4-Terpineol attenuates pulmonary vascular remodeling via suppressing PI3K/Akt signaling pathway in hypoxia-induced pulmonary hypertension rats.

The hyperproliferation of pulmonary arterial smooth muscle cells (PASMCs) plays a pivotal role in pulmonary arterial remodeling (PAR) of hypoxia-induced pulmonary hypertension (HPH). 4-Terpineol is a constituent of Myristic fragrant volatile oil in Santan Sumtang. Our previous study found that Myristic fragrant volatile oil alleviated PAR in HPH rats. However, the effect and pharmacological mechanism of 4-terpineol in HPH rats remain unexplored. Male Sprague-Dawley rats were exposed to hypobaric hypoxia chamber (simulated altitudes of 4500 m) for 4 weeks to establish an HPH model in this study. During this period, rats were intragastrically administrated with 4-terpineol or sildenafil. After that, hemodynamic indexes and histopathological changes were assessed. Moreover, a hypoxia-induced cellular proliferative model was established by exposing PASMCs to 3% O2. PASMCs were pretreated with 4-terpineol or LY294002 to explore whether 4-terpineol targeted PI3K/Akt signaling pathway. The PI3K/Akt-related proteins expression was also accessed in lung tissues of HPH rats. We found that 4-terpineol attenuated mPAP and PAR in HPH rats. Then, cellular experiments showed 4-terpineol inhibited hypoxia-induced PASMCs proliferation via down-regulating PI3K/Akt expression. Furthermore, 4-terpineol decreased the p-Akt, p-p38, and p-GSK-3ß protein expression, as well as reduced the PCNA, CDK4, Bcl-2 and Cyclin D1 protein levels, while increasing levels of cleaved caspase 3, Bax, and p27kip1in lung tissues of HPH rats. Our results suggested that 4-terpineol mitigated PAR in HPH rats by inhibiting the proliferation and inducing apoptosis of PASMCs through suppression of the PI3K/Akt-related signaling pathway.

Kaempferol ameliorates pulmonary vascular remodeling in chronic hypoxia-induced pulmonary hypertension rats via regulating Akt-GSK3ß-cyclin axis.

Excessive proliferation of pulmonary artery smooth muscle cells (PASMCs) is considered a major contributor to elevated pulmonary vascular resistance and a key mechanism of vascular remodeling in hypoxia-induced pulmonary hypertension (HPH). Kaempferol is a natural flavonoid compound and can be derived from numerous common medicinal herbs and vegetables, which exhibit antiproliferative and proapoptotic properties, however, the effects of kaempferol on vascular remodeling in HPH remain unexplored. In this study, SD rats were placed in a hypobaric hypoxia chamber for four weeks to establish a pulmonary hypertension model and given either kaempferol or sildenafil (an inhibitor of PDE-5) during days 1-28, after which the hemodynamic parameter and pulmonary vascular morphometry were assessed. Furthermore, primary rat PASMCs were exposed to hypoxic conditions to generate a cell proliferation model, then incubated with either kaempferol or LY294002 (an inhibitor of PI3K). Immunoblotting and real-time quantitative PCR assessed the protein and mRNA expression levels in HPH rat lungs and PASMCs. We found that kaempferol reduced pulmonary artery pressure and pulmonary vascular remodeling, and alleviated right ventricular hypertrophy in HPH rats. The mechanistic analysis demonstrated that kaempferol reduced the protein levels of phosphorylation of Akt and GSK3ß, leading to decreased expression of pro-proliferation (CDK2, CDK4, Cyclin D1, and PCNA) and anti-apoptotic related proteins (Bcl-2) and increased expression of pro-apoptosis proteins (Bax and cleaved caspase 3). These results collectively demonstrate that kaempferol ameliorates HPH in rats by inhibiting PASMC proliferation and pro-apoptosis via modulation of the Akt/GSK3ß/CyclinD axis.

Analysis of chemical constituents in the extract and rat serum from the chloroform extract of Oxytropis falcata Bunge by HPLC-MS.

Analysis of the constituents of the chloroform extract of Oxytropis falcata Bunge (CEOF), a traditional Tibetan medicine, in rat's serum after oral administration, has been performed by HPLC-MS. We have identified 10 compounds in CEOF and 11 bioactive ingredients from rat's serum after given CEOF. Six bioactive ingredients from rat's serum are matched with original form of the compounds of CEOF. Other five bioactive ingredients were seemed to be respectively metabolites. HPLC-MS is rapid, sensitive method and suitable for identification of bioactive components absorbed into blood of CEOF providing information for further research of pharmacological mechanism.

