ThermomiR-377-3p-induced suppression of Cirbp expression is required for effective elimination of cancer cells and cancer stem-like cells by hyperthermia.

BACKGROUND: In recent years, the development of adjunctive therapeutic hyperthermia for cancer therapy has received considerable attention. However, the mechanisms underlying hyperthermia resistance are still poorly understood. In this study, we investigated the roles of cold­inducible RNA binding protein (Cirbp) in regulating hyperthermia resistance and underlying mechanisms in nasopharyngeal carcinoma (NPC). METHODS: CCK-8 assay, colony formation assay, tumor sphere formation assay, qRT-PCR, Western blot were employed to examine the effects of hyperthermia (HT), HT + oridonin(Ori) or HT + radiotherapy (RT) on the proliferation and stemness of NPC cells. RNA sequencing was applied to gain differentially expressed genes upon hyperthermia. Gain-of-function and loss-of-function experiments were used to evaluate the effects of RNAi-mediated Cirbp silencing or Cirbp overexpression on the sensitivity or resistance of NPC cells and cancer stem-like cells to hyperthermia by CCK-8 assay, colony formation assay, tumorsphere formation assay and apoptosis assay, and in subcutaneous xenograft animal model. miRNA transient transfection and luciferase reporter assay were used to demonstrate that Cirbp is a direct target of miR-377-3p. The phosphorylation levels of key members in ATM-Chk2 and ATR-Chk1 pathways were detected by Western blot. RESULTS: Our results firstly revealed that hyperthermia significantly attenuated the stemness of NPC cells, while combination treatment of hyperthermia and oridonin dramatically increased the killing effect on NPC cells and cancer stem cell (CSC)­like population. Moreover, hyperthermia substantially improved the sensitivity of radiation­resistant NPC cells and CSC­like cells to radiotherapy. Hyperthermia noticeably suppressed Cirbp expression in NPC cells and xenograft tumor tissues. Furthermore, Cirbp inhibition remarkably boosted anti­tumor­killing activity of hyperthermia against NPC cells and CSC­like cells, whereas ectopic expression of Cirbp compromised tumor­killing effect of hyperthermia on these cells, indicating that Cirbp overexpression induces hyperthermia resistance. ThermomiR-377-3p improved the sensitivity of NPC cells and CSC­like cells to hyperthermia in vitro by directly suppressing Cirbp expression. More importantly, our results displayed the significantly boosted sensitization of tumor xenografts to hyperthermia by Cirbp silencing in vivo, but ectopic expression of Cirbp almost completely counteracted hyperthermia-mediated tumor cell-killing effect against tumor xenografts in vivo. Mechanistically, Cirbp silencing-induced inhibition of DNA damage repair by inactivating ATM-Chk2 and ATR-Chk1 pathways, decrease in stemness and increase in cell death contributed to hyperthermic sensitization; conversely, Cirbp overexpression-induced promotion of DNA damage repair, increase in stemness and decrease in cell apoptosis contributed to hyperthermia resistance. CONCLUSION: Taken together, these findings reveal a previously unrecognized role for Cirbp in positively regulating hyperthermia resistance and suggest that thermomiR-377-3p and its target gene Cirbp represent promising targets for therapeutic hyperthermia.

Efficacy of needling acupoints of Guanyuan (CV4), Sanyinjiao (SP6), Zusanli (ST36), Pishu (BL20), Shenshu (BL23), Zigong (EX-CA1) on expression of p38 mitogen-activated protein kinase in ovarian tissue in rats with premature ovarian failure induced by cyclophosphamide.

OBJECTIVE: To investigate the efficacy of needling acupoins of Guanyuan (CV4), Sanyinjiao (SP6), Zusanli (ST36), Pishu (BL20), Shenshu (BL23), Zigong (EX-CA1) on the expression of p38 mitogen-activated protein kinase (p38MAPK) in ovarian tissue in rats with premature ovarian failure induced by cyclophosphamide, and to study the underlying mechanism. METHODS: Forty specific pathogen free female Sprague-Dawley rats were randomly divided into the blank group, the model group, the acupuncture group, the Western Medicine group and the Western Medicine combined with acupuncture group. Except the blank group, the rest of the rats were given with cyclophosphamide for 14 d to establish premature ovarian failure model. No intervention was conducted in the blank group and model group; the acupuncture group was given with acupuncture daily; the Western Medicine group was given with estradiol valerate (0.09 mg/kg) by intragastrical gavage daily; the combination group was given with acupuncture combined with estradiol valerate (0.09 mg/kg) daily. Each group was intervened in continuously for 14 d. After the last treatment, the levels of estradiol (E2), follicle stimulating hormone (FSH) and luteinizing hormone (LH) were detected by enzyme-linked immunosorbent assay, then the ovarian tissue was dissected. Western blot was used to detect the expression of p38MAPK protein. RESULTS: Compared with the blank group, E2 in the serum of rats in the model group was significantly decreased (P < 0.05), FSH and LH were significantly increased (P < 0.05), and the expression of p38MAPK protein in the ovarian tissue of the rats was significantly increased (P < 0.05). Compared with the model group, E2 in the serum of the acupuncture group, Western Medicine group and the combination group were significantly increased (P < 0.05), FSH and LH levels were significantly decreased (P < 0.05), and the expression of p38MAPK protein in the ovarian tissue of the rats was significantly decreased (P < 0.05). However, there was no statistically significant difference between the Western Medicine group and the acupuncture group (P > 0.05). CONCLUSIONS: Acupuncture has the same effect as estrogen in interfering POF caused by cyclophosphamide, and its mechanism may be related to inhibiting the expression of p38MAPK protein in ovarian tissue and affecting the activation of p38MAPK signaling pathway.

