478K-D614G, and L452R-T478K-D614G variants of the SARS-CoV-2 pseudotyped lentivirus. It also impeded the interaction between ACE2 and the receptor-binding domain (RBD) presenting N501Y-K417N-E484A-G339D-Q493R-G496S-Q498R and L452R-T478K mutations. Glycyrrhizic acid, a major compound in GB-2, also hindered the entry of the Omicron variant (BA.1) of the SARS-CoV-2 pseudotyped lentivirus by obstructing the binding between ACE2 and the RBD presenting the N501Y-K417N-E484A-G339D-Q493R-G496S-Q498R mutation. To sum up, these findings suggest that GB-2 can decrease ACE2 and TMPRSS2 expression in oral mucosal cells. Both glycyrrhizic acid and GB-2 were found to reduce the entry efficiency of the Omicron variant (BA.1) of the SARS-CoV-2 pseudotyped lentivirus and block the binding between ACE2 and the RBD with the N501Y-K417N-E484A-G339D-Q493R-G496S-Q498R mutation. This evidence implies that GB-2 might be a potential candidate for further study as a preventative measure against SARS-CoV-2 infection.

Nasal irrigation with licorice extract (Glycyrrhiza glabra) in treating nasal polyps by reducing fibroblast differentiation and extracellular matrix production in TGF-ß1-stimulated nasal polyp-derived fibroblasts by inhibiting the MAPK/ERK-1/2 pathway - an in vitro and in clinic study.

BACKGROUND: To date, treating nasal polyps (NPs) is still a medical challenge. However, we have developed an innovative therapy using licorice extract (LE: Glycyrrhiza glabra) to treat rhinitis and sinusitis via nasal irrigation and have discovered that it significantly affects treatment of NPs. HYPOTHESIS/PURPOSE: This study investigated the mechanism of LE on NPs. STUDY DESIGN: NPs were collected from three patients using tissue biopsies before and 2 weeks after nasal irrigation with licorice for histopathological analysis. Additionally, NPs from two patients were collected, and nasal polyp-derived fibroblasts (NPDF) were isolated and cultured. METHODS: The TGF-ß1-stimulated NPDF model was used to examine the effect of LE on fibroblast differentiation (biomarker: α-SMA), the consequent production of extracellular matrix (ECM; biomarkers: fibronectin, FBN), and the functional signaling pathway. RESULTS: Immunohistochemistry (IHC) revealed that the number of eosinophils and the expression of α-SMA and interstitial collagen of polyps after licorice treatment significantly decreased. Additionally, RT-PCR, western blotting, and immunofluorescence (IF) showed that α-SMA and FBN expressions were significantly increased in the NPDF, which was stimulated by TGF-ß1, and LE dose-dependently could effectively reduce this effect. Furthermore, western blotting showed that LE could attenuate α-SMA and FBN expressions by preventing the signaling pathway of MAPK/ERK-1/2, which IHC and IF further confirmed. In addition, LE effectively suppressed the cell migration of NPDF, which is related to polyp expansion. CONCLUSION: LE is clinically used to treat sinusitis with NPs through nasal irrigation, which significantly reduces the size of NPs. This effect could attenuate fibroblast differentiation, ECM production and cell migration, and one of the functional mechanisms may be through inhibition of the MAPK/ERK-1/2 signaling pathway. TRIAL REGISTRATION: ISRCTN (No. 51425529) registered on 17/04/2020 (retrospectively registered) - http://www.isrctn.com/ISRCTN51425529.

GB-2 blocking the interaction between ACE2 and wild type and mutation of spike protein of SARS-CoV-2.

