The purpose of this study was to determine whether a novel mixture consisting of Panax ginseng (PG) and Salvia miltiorrhiza bunge (SM) could attenuate the deleterious effects of lipopolysaccharides (LPS) on myocardial function. Twenty-four male rats were weight-matched and assigned into three groups: 1) placebo control (Con- trol), 2) placebo with acute LPS challenge (LPS; 0.5 mg LPS/kg), and 3) a GD mixture (PG: 150 mg/kg; SM: 150 mg/kg; total dose: 300 mg/kg for a week; 0.5 mg LPS/kg) with LPS challenge (GD+LPS). Animals were treated with either placebo or the GD mixture in accordance with experimental design for another 7 days. On the ex- periment day, the blood samples were collected at base- line, 2 h and 4 h after LPS injection, and the echocar- diographic data were measured at baseline and 3.5 h after LPS injection. One-week GD mixture supplemen- tation significantly preserved declines in left ventricular stroke volume (LVSV) and left ventricular ejection frac- tion (LVEF) after LPS treatment (P < 0.05). The GD mixture significantly diminished LPS-induced secretion of interleukin-6 (IL-6) (P < 0.05) but not that of tumor necrosis factor-alpha (TNF-α), and the LVEF inversely correlated to the increase in circulating IL-6 after LPS challenge (r = -0.513, P = 0.012). The GD mixture had slight but not significant effects on attenuating the LPS- induced in myocardial glyceraldehyde-3-Phosphate de- hydrogenase (GAPDH) expression after LPS challenge. In conclusion, one-week GD mixture supple-mentation significantly preserved left ventricular systolic function after LPS challenge. Moreover, the GD mixture diminished the LPS-induced increase in IL-6 but had no effects on LPS-induced increases in TNF-α.

Key Words: danshen, endotoxin, ginseng, IL-6, systolic dysfunction

Introduction

Sepsis-induced myocardial dysfunction, such as sig- nificant declines in left ventricular stroke volume and ejection fraction, is one of the primary septic shock symptoms (6, 8, 20). Moreover, the presence of cardio- vascular dysfunction has been reported to be highly associated with increased mortality rate in septic patients without cardiovascular impairment (26). The existing evidence suggests that the cardiovascular dysfunction induced by server sepsis possibly account for the increased mortality in patients with severe sepsis. Therefore, de- veloping effective preventive or therapeutic strategies for attenuating cardiovascular dysfunction is critical to the patients with sepsis.

Lipopolysaccharide (LPS), a glycolipid endo- toxin found in the outer membrane of Gram-negative bacterial cell walls, has been shown to impair myo- cardial functions (6, 33). This substance can induce very strong inflammatory response and acts as a powerful trigger of several inflammatory mediators, such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), and very small amounts of LPS are able to elicit the releasing of these cytokines (12). Furthermore, LPS increases reactive oxygen species (ROS) and causes intracellular protein degradation in rat heart tissue

(3, 13). The excess protein oxidation and cell mem- brane impairment may take place in cardiomyocyte in response to oxidative challenge (43), and protein turnover subsequently increases (13). The inducible effects of LPS on inflammation initiates a sequence of cellular events (e.g. elevating oxidative stress, impairing sarcolemmal integrity, and myofibrillar damage in cardiomyocytes) that result in decreased myocardial contractile efficiency and left ventricular dysfunction (2, 22, 24).

Panax ginseng (ginseng) and Salvia miltiorrhiza bunge (danshen) are very popular herbal medicine in Asian and oriental countries (10, 30). In Chinese medicine, Panax ginseng is a common booster for alleviating phys- ical strength, improving blood circulation, accelerating recovery, and delaying the occurrence of fatigue (36). Panax ginseng consists of several primary ginse- nosides (e.g. Rg1, Rb1, and Rd), which predomi- nantly account for its pharmacological effects (35). Rb1, a primary constitute in the Panax ginseng, is capable of suppressing LPS-induced lung injury in rats (18, 39). Panax ginseng extract also enhances myo- cardial contractility in cardiac cells from STZ-diabetic rats (19). Moreover, several lines of evidence also reveal that ginseng administration has protective effects on attenuating myocardial ischemia-reperfusion dam- age (11, 32). These phenomena suggest the possible protective benefits of Panax ginseng for the cardio- vascular system.