Inhibitory effect of Zanthoxylum bungeanum seed oil on ovalbumin­induced lung inflammation in a murine model of asthma.

The present study aimed to investigate the therapeutic efficacy of Zanthoxylum bungeanum seed oil (Z. seed oil) to alleviate airway inflammation in asthmatic mice. The asthmatic mice were treated with vehicle, ovalbumin (OVA), or OVA + Z. seed oil (2 g/kg) for between 24 h and 14 days. Following treatment, inflammatory cell infiltration and pulmonary tissue damage were assessed by hematoxylin and eosin staining, and immunohistochemistry. The expression levels of pro­inflammatory cytokines, chemokines, adhesion molecules and mitogen activated protein kinase signaling proteins were measured by enzyme­linked immunosorbent assays, reverse transcription quantitative­polymerase chain reaction and western blot analysis. In asthmatic mice, administration of Z. seed oil attenuated lung tissue injury and airway remodeling, and inhibited the infiltration of leukocytes and eosinophils into the airway by reducing the expression levels of inflammatory cytokines and chemokines compared with OVA­treated mice (P<0.05). Z. seed oil also reduced the levels of inflammatory chemokine and adhesion molecules via downregulation of extracellular signal­regulated kinase and activation of c­JUN N­terminal kinase in the Z. seed­treated mice compared with OVA­treated mice (P<0.05). Thus, data from the present study indicates that Z. seed oil can suppress pulmonary inflammation and tissue injury during asthma, and suggests that it may be used to effectively treat allergen­induced asthma.

[Regulative effects of aquaporin 4 expression by rhein in rhubarb to intestinal epithelial cell line LoVo].

OBJECTIVE: To investigate the effects of rhein on regulating aquaporin4 (AQP4) to LoVo cells cultured with RPMI-1640 medium containing rhein. METHODS: LoVo cells were cultured with RPMI-1640 medium containing different concentration rhein for 24 hours and were cultured with RPMI-1640 containing rhein (20 mg/L) for different time. Four groups were assigned as LoVo cells were cultured respectively with RPMI-1640 medium containing different concentration rhein (40, 20, 10 mg/L and control group), while six groups were assigned as LoVo cells were cultured with RPMI-1640 medium containing rhein (20 mg/L) for different time (3, 6, 12, 24, 48 h and control group). The location of AQP4 protein in LoVo cells was definited by immuocytochemistry dying. Western blotting and semiquantitative RT-PCR were adopted to detect the relative expression of AQP4 protein and mRNA. RESULTS: AQP4 was located mainly in plasma membrane of LoVo cells while partly in cytoplasm. The relative expression of AQP4 protein and mRNA decreased with the increasing of rhein concentration; there was no significant difference of the relative expression of AQP4 in 10 mg/L group compared with that in control group, but it decreased significantly in 40 and 20 mg/L groups. The relative expression of AQP4 in 3 and 6 h groups was lower than that in control group but there was no statistical significance, however that in 12, 24, 48 h groups was lower significantly compared with that in control group. CONCLUSION: Rhein can inhibit the genetic transcription and the translation of AQP4 gene in LoVo cells, which demonstrates that the change of AQP4 expression regulated by rhein may be related to the cathartic effect of rhubarb.

[Novel exploration of cathartic pharmacology induced by rhubarb].

Rhubarb is well-known for its cathartic effect, and this cathartic effect, which is closely correlated with "whter" of traditional Chinese medicine (TCM), is brought into play in colon. Recent researches about the relation between formation and effects have identified that the anthraquinone glycosides with 1,8-dio-hydroxy and without hydroxyl in the 2, 3, 6, 7 location, such as emodin, rhein, chrysophanol, et al, can bring about fairly obvious effects of "Watery Diarrhea". Aquaporins (AQPs) are expressed abundantly in colonic epithelial cells, and the abnormal expression of AQPs can lead to the less absorption of water in colon and/or the more secretion of intestinal juice, which suggest that AQPs might be one kind of the effector molecules, which some drugs playing pharmacologic actions in colon depend on. This assumption provides a novel field of vision. Is this "Watery Diarrhea" effect induced by rhubarb concerned with the location alteration or the expression change of AQPs. We deduce that the regulative effects of AQPs by rhubarb in colon might provide a new pharmacologic explation about the cathartic effect through the exploration of TCM and Chinese herbal drugs, with TCM theory and the analysis of data about efficiency and pharmacologic researches of rhubarb and the researches of AQPs. This deduction might be used to reveal why rhubarb can bring about multi-efficiency.