Since the start of the outbreak of coronavirus disease 2019 in Wuhan, China, there have been more than 150 million confirmed cases of the disease reported to the World Health Organization. The beta variant (B.1.351 lineage), the mutation lineages of SARS-CoV-2, had increase transmissibility and resistance to neutralizing antibodies due to multiple mutations in the spike protein. N501Y, K417N and E484K, in the receptor binding domain (RBD) region may induce a conformational change of the spike protein and subsequently increase the infectivity of the beta variant. The L452R mutation in the epsilon variant (the B.1.427/B.1.429 variants) also reduced neutralizing activity of monoclonal antibodies. In this study, we discovered that 300 µg/mL GB-2, from Tian Shang Sheng Mu of Chiayi Puzi Peitian Temple, can inhibit the binding between ACE2 and wild-type (Wuhan type) RBD spike protein. GB-2 can inhibit the binding between ACE2 and RBD with K417N-E484K-N501Y mutation in a dose-dependent manner. GB-2 inhibited the binding between ACE2 and the RBD with a single mutation (K417N or N501Y or L452R) except the E484K mutation. In the compositions of GB-2, glycyrrhiza uralensis Fisch. ex DC., theaflavin and (+)-catechin cannot inhibit the binding between ACE2 and wild-type RBD spike protein. Theaflavin 3-gallate can inhibit the binding between ACE2 and wild-type RBD spike protein. Our results suggest that GB-2 could be a potential candidate for the prophylaxis of some SARS-CoV-2 variants infection in the further clinical study because of its inhibition of binding between ACE2 and RBD with K417N-E484K-N501Y mutations or L452R mutation.

Dihydroisotanshinone I induced ferroptosis and apoptosis of lung cancer cells.

Danshen (Salvia miltiorrhiza Bunge) is broadly utilized in traditional Chinese medicine for lung cancer. However, it's exact effort and mechanism on lung cancer is fully unclear. In this study, we found that dihydroisotanshinone I (DT), a pure compound extracted from danshen, can inhibit the growth of A549 cells and H460 cells. DT also induced apoptosis and ferroptosis in these lung cancer cells. DT also blocking the protein expression of GPX4 (Glutathione peroxidase 4). For in vivo study, DT treatment can inhibit metastasis of A549 cells in the nude mice model without adverse effects on mice. In conclusion, DT inhibited the growth of lung cancer cells through apoptosis and ferroptosis and inhibited metastasis of A549 cells in the nude mice model. Further studies are warranted to validate the findings of this study.

Nasal irrigation with Glycyrrhiza glabra extract for treatment of allergic rhinitis - A study of in vitro, in vivo and clinical trial.

ETHNOPHARMACOLOGICAL RELEVANCE: Glycyrrhiza glabra, a family of licorice and a traditional Chinese medicine with sweet taste and favorable smell, has anti-inflammatory, anti-allergic and immunomodulatory functions. AIM OF THE STUDY: We developed a licorice extract (LE) by using glycyrrhiza glabra and administered it through nasal irrigation to treat allergic rhinitis (AR). MATERIALS AND METHODS: LE was prepared into extract powder, and the anti-inflammatory effect of the LE was evaluated by calcium ionophore-induced activated mast cell model (in vitro). Then, local passive anaphylaxis assays were applied to investigate the anti-IgE-mediated allergic reaction of the LE in mice (in vivo). A developed LE was administered through nasal irrigation to treat AR in clinic settings. A total of 60 participants diagnosed with AR were included in this clinical trial; they were randomly assigned to three interventions: licorice nasal irrigation (LNI), corticosteroid nasal irrigation (CNI), and saline nasal irrigation (SNI). They performed nasal irrigation once a day for 1 month. Both subjective questionnaires (22-item Sino-Nasal Outcome Test [SNOT-22] and visual analog scale [VAS]) and objective examinations (acoustic rhinometry and nasal endoscopy) were used for effectiveness assessments. RESULTS: All three interventions could improve SNOT-22 scores, but the effects of LNI and CNI were more significant. According to VAS scores for nasal blockage, rhinorrhea, sneezing, nasal pruritus, postnasal discharge, and olfactory disturbance, the effect of LNI was superior to those of CNI and SNI. The results of rhinometry revealed that LNI significantly improved nasal resistance. Endoscopic analysis showed that both LNI and CNI, but not SNI, could significantly improve turbinate hypertrophy. Moreover, the best procedural comfort was found for LNI, which had no side effects or complications during the trial. CONCLUSIONS: LNI is a natural, safe, and innovative therapy that can effectively treat AR. Its effect is superior to those of CNI and SNI, and it has greatly improved procedural comfort.

