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Protective Effect of Lonicerae Flos Aqueous Extracts on a Pressure Overload-induced Heart Failure Model

Objectives: Lonicerae flos (LF), a dried flower part of Lonicera japonica Thunb., has been widely used in Korean medicine as anti-inflammatory and antioxidative agent. The purpose of this study was to determine the cardioprotective effects of LF, through potential antioxidant effects, on the pressur...

because of its high prevalence, mortality, morbidity, and cost of care 2 . HF can be caused by disease such as ischemic heart disease, hypertension, valve or myocardium structural defects and arrhythmias 3 .
The classic clinical syndrome of HF is characterized by fluid retention leading to pulmonary congestion and peripheral edema and by low cardiac output, that combined may cause a severe limitation of exercise capacity 4 . Conventional treatment of HF involves oxygen therapy, diet, angiotensin-convertising enzyme inhibitors (ACEI), diuretics, anti platelets or beta-blockers, etc 5 . Despite existing therapies for HF, the current 1-year mortality rate after diagnosis of symptomatic HF remains at 25% to 40% 6 . Accordingly, there are a few efforts in domestic and foreign to find other treatment for HF. However, there are little research on the mechanisms how these treatment can control heart damage and on optimal stage to use.
Lonicerae Flos (Lonicerae japonica Thunb, LF), also called Jinyinhua (金銀花) is a widely used herb prescribed in many Korean herbal formulas against gastroesophageal reflux disease, lung disease, carcinoma, etc. Pharmacological studies show that LF possessed various actions, such as anti-inflammatory, antiviral, antidiabetic, antiallergic, and antioxidants [7][8][9] . Because LF has beneficial effect on oxidative damages and cytoprotective action, we hypothesized that LF could also have positive effects in PO induced HF by antioxidant mechanism. And therefore there are no studies about effects of LF on heart.
So this study performed for the cardioprotective effects of LF on the PO-induced HF by TAC in mice, with antioxidant effects. The changes on the mortality, body weight, heart weights, indicators of antioxidant defense system and histopathology of heart were investigated.

Animals and husbandry
One hundred 6-week old male SPF/VAF inbred C57BL/6NCrljOri [C57BL/6] mice (OrientBio, Seungnam, Korea) were used after acclimatization for 7 days. In this study, forty transverse aortic constriction (TAC) and eight sham operated mice were sorted out based on the weights at two weeks after TAC surgery (mean weights: 21.60±0.74 g), and divided into six groups (Table 1).

Preparations and administration of test materials
Three different dosages, 50, 25 and 12.5 mg of LF were directly dissolved in distilled water 1 ml, and administered in a volume of 10 ml/kg as equivalence to 500, 250 and 125 mg/kg using gastric gavages by oral, once a day for 14 days from 14 days after TAC operation, and resveratrol also dissolved in distilled water and orally administered at a dose level of 10 mg/kg, in a volume of 10 ml/kg. In sham and TAC control mice, instead of test materials, same volumes of distilled water as vehicle were administered once a day for 14 days from 14 days by oral after TAC surgery, respectively (Table 1)

TAC operation
The mice were intubated with tubing by oral and ventilated at 0.2 mL tidal air volume. A 3 mm center thoracotomy was treated. The transverse aortic arch was tied (7-0 Prolene) between the innominate artery and left common carotid artery using an overlying 28-gauge needle. After that, the needle was eliminated, leaving a detached region of stenosis. The chest was closed. The pneumothorax was clearly evacuated. Some mice underwent a sham operation in which the aortic arch was visualized but not ligated.

Mortalities
Based on the Functional Observational Battery (FOB), all abnormal mortalities were represented before and after administration twice a day during the first day of administration to the last day of drug treatment.

Body weight measurements
Changes in weight of body were evaluated at one day before initial resveratrol or LF administration, the day of first administration, 1, 7, 13 and 14 days after initial test material administration with an automatic electronic balance (Precisa Instrument, Dietikon, Switzland).

Heart weight measurements
At sacrifice, the heart weights in all survived animals were evaluated at gram levels, separately with an automatic electronic balance (Precisa Instrument, Dietikon, Switzland).
To reduce individual differences, the relative weights (percentage of body weights) were calculated using weight of body at sacrifice and absolute weight of heart as following equation : (absolute weights of heart/weight of body at sacrifice)×100.

Analysis of Antioxidant defence system
The total protein amount for the determination of enzyme antioxidant, GSH, and lipid peroxidation assays was refered to the Lowry protein assay.

Histological process
Approximately corresponding regions of individual hearts, taken from survived mice at sacrifice (five in each group), were obliquely cut based upon the ventricles as one part of each heart. To investigate more specific changes, total thicknesses of endocardium to pericardium (μm/heart) in left ventricle (LV), mean numbers of lytic necrotic muscle fibers of heart (myofibers/1000 myofibers) and diameters of muscle fibers of heart (μm/fiber) were evaluated using a computer-assisted progrom (iSolution FL ver 9.1, IMT i-solution INC., Quebec, Canade) for image anlysis under H&E stain with the mean percentages of perivascular and interstitial collagen fiber occupied parts in LV (%/mm 2 of field) under Sirius red (SR) stain.