Srolo Bzhtang reduces inflammation and vascular remodeling via suppression of the MAPK/NF-κB signaling pathway in rats with pulmonary arterial hypertension.

ETHNOPHARMACOLOGICAL RELEVANCE: Srolo Bzhtang (SBT), which consists of Solms-laubachia eurycarpa, Bergenia purpurascens, Glycyrrhiza uralensis, and lac secreted by Laccifer lacca Kerr (Lacciferidae Cockerell), is a well-known traditional Tibetan medicinal formula and was documented to cure "lung-heat" syndrome by eliminating "chiba" in the ancient Tibetan medical work Four Medical Tantras (Rgyud bzhi). Clinically, it is a therapy for pulmonary inflammatory disorders, such as pneumonia, chronic bronchitis, and chronic obstructive pulmonary disease. However, whether and how SBT participates in pulmonary arterial hypertension (PAH) is still unclear. AIM OF THE STUDY: We aimed to determine the role of SBT in attenuating pulmonary arterial pressure and vascular remodeling caused by monocrotaline (MCT) and hypoxia. To elucidate the potential mechanism underlying SBT-mediated PAH, we investigated the changes in inflammatory cytokines and mitogen-activated protein kinase (MAPK)/nuclear factor-kappa B (NF-κB) signaling pathway. MATERIALS AND METHODS: MCT- and hypoxia-induced PAH rat models were used. After administering SBT for four weeks, the rats were tested for hemodynamic indicators, hematological changes, pulmonary arterial morphological changes, and the levels of interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α in serum and lung tissues. Protein expression of the MAPK/NF-κB signaling pathway was determined using western blotting. RESULTS: SBT reduced pulmonary arterial pressure, vascular remodeling, and the levels of inflammatory cytokines induced by MCT and hypoxia in rats. Furthermore, SBT significantly suppressed the MAPK/NF-κB signaling pathway. CONCLUSIONS: To our knowledge, this is the first study to demonstrate that SBT alleviates MCT- and hypoxia-induced PAH in rats, which is related to its anti-inflammatory actions involving inhibition of the MAPK/NF-κB signaling pathway.

Mechanism of Traditional Tibetan Medicine Grubthobrildkr Alleviated Gastric Ulcer Induced by Acute Systemic Hypoxia in Rats.

Objective: This study was aimed at investigating the potential mechanism of Grubthobrildkr (GTB) on systemic hypoxia-induced gastric ulcers in rats and at detecting the chemical profile of GTB. Methods: Male Sprague-Dawley rats were separated into control, hypoxia, hypoxia+omeprazole, and hypoxia+GTBs (0.25, 0.5, and 1.0 g·kg-1·d-1) groups. Systemic hypoxia was created in a hypobaric chamber to simulate 5000 m high altitude by adjusting the inner pressure and oxygen content for 6 days. After that, the ulcer index, pH, and volume of gastric juice were assessed. The levels of endothelin-1 (ET-1), gastrin (GAS), motilin (MTL), phospholipase A2 (PLA2), and prostaglandin E2 (PGE2) were detected by ELISA. The expression level of hydrogen potassium ATPase (H+-K+-ATPase), cyclooxygenase-1 (COX-1), and cyclooxygenase-2 (COX-2) was tested by western blotting. Chemical profile of GTB was revealed by UHPLC-Q-exactive hybrid quadrupole-orbitrap mass (UHPLC-Q-Orbitrap MS). Results: GTB decreased the ulcer index in rats under hypoxia for six days, which was related to increased pH and volume of gastric juice, enhanced MTL and PGE2 levels, and decreased ET-1 and PLA2 levels of gastric mucosa. Furthermore, GTB decreased the level of H+-K+-ATPase and COX-2 while increased COX-1 levels in gastric mucosal tissue. 44 constituents were identified by UHPLC-Q-Orbitrap MS in GTB. Conclusion: GTB exerted a gastroprotective effect to alleviate gastric ulceration induced by acute systemic hypoxia in rats. The effect of GTB increasing the volume and pH of gastric juice in rats under acute systemic hypoxia could be regulated by gastrointestinal hormones, including MTL and ET-1. Mechanically, gastrointestinal protection of GTB was based on inhibition of the protons pumping H+-K+-ATPase and regulation of prostaglandin family in rats.

The Mechanism of Volatile Oil of Rhodiola tangutica against Hypoxia-Induced Pulmonary Hypertension in Rats Based on RAS Pathway.