Salvia miltiorrhiza bunge is a generally used herb for the treatment of cardiovascular disease and hy- pertension (41). The major pharmacological compo- nents of Salvia miltiorrhiza bunge root are tanshinone IIA and salvianolic acid B (25). More recently, Salvia miltiorrhiza aqueous extract has been demonstrated to attenuate oxidative stress and myocardial ischemia-rep- erfusion injury in rats (11, 32). Moreover, the extract of Salvia miltiorrhiza bunge root also has in vivo anti- inflammatory activity through suppressing pro- inflammatory molecule expression, including cy- clooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) (15). These results provide a clear scientific evidence for the anti-inflammatory use of danshen in Chinese medicine.

In light of the distinct benefits of Panax ginseng and Salvia miltiorrhiza bunge on attenuating cell damage or quenching inflammation, it raises a possibility that the combination of these two herbs possibly produces better benefits than along. Therefore, we hypothesized that one-week administration of Panax ginseng and Salvia miltiorrhiza bunge mixture would attenuate the cardiac systolic dysfunction response to LPS challenge. We also hypothesized that the improvement would be due to the anti-inflammatory action and cellular pro- tective effects of these two herbs. Therefore, the pur- pose of this study was to determine whether a novel

mixture consisting of Panax ginseng and Salvia miltio- rrhiza bungecould attenuate the deleterious effects of LPS on inducing myocardial dysfunction and circu- lating pro-inflammatory cytokines.

Materials and Methods

Plant Extract and Chemicals

All the chemicals used in this study were obtained from Sigma-Aldrich Inc., USA. The mixture of Panax ginseng and Salvia miltiorrhiza (GD mixture) was obtained from Brion Research Institute, Sun Ten Pharmaceutical Co., LTD, Taiwan. High-performance liquid chroma- tography (HPLC) spectrometry method was used to analyze the functional ingredient of Panax ginseng and Salvia miltiorrhiza (5, 14). The separation column employed was Cosmosil 5C18-MS II (5 ∝m, 4.6 I.D. 5C18-M, Nacalaitesque). The recoded wavelength for ginseng and danshen was 203 nm and 280 nm respectively. The results of chromatographic quantification of func- tional ingredient in supplement have showed in Fig. 1. The functional ingredient in Panax ginseng included 2.24, 1.13, and 1.47 mg/g for Rb1, Re, and Rg1. The major functional ingredient of Salvia miltiorrhiza is Salvi- anolia acid B 28.2 mg/g and Tanshinone IIA 0.6 mg/g.

Animal Care and Maintenance

Twenty-four male Sprague-Dawley rats (SD rats) were maintained at temperature-controlled animal room (22 ± 2°C) with 50% humidity and a photoperiod of 12 h light and 12 h dark cycle. We housed all the rats in the polypropylene cages under hygienic conditions and they were allowed to freely access standard labo- ratory chow (PMI Nutrition International, Brentwood, MO, USA) and water. All experimental protocols were approved by Institutional Animal Care and Use Com- mittee (IACUC No. UT104002) of the University of Taipei.

Experimental Design and Treatment

Twenty-four male SD rats were weight-matched and assigned into three groups: [1] placebo control (Control; n = 8), [2] placebo with acute LPS challenge (LPS;

0.5mg LPS/kg; n = 8), and [3] a GD mixture (PG: 150 mg/kg; SM: 150 mg/kg; total dose: 300 mg/kg for a week; 0.5 mg LPS/kg; n = 8) with LPS challenge (GD+ LPS; n = 8). After one-week acclimation, all animals were treated with either placebo or the GD mixture for another 7 days in accordance with experimental design. The blood samples were collected in tube with ethylenedi- aminetetraacetic acid (EDTA) (24 mg/ml, pH 7.4) at baseline, 2 h and 4 h after LPS injection, and the echocar- diographic data were measured at baseline and 3.5 h after

LPS injection. Immediately after 4 h blood sample col- lection, we dissected them to collect heart tissues (left ventricle).