Statistical analyses
All Data were displayed as mean±standard deviations (SD). Multiple comparison tests for the different dose groups were prosecuted. Variance homogeneity was examined using the Levene test. If the Levene test exhibited no significant deviations from variance homogeneity, the obtained data were analyzed by one way ANOVA test and least-significant differences (LSD) multi-comparison test to determine which pairs of group comparison were clearly different. When great deviations from variance homogeneity were detected at Levene test, Kruskal-Wallis H test, a non-parametric comparison test, was done. If an important difference was detected in the Kruskal-Wallis H test, the Mann-Whitney U (MW) test was conducted to determine the specific pairs of group comparison that are considerably different. Statistical analyses were conducted using SPSS for Windows (Release 14.0K, IBM SPSS Inc., Armonk, NY, USA). Ⅲ . Results

Survivability
Unscheduled mortalities were not demonstrated in sham-operated control mice throughout the whole experimental periods, but three mice (3/8; 37.5%) in TAC control died within 14 days of experimental periods; each of one mouse died at 3, 8 and 10 days after initial administration, respectively. In addition, one mouse (1/8; 12.5%) administered with resveratrol 10 mg/kg were died at 6 days after initial administration, and also one mouse (1/8; 12.5%) at 7 days after initial administration in LF 250 mg/kg treated mice. Two mice (2/8; 25.0%) among LF 125 mg/kg treated mice died; each of one at 10 and 11 days after initial administration. However, no unscheduled mortalities were demonstrated in LF 500 mg/kg treated mice throughout the whole 14 days of experimental periods in this experiment (Table 2).  Values are expressed as Mean±S.D. of variable numbers of the animals according to mortality (see Table 2), g. TAC : transverse aortic constriction, LF : Lonicerae flos aqueous extracts and in resveratrol 10 mg/kg treated mice (Fig. 1, 2). Fig. 1. The representative gross images of heart, selected from sham or TAC operated mice. In addition, the heart CAT activities in resveratrol 10 mg/kg treated mice were also significantly (p<0.01) increased as compared to TAC control mice (   Fig 3).

2) Changes on the mean numbers of lytic necrotic cardiac muscle fibers in left ventricle
In  Fig 3).

3) Changes on the diameters of cardiac muscle fibers in left ventricle
In TAC control mice, markedly (p<0.01) increased mean diameters of cardiac muscle fibers were demonstrated. However, 14 days sustained oral treatment of resveratrol 10 mg/kg, LF 500, 250 and 125 mg/kg in TAC-operated mice significantly (p<0.01) inhibited the TAC related increases of the diameters of muscle fibers in heart, respectively.

4) Changes on the mean percentages of perivascular collagen fiber occupied parts in left ventricle
The mean percentages of perivascular collagen fiber occupied parts in LV were significantly (p<0.01) increased in TAC control mice. These TAC-related perivascular fibrosis were dose-dependently and markedly (p<0.01 or p<0.05) restored by LF treatment. Furthermore, the mean percentages of perivascular collagen fiber occupied parts in LV were significantly (p<0.01) decreased in resveratrol 10 mg/kg treated group ( Table 5, Fig 3).

5) Changes on the mean percentages of interstitial collagen fiber occupied parts in left ventricle
The mean percentages of interstitial collagen fiber occupied parts in LV were significantly and markedly (p<0.01) increased in TAC control mice. These TAC-related interstitial fibrosis were dose-dependently and markedly (p<0.01 or p<0.05) restored by LF treatment. Furthermore, the mean percentages of interstitial collagen fiber occupied parts in LV were markedly (p<0.01) decreased in resveratrol 10 mg/kg treated group (

Ⅳ. Discussion
Resveratrol, a polyphenol with antioxidant, anti-apoptotic, anti-inflammatory and metabolic properties was recently identified as an activator of energy metabolism 10 . In the heart, resveratrol decreases pressure overload-induced hypertrophy and contractile dysfunction 11 . Because of its positive effects on vascular function and energy metabolism 12 , we used it as a reference drug.
In the present study, the cardioprotective effects of LF, a dried flower part of Lonicerae japonica Thunb. which has been widely used as anti-inflammatory and antioxidative agent in Korean medicine 7,8,13 , were observed on the PO HF induced by TAC in C57BL/6 mice 14-16 through possible antioxidant effects. In this experiment, resveratrol 10 mg/kg, LF 500, 250 and 125 mg/kg were administered in mouth, once a day during 14 days, from 14 days after TAC surgery. And the changes in the mortality, body weights, heart weights, heart histopathology, and antioxidant defense system of the heart were analyzed 14 . The heart MDA contents were observed for lipid peroxidation, GSH contents, CAT and SOD activities were observed for antioxidant effects 17,18 , and also the overall thicknesses of LV from endocardium to pericardium, mean numbers of diameters of cardiac muscle fibers and lytic necrotic cardiac muscle fibers were calculated for histomorphometrical analysis with the mean percentages of interstitial and perivascular collagen fiber occupied parts in LV, respectively 14,16 .
The dosages of LF were selected according to the results of previous animal studies 7,8 . In this experiment, the results were compared to resveratrol, in which influential cardioprotective effects on TAC mice model were confirmed at a dose level of 10 mg/kg through antioxidant effects, as reference in this experiment 14 .
Corresponding to the previous studies 14 22 . Among them, it is also reported that chlorogenic acid are well distributed into the heart after oral administration in rats 30 . For more detail mechanism studies, screening of the biological active compounds in LF is necessary.

Ⅴ. Conclusion
The results obtained in the present study suggest that oral administration of LF favorably alleviates PO-induced HF by TAC, through enhancement of heart antioxidant defense system, at the very least in a condition of this experiment. The overall effects of LF 250 mg/kg were similar to those of resveratrol 10 mg/kg. However, LF is usually prescribed in mixed form with various herbal medicine in clinical field, and optimal stage of use in heart damage is yet unknown. Therefore, more diverse but detail mechanism studies of LF should be conducted in future with functional restoration in heart damages.