Materials and Methods: Seventy-five male Sprague-Dawley (SD) rats were separated into control (Ctr), hypoxia (Hyp), and Hyp+VORA treatment (100 mg/kg/d, 80 mg/kg/d, and 40 mg/kg/d) groups in random. To achieve the chronic hypoxia condition, rats were kept inside the hypobaric chamber with automatically adjusted inner pressure as well as oxygen content equal to those of 4500 m in altitude for 4 continuous weeks. After 4 weeks, the rats' physiological parameters were determined (mean pulmonary artery pressure (mPAP); right ventricular hypertrophy index (RVHI)). Based on hematoxylin and eosin (HE) staining and transmission electron microscope (TEM), morphological features of their lung tissues were also analyzed. Proliferation of pulmonary arterial smooth muscle cells (PASMCs) was detected by MTS Cell Proliferation Colorimetric assay. The levels of glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD) in PASMCs were detected through corresponding kits, respectively. The protein levels in PASMCs and HPH rats were evaluated by Western blot (WB). Chemical components of VORA were detected through gas chromatography-mass spectrometer (GC-MS). Results: After induced by hypoxia for 4 weeks, the mPAP and RVHI levels were increased significantly in hypoxia group in contrast to the Ctr group, indicating the establishment of HPH rat model. The subsequent administration of VORA decreased the mPAP and RVHI level. The vascular wall thickness and lumen size were also decreased after treated by VORA compared with Hyp group. Meanwhile, VORA suppressed the proliferation and oxidant stress in PASMCs. Therefore, the effect of VORA on decreasing vascular wall thickening and lumen size could be related to its antiproliferation effect on PASMCs. In addition, compared to the Hyp group, VORA downregulated the ACE, AngII, and AT1R protein expressions but increased ACE2 and MAS protein expressions (P < 0.05). A total of 48 constituents in VORA were identified by GC-MS in comparison with reference standards as well as the reference pieces of literatures. Conclusions: HPH rat model as established based on the significant increased mPAP and RVHI. VORA presented a significant antihypoxia function plus an inhibiting effect on PASMC proliferation induced by hypoxia. Moreover, VORA treatment inhibited oxidative stress among PASMCs. With regard to the mechanism, VORA reduced ACE, AngII, and AT1R protein expressions but increased ACE2 and MAS protein expressions. There were 48 constituents in VORA identified by GC-MS.

Tsantan Sumtang Restored Right Ventricular Function in Chronic Hypoxia-Induced Pulmonary Hypertension Rats.

Background: Tsantan Sumtang originated from Four Tantras, which consisted of Choerospondias axillaris (Roxb.) B. L. Burtt and A. W. Hill, Santalum album L., and Myristica fragrans Houtt. The three herbs are in ratio 1:1:1. This medication is widely used for cardiovascular diseases. Aims: The purpose of this study was to explore the effect of Tsantan Sumtang on right ventricular (RV) function in hypoxia-induced pulmonary hypertension (HPH) rats and investigate the underlying mechanism. Methods: Sixty male Sprague-Dawley (SD) rats were divided into control, hypoxia, and hypoxia + Tsantan Sumtang (1.0, 1.25, and 1.5 g•kg-1•d-1) groups. Chronic hypoxia was induced by putting the rats inside a hypobaric chamber for four weeks and adjusting the inner pressure and oxygen content to match an altitude of 4500 m. Echocardiography was used to assess RV function and right ventricular-pulmonary arterial (RV-PA) coupling. The physiological parameters of the animals were also evaluated. Morphological characteristics of RV were assessed by hematoxylin and eosin (H&E) staining and TEM. Masson's trichrome staining, immunohistochemical staining, western blotting, and TUNEL assay were used to assess fibrosis and apoptosis levels. The antioxidant and anti-apoptosis properties of Tsantan Sumtang were also evaluated. The effect of Tsantan Sumtang on ROCK signaling pathway was evaluated using real-time quantitative PCR and western blotting. Results: We established an HPH rat model as indicated by the significant increases in the physiological parameters of the rats. Tsantan Sumtang showed a significant cardiac-protective function and an improved effect on RV-PA coupling. Moreover, Tsantan Sumtang treatment inhibited fibrosis and alleviated apoptosis and oxidative stress in RV. In terms of mechanism, Tsantan Sumtang reduced the expression of ROCK (ROCK1, ROCK2) in RV, inhibited cardiac remodeling-related transcription factors (NFATc3, P-STAT3), and regulated apoptosis-related proteins. Conclusion: Tsantan Sumtang was able to restore RV function, improve RV-PA coupling, recover hemodynamic and hematological indexes, and protect RV against structural maladaptive remodeling in the HPH rats. These findings demonstrated that Tsantan Sumtang protects the function of RV in HPH rats. The antioxidant and anti-apoptosis properties of Tsantan Sumtang may be responsible for inhibiting the ROCK signaling pathway.