Echocardiography

All rats received echocardiography measurements at baseline and 3.5 h after LPS or vehicle injection. Any extra disturbance, which caused higher physiological pressure, was avoided during the process. Before each measurement, rats were sedated with pentobarbital (50 mg/kg). Then, we shaved the thorax of rats and placed them in supine position for measuring with multipurpose ultrasound system platform (Acuson Sequoia C512, SIEMENS, Munich, Germany). In short, in short-axis view, transducer was used to illustrate the image of echocardiography using 2-dimensional mode of the left ventricle. Data was measured on cardiac geometry and function, including interventricular septum (IVS), left ventricular posterior wall (LVPW), fractional short- ening (FS), LV ejection fraction (LVEF), and LV stroke volume (LVSV) as based on American Society of Echocardiology leading-edge method (27, 31).

Serum Levels of IL-6 and TNF-α

Whole blood samples were collected into tubes containing ETDA and then centrifuged at 3000 ⋅ g for 10 min. The supernatant was used to measure for IL-6 (BioLegend, San Diego, CA, USA) and TNF-α (BioLegend, San Diego, CA, USA) by commercially available kit according to the manufactures’ instructions. Firstly, sample was diluted based on preliminary test of IL-6 and TNF-α to fit in standard curve provided form commercial kits. One day prior to running the enzyme-linked immunosorbent assay (ELISA), plates were incubated with coating buffer and antibody from kit. On the day of ELISA running, we washed plate with wash buffer firstly, 1X assay diluent was added to block non-specific binding and reduce background. After incubation of 1 h at room temperature (RT), the plate was washed to remove assay diluents. Then, either sample or standard were added into well and incubated for 2 h. We washed all wells again after sample/standard incubation and added diluted Avidin-horseradish peroxidase (HRP) solution to each well. As a final wash, we pipetted wash buffer into well again. Immediately after last wash, we added freshly mixed tetramethylbenzidine (TMB) substrate solution and incubated in the dark for 25 min. We added stop solution immediately after last incubation and used ELSIA reader (TecanGENios Microplate Reader, Switzerland) to read the optical

absorbance. TNF-α and IL-6 were measured by ELISA reader at different wavelength accordingly.

Protein Assay and Western Blotting

Left ventricle tissue was weighed for 50 mg and homoge- nized with 400 ∝l Radioimmunoprecipitation assay (RIPA) buffer consisting of 150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 50 mM Tris-HCl at pH 8.0 and protease inhibitors cocktail. The homogenate was then centrifuged at 14000 ⋅ g for 10 min at 4°C. The su- pernatants were aliquoted into several tubes for further assay (9). Afterward, we assayed protein content by Broadford protein assay. Firstly, bovine serum al- bumin (BSA) served as standard and diluted into 15, 30, 62.5, 125, 250, 375, and 500 μg/ml with water as a standard curve. Secondly, samples were diluted to fit in this standard curve. Lastly, we pipetted sample or standard in duplicate on 96-well plate and added 1X dye reagent to each well. Reader (TecanGENios Microplate Reader, Switzerland) were used to measure the absorbance at 595 nm. Supernatants from aliquot were adjusted to same concentration (2 μg/μl) with 4X SDS sample buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, 0.5 M Tris-HCl) based on the result of protein content. Next, all samples were run at 10% gel of SDS-PAGE to separate different protein by molecular weight. The resolved proteins were transferred to PVDF membrane using a wet transfer unit. The membranes were then blocked in 7% non-fat dry milk in Tris-Tween-buffered saline (TTBS). Subsequently, each membrane was separated according to different molecular weight and incubated with different primary antibodies overnight and secondary antibodies for 1 h. Concentration of each primary and secondary antibody were adjusted accordingly (primary antibody: glyc- eraldehyde-3-Phosphate dehydrogenase (GAPDH), 1:2000, GeneTex; secondary antibody: anti- rabbit, 1:2000, Abcam). Three times of 5 min washing in TTBS were required between steps. Finally, the blot was visualized in Amersham enhanced chemilumi- nescence (ECL) Prime Western Blotting Detection Reagent (RPN2232, GE Healthcare, Milwaukee, WI, USA). Imaging was captured using ChemiDoc system (BioRad, Hercules, CA, USA). Image LabVersion 5.2.1 (BioRad) was used to quantify the expression of different proteins.