Anxiolytic effect of an extract of Salvia miltiorrhiza Bunge (Danshen) in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza Bunge (Danshen), a traditional Chinese medicine, has demonstrated in modern studies for its pharmacological activities in treatments of CNS disorders like insomnia, dysphoria. However, its application on anxiolytic effect from the ethanol extract of Salvia miltiorrhiza Bunge (SMEtOH) has not yet been reported. MATERIALS AND METHODS: This study investigated the anxiolytic effect of the SMEtOH using the elevated plus-maze test (EPM) and the hole-board test (HBT) with diazepam and buspirone as positive controls. Also, the spontaneous locomotor activity of mice had been investigated in the open field. Further, we have illustrated the anxiolytic mechanisms of SMEtOH with its influencing upon GABAergic and/or serotonergic nervous systems via a method that SMEtOH was co-administered with flumazenil, a benzodiazepine (BZD) antagonist, or a drug (WAY-100635), a selective 5HT1A receptor antagonist. RESULTS: In hole-board test, results presented that SMEtOH increased head-dip counts and duration time. On the other hand, a decrease in spontaneous locomotor activity was observed. In the EPM test, SMEtOH increased the percentage of open-arm entries and the percentage of time spent in open arms. However, when SMEtOH co-administered with flumazenil or WAY-100635, the anxiolytic effect of SMEtOH was significantly counteracted. CONCLUSION: From these results, we can conclude that the anxiolytic mechanism of SMEtOH is exerted through an activation of the BZD and 5HT1A receptors.

GB-2 inhibits ACE2 and TMPRSS2 expression: In vivo and in vitro studies.

After the first case of Coronavirus disease 2019 (COVID-19) was reported in Wuhan, COVID-19 has rapidly spread to almost all parts of world. Angiotensin converting enzyme 2 (ACE2) receptor can bind to spike protein of SARS-CoV-2. Then, the spike protein of SARS-CoV-2 can be cleaved and activated by transmembrane protease, serine 2 (TMPRSS2) of the host cells for SARS-CoV-2 infection. Therefore, ACE2 and TMPRSS2 are potential antiviral targets for treatment of prevention of SARS-CoV-2 infection. In this study, we discovered that 10-250 µg/mL of GB-2, from Tian Shang Sheng Mu of Chiayi Puzi Peitian Temple, can inhibit ACE2 mRNA expression and ACE2 and TMPRSS2 protein expression in HepG2 and 293 T cells without cytotoxicity. GB-2 treatment could decrease ACE2 and TMPRSS2 expression level of lung tissue and kidney tissue without adverse effects, including nephrotoxicity and hepatotoxicity, in animal model. In the compositions of GB-2, we discovered that 50 µg/mL of theaflavin could inhibit protein expression of ACE2 and TMPRSS2. Theaflavin could inhibit the mRNA expression of ACE2. In conclusion, our results suggest that GB-2 and theaflavin could act as potential compounds for ACE2 and TMPRSS2 inhibitors in the further clinical study.

The potential chemical structure of anti-SARS-CoV-2 RNA-dependent RNA polymerase.

An outbreak of coronavirus disease 2019 (COVID-19) occurred in Wuhan and it has rapidly spread to almost all parts of the world. For coronaviruses, RNA-dependent RNA polymerase (RdRp) is an important polymerase that catalyzes the replication of RNA from RNA template and is an attractive therapeutic target. In this study, we screened these chemical structures from traditional Chinese medicinal compounds proven to show antiviral activity in severe acute respiratory syndrome coronavirus (SARS-CoV) and the similar chemical structures through a molecular docking study to target RdRp of SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV). We found that theaflavin has a lower idock score in the catalytic pocket of RdRp in SARS-CoV-2 (-9.11 kcal/mol), SARS-CoV (-8.03 kcal/mol), and MERS-CoV (-8.26 kcal/mol) from idock. To confirm the result, we discovered that theaflavin has lower binding energy of -8.8 kcal/mol when it docks in the catalytic pocket of SARS-CoV-2 RdRp by using the Blind Docking server. Regarding contact modes, hydrophobic interactions contribute significantly in binding and additional hydrogen bonds were found between theaflavin and RdRp. Moreover, one π-cation interaction was formed between theaflavin and Arg553 from the Blind Docking server. Our results suggest that theaflavin could be a potential SARS-CoV-2 RdRp inhibitor for further study.

Danshen Improves Survival of Patients With Breast Cancer and Dihydroisotanshinone I Induces Ferroptosis and Apoptosis of Breast Cancer Cells.

