Establishment and evaluation of rat model of pulmonary arterial hypertension with heart-kidney yang deficiency syndrome

A rat model of pulmonary arterial hypertension with heart-kidney yang deficiency syndrome was established using monocrotaline(MCT)plus hydrocortisone. The study aimed to investigate the correlation between traditional Chinese medicine(TCM)syndrome and the pathological progression of pulmonary arterial hypertension to provide experimental evidence for TCM therapeutic research.

Thirty Sprague-Dawley rats were randomly divided into the blank control group, pulmonary arterial hypertension group, and pulmonary arterial hypertension with heart-kidney yang deficiency syndrome group using the random number table method, with 10 rats in each group. Rats in the blank control group were injected with equal-volume normal saline. The pulmonary arterial hypertension group received an intraperitoneal injection of 60 mg/kg MCT once daily for 28 consecutive days to induce pulmonary arterial hypertension. On the basis of MCT administration, rats in the pulmonary arterial hypertension with heart-kidney yang deficiency syndrome group were subcutaneously injected with 20 mg/kg hydrocortisone once daily for 14 consecutive days starting from the 15th day of modeling. Twenty-eight days after modeling, the syndromic characteristics of the kidney-yang deficiency of rats were scored based on vital signs (rectal temperature), general appearance (body mass, auricle color, nail color, tail color, ocular state and color, tongue color, tongue fluid, and hair sparsity and hair luster), and behavioral performances (duration of hunching and curling, and duration of huddling). An enzyme-linked immunosorbent assay (ELISA)was used to detect blood and urine biochemical indicators including blood urea nitrogen(BUN), serum creatinine (Scr), urine total protein (U-TP), and urine creatinine (Ucr) for renal function assessment. ELISA was also applied to determine triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH), adrenocorticotropic hormone (ACTH), corticosterone (CORT), estradiol (E₂), testosterone (T), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), and cAMP/cGMP ratio to evaluate the manifestations of kidney yang deficiency. A small animal ultrasound imaging system was adopted to measure right ventricular anterior wall thickness (RVWT), main pulmonary artery diamete(MPAD), anteroposterior diameter, left-right diameter and superoinferior diameter of right ventricle(RV), peak systolic velocity of main pulmonary artery(Vpa), tricuspid annular systolic peak velocity (s′), tricuspid annular plane systolic excursion (TAPSE) and tricuspid regurgitation. Combined with right ventricular systolic pressure (RVSP) detected by a multi-channel physiological recorder, cardiac function of rats was assessed. Blood gas analyzer was used to detect potential of hydrogen(pH), partial pressure of carbon dioxide (PCO₂), partial pressure of oxyge (PO₂), oxygen saturation(SaO₂), actual bicarbonate (HCO₃^-), total carbon dioxide(TCO₂), arterial-alveolar oxygen partial pressure ratio (a/A) and lactic acid (Lac). ELISA was performed to detect vasoactive marker endothelin-1(ET-1)and myocardial functional marker N-terminal pro-B-type natriuretic peptide(NT-proBNP). Organ coefficients and right ventricular hypertrophy index(RVHI)were calculated to evaluate the degree of myocardial injury. HE staining was performed to observe the pathological morphology of myocardial and pulmonary arterial tissues. Meanwhile, the percentage of vessel wall thickness to external diameter(WT) and the percentage of vessel wall area to total cross-sectional area (WA) of pulmonary arteries were measured to quantitatively evaluate the pathological structural changes of pulmonary arteries. Transmission electron microscope was used to assess cellular morphological changes.

Compared with the blank control group, rats in the pulmonary arterial hypertension with heart-kidney yang deficiency syndrome group exhibited decreased rectal temperature, visible distinct auricular blood vessels, increased tongue fluid, and reduced body weight starting from day 14 after modeling, and developed moderate heart-kidney yang deficiency on day 28. The score of the syndromic characteristics of the kidney-yang deficiency increased(P<0.01). In terms of renal function, compared with the blank control group and pulmonary arterial hypertension group, BUN, Scr, and U-TP were elevated, whereas Ucr was reduced in the pulmonary arterial hypertension with heart-kidney yang deficiency syndrome group(P<0.01). For kidney yang deficiency related indicators, compared with the blank control group and pulmonary arterial hypertension group, the levels of T3, T4, TSH, ACTH, CORT, T, and cGMP were decreased, while E₂, cAMP, and cAMP/cGMP ratio were elevated in the pulmonary arterial hypertension with heart-kidney yang deficiency syndrome group (P<0.01). In terms of cardiac function, both in the pulmonary arterial hypertension group and pulmonary arterial hypertension with heart-kidney yang deficiency syndrome group, the anteroposterior, left-right, and superior-inferior diameters of the RV, together with RVWT, MPAD, RVSP, RVHI, ET-1, and NT-proBNP levels, were higher, while Vpa, s′, and TAPSE were lower than those in the blank control group (P<0.01). Blood gas analysis showed that, compared with the blank control group, the pH value, PO₂, and SaO₂ in abdominal aortic blood decreased in both model groups, while Lac increased. Additionally, Lac was further higher in the pulmonary arterial hypertension with heart-kidney yang deficiency syndrome group than that in the pulmonary arterial hypertension group (P< 0.01). Organ coefficients revealed that the cardiac, pulmonary, and renal coefficients of the two model groups were increased compared with the blank control group(P<0.01). HE staining and transmission electron microscopy results revealed that pathological damage of myocardium, pulmonary arteries and lung tissues, pulmonary arterial remodeling as well as mitochondrial damage were observed in both model groups. Besides, both WT and WA levels were increased (P<0.01).

Combined administration of MCT and hydrocortisone can alter renal function and kidney-yang deficiency-related phenotypic indicators in rats, successfully establishing a disease-syndrome combined model of pulmonary arterial hypertension complicated with heart-kidney yang deficiency syndrome. This model simultaneously presents pathological injuries of pulmonary arterial hypertension and typical manifestations of kidney-yang deficiency, which provides a stable and reliable experimental platform for further exploring the pathological mechanism of heart-kidney yang deficiency syndrome and evaluating the efficacy of TCM against pulmonary arterial hypertension.