Statistical Analysis

All data obtained from present study was analyzed by statistical product and service solutions (SPSS) statistical software (SPSS Inc, Chicago, IL, USA) for all statistical analyzes and shown as mean ± SE. The percentage change between baseline and different time-point in all measured value were calculated

as [(value at different time-point – baseline value)/ baseline value] ⋅ 100%. In some cases with normal distribution, the treatment means were tested for homogeneity by the analysis of variance (ANOVA) and the difference between specific means was tested for significance by Duncanʼs multiple-range test. For plasma IL-6 and TNF-α, the Kruskal-Wallis one-way ANOVA was used to compare average differences among the three groups. The level of significance for all analyze was set at P < 0.05.

Results

Food Intake and Body Weight

The average body weight at baseline was 157.3 ± 7.0 g. On the experiment day, the body weight of Control, LPS, and GD+LPS groups were 237.4 ± 8.5 g, 235.4 ±

4.0g, and 235.8 ± 4.2 g, respectively. Supplementation of purified mix herbs did not alter food intake. The av- erage daily food intake of Control, LPS, and GD+LPS groups during the supplementation periods were 32.0 ± 1.3,

37.3± 1.9, and 37.4 ± 2.7 g, respectively. There were no significant differences in food intake and body weight among each group, indicating that there were no negative effects of this herb mixture on appetite and body weight in these animals.

Cardiac Function

The measurements of cardiac function were shown in Table 1. The baseline values, 2-dimentional cardiac di- mensions, area, and calculated values during diastolic and systolic period were not significantly different among groups. After two weeks, following M mode investigation and calculation, diastolic left ventricular dimension (LVDd) (P = 0.001), systolic left ventricular dimension (LVDs) (P = 0.008), systolic LVPW (LVPWs) (P = 0.057), left ventricular end diastolic volume (LVEDV) (P = 0.001), left ventricular end systolic volume (LVESV) (P

=0.028), and LVSV (P = 0.007) increased significantly. Table 1 showed that, 3.5 h after LPS injection, LPS sig- nificantly reduced diastolic interventricular septum (IVSd) (P < 0.05), systolic interventricular septum (IVSs) (P < 0.01), left ventricular dimension fraction shortening (LVDFS) (P < 0.01), LVEF (P < 0.05), and increased LVDs (P < 0.05), LVESV (P < 0.05). The GD mixture partially reversed LVDFS and LVEF but did not alter the deleterious effects of LPS on decreasing IVSd, IVSs, LVDs, and LVESV. One-week of the GD mixture supplementation significantly increased in LVSV by 24.16% above baseline in the Control group. However, the increase of LVSV was unchanged in the LPS group compared to baseline value, whereas the increase of LVSV were sustained at higher level when compared with the GD mixture provided under LPS challenge (+15.50%) (Fig. 2A). The LVEF

A

Change in Stroke Volume (%)

40

30

20

10

0

B

			6
		(%)	4
		Fraction	2
			0
		Ejectionin	-2
			-6
			-4
		Change	-10
			-8
Control	LPS	GD+LPS	-12

*

**

also showed similar protective effects as the changes in LVSV. LPS injection resulted in a significant decrease in LVEF (-8.18%), but the GD mixture slightly reversed the LPS-induced decline in LVEF (-5.1%) (Fig. 2B).