Danshen (salvia miltiorrhiza Bunge) is widely used in traditional Chinese medicine. However, it is definite clinical effort and mechanism on breast cancer is unclear. In our study, we used the real-world database to investigate in vivo protective effort of danshen in the breast cancer patients through using population-based data from the Taiwan National Health Insurance Research Database (NHIRD). In vitro, human breast cancer cells (MCF-7 cells and MDA-MB-231 cells) were used to investigate the effect and the underlying mechanism through XTT assay, flow cytometry, glutathione peroxidase (GPX) activity assay, GSH (reduced glutathione)/GSSG (oxidized glutathione), malondialdehyde (MDA), and western blot analysis. The in vivo effect was investigated through a xenograft nude mouse model. We found that dihydroisotanshinone I (DT), a pure compound present in danshen, can inhibit the growth of breast carcinoma cells, including MCF-7 cells and MDA-MB-231 cells. Moreover, DT induced apoptosis and ferroptosis in these breast cancer cells. DT also repressed the protein expression of GPX4 (Glutathione peroxidase 4). For in vivo study, DT treatment also significantly inhibited the final tumor volume without adverse effects in a xenograft nude mouse model. In conclusion, danshen has protective efforts in breast cancer patients, which could be attributed to DT through inducing apoptosis and ferroptosis of breast cancer cells.

Antrodia cinnamomea extract inhibits the proliferation of tamoxifen-resistant breast cancer cells through apoptosis and skp2/microRNAs pathway.

BACKGROUND: Breast cancer is the most common cancer in women and affects 1.38 million women worldwide per year. Antiestrogens such as tamoxifen, a selective estrogen receptor (ER) modulator, are widely used in clinics to treat ER-positive breast tumors. However, remissions of breast cancer are often followed by resistance to tamoxifen and disease relapse. Despite the increasing understanding of the resistance mechanisms, effective regimens for treating tamoxifen-resistant breast cancer are limited. Antrodia cinnamomea is a traditional medicinal mushroom native only to Taiwan. In this study, we aimed to examine in vitro effect of antrodia cinnamomea in the tamoxifen-resistant cancer. METHODS: Antrodia cinnamomea was studied for its biological activity against proliferation of tamoxifen-resistant breast cancer by XTT assay. Next, the underlying mechanism was studied by flow cytometry, qPCR and Western's blotting assay. RESULTS: Our results revealed that the ethanol extract of antrodia cinnamomea (AC) can inhibit the growth of breast cancer cells, including MCF-7 cell and tamoxifen-resistant MCF-7 cell lines. Combination treatment with AC and 10- 6 M tamoxifen have the better inhibitory effect on the proliferation of tamoxifen-resistant MCF-7 cells than only AC did. AC can induce apoptosis in these breast cancer cells. Moreover, it can suppress the mRNA expression of skp2 (S-phase kinase-associated protein 2) by increasing the expressions of miR-21-5p, miR-26-5p, and miR-30-5p in MCF-7 and tamoxifen-resistant MCF-7 cells. CONCLUSIONS: These results suggest that the ethanol extract of antrodia cinnamomea could be a novel anticancer agent in the armamentarium of tamoxifen-resistant breast cancer management. Moreover, we hope to identify additional pure compounds that could serve as promising anti-breast cancer candidates for further clinical trials.

Danshen improves survival of patients with advanced lung cancer and targeting the relationship between macrophages and lung cancer cells.

In traditional Chinese medicine, Salvia miltiorrhiza Bunge (danshen) is widely used in the treatment of numerous cancers. However, its clinical effort and mechanism in the treatment of advanced lung cancer are unclear. In our study, the in vivo protective effort of danshen in patients with advanced lung cancer were validated using data from the National Health Insurance Research Database in Taiwan. We observed in vitro that dihydroisotanshinone I (DT), a bioactive compound in danshen, exerts anticancer effects through many pathways. First, 10 µM DT substantially inhibited the migration ability of lung cancer cells in both macrophage and macrophage/lung cancer direct mixed coculture media. Second, 10 µM DT repressed the phosphorylation of signal transducer and activator of transcription 3 (STAT3), the protein expression of S-phase kinase associated protein-2 (Skp2), and the mRNA levels of STAT3-related genes, including chemokine (C-C motif) ligand 2 (CCL2). In addition, 10 µM DT suppressed the macrophage recruitment ability of lung cancer cells by reducing CCL2 secretion from both macrophages and lung cancer cells. Third, 20 µM DT induced apoptosis in lung cancer cells. Furthermore, DT treatment significantly inhibited the final tumor volume in a xenograft nude mouse model. In conclusion, danshen exerts protective efforts in patients with advanced lung cancer. These effects can be attributed to DT-mediated interruption of the cross talk between lung cancer cells and macrophages and blocking of lung cancer cell proliferation.