The Role Of Sodium Tanshinone ⅡA Sulfonate Treats Pulmonary Arterial Hypertension

【Background】Pulmonary arterial hypertension (PAH) is a disease of pulmonary vascular, which is characterized by proliferation and remodeling in the vascular wall. Chronic hypoxia is thought to be an important factor contributing to the development of PAH. Extensively researches showed that intracellular Ca2+ level plays an essential role in the process of distal pulmonary artery constriction and proliferation.What's more, we and other groups have previously found that CH elevated basal [Ca2+]i in PASMCs due in large part to enhanced store-operated Ca2+ entry (SOCE) through store-operated Ca2+ channels (SOCC). SOCC are thought to be composed of canonical transient receptor potential (TRPC) protein family of which TRPC1 and TRPC6 were found to be selectively upregulated by chronic hypoxia in both mRNA and protein level in our previous work.At the same time, hypoxia can increase expression of various pro- inflammatory cytokines around pulmonary vessel and lung, such as TNF-α,IL-1βand IL-6. Overexpression of these cytokines plays an important role in the phenotype of vascular wall cells, including cell proliferation and matrix protein expression, as well as response to vasoconstrictor and the vasodilator vascular changes. In particular, IL-6 is directly involved in the process of COPD and pulmonary hypertension.Recent studies showed that sodium tanshinoneⅡA sulfonate (STS), a water-soluble derivative of tanshinoneⅡA which is an active component of Danshen, had protective effects on chronic hypoxic pulmonary hypertension including lowering pulmonary artery pressure (PAP) and decreasing the extent of pulmonary artery thickness and right ventricular hypertrophy. However, the underlying mechanisms remain unclear.【Objective】Chronic hypoxic pulmonary hypertension model was established to: measure the expression of TRPC1,6 in PASMCs and pulmonary arteries from CHPH rat; study IL-6 gene expression in lung tissue and IL-6 concentration in blood serum ; estimate the effect of STS on basal [Ca²⁺]i and SOCE induced by chronic hypoxia in PASMCs and to make sure whether STS has any regulating effect on TRPC1,6 and IL-6, aiming to obtain evidence for further discussion of the CHPH mechanism.【Methods】Rat distal PASMCs were isolated and cultured by collagenase digestion and characterized by morphological features and specific immunofluorescenceα-actin staining. Rat distal PASMCs [Ca²⁺]i were measured using Incyte intracellular Ca²⁺ concentration system. PASMCs were treated by STS of several different concentrations. Both mRNA and protein levels of TRPC1,6 were detected. CHPH rats were obtained using a hypoxic (10%O2) chamber by keeping them in the chamber for 21 days.Animals were treated by STS (10 or 30 mg/kg body weight/day) or equal volume of normal saline intraperitoneal injection before exposed to hypoxia for 3 weeks. Normoxic control animals were put under room air condition and handled accordingly.Pulmonary hypertension parameters were assessed. Pathological and immunofluorescenceα-actin staining slices were observed under light microscope and fluorescence laser scanning confocal microscope separately. PASMCs were isolated from CHPH rats or normal rats. mRNA and protein levels of TRPC1,6 were detected by real-time PCR and western blot. IL-6 gene expression in lung tissue and IL-6 concentration in blood serum were measured by real-time PCR and ELISA.【Results】It was shown that hypoxia increased TRPC1,6 expression in both mRNA and protein levels, triggered Ca²⁺ release from SR and subsequently led to enhanced SOCE in PASMCs. STS inhibited the mRNA and protein upregulation of TRPC1,6 induced by hypoxia in PASMCs. In addition, PASMCs cultured under hypoxic conditions and treated with STS had a weakened response to restoration of extracellular [Ca²⁺]i。MPAP,RVSP and RV/(LV+S) were all increased in rats exposed to chronic hypoxia. The pulmonary arteries isolated from CHPH rats were thicker than that from normal rats.On the contrary, the rats under normoxic condition treated with STS were not altered in mPAP,PVSP,RV/(LV+ S) and vascular appearences. Daily application of STS before exposed to hypoxia for 21 days significantly decreased mPAP,PVSP and RV/(LV+ S) compared with those of CHPH rats. Besides, hypoxia increased TRPC1 and TRPC6 mRNA expression in vivo. IL-6 gene expression in lung tissue and IL-6 concentration in blood serum also increased under chronic hypoxia. Treated with STS significantly suppressed these increasing in chronic hypoxic rats.【Conclusion】1. Chronic hypoxia increased mRNA and protein expression of TRPC1,6 in PASMCs and increased SOCE.2. STS has protective inhibiting effects on chronic hypoxia induced increasing in mRNA and protein levels of TRPC1,6 and SOCE in PASMCs.3. STS has protective inhibiting effects on chronic hypoxia induced increasing in mRNA and protein levels of TRPC1,6 and SOCE in distal pulmonary artery isolated from chronic hypoxic rats.4. STS inhibited hypoxia-induced increasing of IL-6 gene expression in lung tissue. 5. All the above conclusion indicates that STS may have the potential to become a new effective and possible method for the clinical diagnosis and therapy of CHPH.