Inflammation Markers (IL-6 and TNF-α)

IL-6 concentration in LPS and GD+LPS groups sig- nificantly (P < 0.01) increased at 2 h and 4 h com- pared to Control. However, GD+LPS group had lower IL-6 values than LPS group at 2 h after LPS adminis- tration (Fig. 3A). As consider the individual variance, we calculated the response fold above baseline, and found that the responsiveness of IL-6 after LPS

124

A

	6000	Control (n = 8)
	5000	LPS (n = 8)
(pg/ml)IL6		GD+LPS (n = 8)
	3000
	4000
Plasma	2000
	0	Baseline	0	2	4

Time (h)

Tsai, Chuang, Lin, Chen, Chen and Liao

B

LPS	300	Control (n = 8)	*
to	250	LPS (n = 8)
		GD+LPS (n = 8)
IL6	200
of
	150		+
Responsiveness				* *
	100		*
	50
	0	0	2	4

Time (h)

C 100		2D Graph 1
				r = -0.513
	95
(%)				p = 0.012
	90
Fraction
	85
Ejection	80
	75
	70
	65	2000	4000	6000
	0

Plasma IL6 (pg/ml)

A 5000

(pg/ml)α	4000
Plasma TNF	3000
	2000
	0

B 100

Control (n = 8)			LPS
LPS (n = 8)
GD+LPS (n = 8)			TNFα to	80
				60
			of
			Responsiveness	40
				20
Baseline	0	2	4	0

Time (h)

Control (n = 8)
LPS (n = 8)	*
GD+LPS (n = 8)

*

0	2							4
				Time(h)

challenge had significantly lower in GD+LPS group than LPS group (LPS: +220 folds vs. GD+LPS: +101 folds; P < 0.05; Fig. 3B). Moreover, there was a significant negative relationship between circulating IL-6 levels at 2 h after LPS injection and LVEF at 3.5

hafter LPS challenge (r = -0.513, P = 0.012) (Fig. 3C). However, no significant correlations were observed be- tween circulating IL-6 levels at 2 h after LPS injection and LVSV at 3.5 h after LPS challenge.

The trend of responses of circulating TNF-α level to LPS challenge was similar to IL-6. The circulating TNF-α level was increased significantly in LPS and GD+LPS groups at 2 h, thereafter was decreased to the level of Control group at 4 h (Fig. 4A), whereas there were no differences between LPS and GD+LPS groups. Furthermore, compared with baseline, the fold changes of TNF-α level was not different between LPS and GD+LPS groups at 2 h after LPS injection, whereas the absolute values of TNF-α for GD+LPS group was lower than LPS group (GD+LPS : +25.51 folds vs. LPS: +51.07 folds) (Fig. 4B).

Protein Concentration and Expression of GAPDH

We performed the protein assay to certify the protein amount in equal weight of each sample to check whether LPS can result in proteolysis. The results showed in Fig. 5. The average protein contents were 0.96 mg in 50 mg left ventricular tissue. LPS resulted protein content loss to 0.71 mg, and GD+LPS maintain protein content at 1.0 mg. However, there were no significant differences among three groups (Fig. 5A). Following equal loading of protein performed in Western blot, the expression of GAPDH decreased by ~28% in LPS and by ~22% in GD+LPS after LPS challenge compared to Control group, but no significance was found among three group (Fig. 5B).

Discussion

To test whether a novel GD mixture consisting of Panax ginseng and Salvia miltiorrhiza bunge has myocardium protective effects after one-week of GD mixture

A1.2

		1.0
	LV	0.8
	mg
	mg Protein/50	0.6
		0.4
		0.2
		0.0

	Control	LPS	GD+LPS
B	C	LPS	GD+LPS
	LPS
	Supplement
	GAPDH
	(37 kDa)
	1.4
107)	1.2
	1.0
×
GAPDH (DensitometricSignal	0.2
	0.8
	0.6
	0.4
	0.0
	Control	LPS	GD+LPS

Fig. 5. Effect of supplement on LPS-altered protein content

(A)and GAPDH expression (B). The left ventricular tissue (50 mg) was homogenized, and the supernatant was performed for protein assay. The equal protein was loaded and performed in Western blot, including revers- ible Ponceau S. staining prior to blot with anti-GAPDH antibody. Value are expressed as mean ± SEM.

supplementation, we administered LPS intraperito- neally to determine the acute changes in myocar- dial functions and underlying mechanisms for the possible benefits. The primary findings of the present study are that one-week supplementation of the GD mixture showed a clear protective effect to preserve the rapid declines in LVSV and LVEF to an acute LPS challenge. Here we observed that the incremental degrees of IL-6 and TNF-α after LPS treatment was sig- nificantly diminished in rats with the GD mixture. Remarkably, we also detected that the LVEF inversely correlated with the increase in circulating IL-6 and TNF-α concentrations at 2 h after LPS injection. However, the GD mixture had no effect on attenuating the in- creases in circulatory TNF-α after acute LPS challenge.

These findings provide evidence that the GD mixture attenuated this acute LPS-induced myocardium dys- function through its anti-inflammatory actions.

LPS has strong deleterious effects on triggering systemic inflammation and impairing cellular functions in many organs, e.g. heart (3, 6, 12, 13, 33). More- over, LPS at doses of 0.1-20 mg/kg has been demon- strated to rapidly increase pro-inflammatory cytok- ines and ROS, thereby leading to acute impairments of vital macromolecules and subsequent cellular injury (4, 21, 39, 40, 42). In the present study, we showed that intraperitoneal administration of LPS at dose of 0.5 mg/kg might cause myocardial injury with the characteristic features of cardiac function impairments, which was confirmed by the marked decreases in LVSV and LVEF. Additionally, the re- sponses of pro-inflammatory cytokines (TNF-α and IL-6) were markedly increased after an acute LPS challenge.

The most striking findings are that one-week provision of GD mixture significantly attenuated LPS- induced decreases in LVSV and LVEF. Furthermore, the responsiveness of IL-6 increased significantly after acute LPS challenge, which was significantly attenu- ated by the GD mixture (Figs. 3, A and B). In East Asian countries, ginseng has been traditionally used to enhance physical condition, improve blood circulation, and ameliorate pathological hemostasis (36). Danshen is a generally used to treat cardiovascular disease and hypertension (41), and it was also reported to have in vivo anti-inflammatory activity through suppressing intracellular pro-inflammatory molecules expression (15). In line with the traditional therapeutic purposes of traditional Chinese medicine, here we provide new scientific evidence that a mixture consisting of ginseng and danshen is capable of alleviating acute myocardial functional impairments through attenuating infla- mmation after LPS challenge.

Acute inflammation involving macrophage activation is critical to immune defense under various external and internal pathogens in the organism (28). The most frequently seen external pathogen, LPS, triggers the plentiful release of several pro-inflammatory cytokines from macrophages including IL-6 (1) and TNF-α (16), which primarily account for the patho- physiology of septic shock, including myocardial dys- function. To evaluate the protective effects of the GD mixture on LPS-induced IL-6 and TNF-α secretions, which are major cytokines associated with inflammation, we initially confirmed its effects on suppressing the release of IL-6 but not TNF-α after LPS injection. Importantly, we observed that the decline in LVEF inversely correlated to the increase in circulating levels of IL-6 at 2 h after LPS treatment (Fig. 3C). Previous evidence reveals that elevated circulating IL-6 level presents in individuals with depressed left ventricular

systolic dysfunction even in the absence of the clinical syndrome of chronic heart failure (29, 34), suggesting IL-6 may play a critical role in the progression of subclinical LV dysfunction. Furthermore, the extract of Salvia miltiorrhiza bunge root shows a clear in vivo anti-inflammatory activity in LPS-treated Raw 264.7 cells (15). Provision of Panax japonicas (Japanese ginseng) at dose of 50-100 mg/kg for 7 days has been reported to improve myocardial function through inhibiting inflammation (38), indicating the anti-inflammatory effects of ginsenosides. More recently, Kim et al. (2016) also demonstrated that ginsenosides can attenuate LPS-induced inflammatory responses in macrophage cells through upregulating intracellular heme oxygenase- 1 (HO-1) expression (17). Taken together, we speculate that the anti-inflammatory effects of GD mixture might ameliorate myocardial functional impairments to LPS challenge through affecting the release of IL-6 from macrophage.

Although the circulating levels of TNF-α were unaffacted by the GD mixture in this study, it has to be noted that Salvianolic acid B, the major pharmacological constititue, shows clear effects on attenuating LPS-induced cardiomyote damage through inhibiting toll-like receptor

4(TLR4)-nuclear factor kappa B (NF-κB)-TNF-α sig- naling pathway (37). Therefore, the protective effects of the GD mixture on preserving myocardial dysfunction possibly mediated through suppressing the intracellular TNF-α signaling cascades but not directly inhibiting its secretion from activated marcophage in response to LPS challenge. However, the involvement of TLR4-NF-κB- TNF-α pathway in the beneficial effects of GD mixture will require further investigation.

A number of recent publications highlighted the action of LPS for inducing intracellular protein oxidative damage and proteolysis (7, 18, 23). Mehlhase et al. (2000) demonstrated that the involvement of oxidative stress in the increased proteolysis and upregulation of proteasomal system in BV-2 cells after a 16 h LPS- treatment (23). Additionally, LPS at dose of 7.5 mg/kg is capable of increasing expression of primary proteolytic ligases (i.e. MafBx and MuRF) by 20-30 folds in rat skeletal muscle tissue (7), implying that the amounts and functions of intracellular proteins could be substantially influenced to this acute challenge. Of note, both contractile proteins and signaling proteins are important for myocardiocyte to perform normal physiological functions, thus the rapid upregulation of proteasomal system and proteolysis might account for the myocardium dysfunction after LPS challenge.

Here we only observed that LPS resulted in a slight decrease in total intracellular protein amount (decreased by ~26%) and GAPDH expression (decreased by ~28%) in left ventricular myocardium (Figs. 5, A and B). The GD mixture showed a slight but not significant ef- fect on preserving total cellular protein levels (total

protein decreased by 4%; GADPH decreased by ~22%). The discrepancy between our and previous findings is probably due to different experimental models, doses and durations of LPS treatment. Presumably, the LPS dose and treatment duration in this study (0.5 mg/kg for 4 h) was relatively lower than those previous reports (7, 23). Therefore, the benefits of the GD mixture on suppressing LPS-induced proteolysis might be masked. These can help to explain why we only observed minor decline in total intracellular protein after LPS injection and why the GD mixture protective effect on preventing protein degradation was relatively lower. Neverthe- less, it has not been investigated whether Panax ginseng or Salvia miltiorrhiza bunge has inhibitory effects on proteasomal system. Future studies are therefore warranted to address the therapeutic effects of the GD mixture in the physiological relevance between LPS-induced myocardium proteolysis and cardiac ventricular dys- function.

In summary, we demonstrate that one-week sup- plementation of the mixture consisting of Panax ginseng or Salvia miltiorrhiza bunge showed a clear protective effect on preserving left ventricular systolic function after acute LPS treatment. Moreover, the GD mixture diminished the LPS-induced increase in IL-6 but had no effects on LPS-induced increases in TNF-α and cellular oxidative damage. These findings provide evidence that the ginseng and danshen mixture attenuates LPS-induced myocardium dysfunction through its anti-inflammatory action.

Acknowledgments

This work was partly supported by the Ministry of Science and Technology, Taiwan, ROC. (MOST-102-2410-H-227- 001-MY2 for L.Y.H., MOST-102-2410-H-845-014-MY3 for T.S.C., MOST-101-2410-H-002-201-MY2 for L.H.F., MOST-102-2410-H-154-005-MY2 for C.C.Y) and by the Chang Gung Memorial Hospital, Taiwan, ROC. (CMRPD1A0413 for C.C.N.).

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