Effects of emodin and baicalein on rats with severe acute pancreatitis

Abstract

AIM: To investigate the therapeutic effects of emodin in combination with baicalein on severe acute pancreatitis (SAP) rats and to explore the mechanism of SAP.

METHODS: A total of 112 SAP rats induced by retrograde injection of 5% sodium taurocholate into the biliary-pancreatic duct, randomly assigned to a untreated group and three treated groups emodin group, combined emodin and baicalein group, and sandostatin group. Meanwhile, another 28 other rats were selected as sham operation (SO) group. There were 28 rats in each group, 8 rats were in 3 and 6 h groups respectively, and 12 rats in 12 h group. At each time-points, survival rates, ascites volumes, pathological lesion scores of pancreas tissues, serum amylase, tumor necrosis factor-α and IL-6 levels were determined as the indexes of therapeutic effects.

RESULTS: The survival rate at 12 h was significantly higher in three treated groups than in untreated group. The ascites volume at 12 h was remarkably less in combined and sandostatin groups than in emodin group, but there was no difference between combined group and sandostatin group (P>0.05). Serum amylase levels at all time-points were significantly lower in three treated groups than in untreated group. However, they had no difference among treated groups (P>0.05). Serum TNF-α were lower in three treated groups than in untreated group at all time points. Among the three treated groups, at 6 h, the TNF-α levels of combination and sandostatin groups were lower than those of emodin group. These was no difference between combined and sandostantin. Serum IL-6 concentration at 3 h were lower in combined and sandostatin groups than in untreated group, but at 6 and 12 h they were lower in all treated groups than in untreated group and the combined and sandostatin groups and in emodin group, no difference was found between combined and sandostatin groups at all time-points (P>0.05). The pathological scores of pancreas at all time points were significantly lower in three treated groups than in the untreated group, and at 6, 12 h, the scores of combined and sandostatin groups were lower than in emodin group. There was no difference between combined and sandostatin groups (P>0.05).

CONCLUSION: Combination of emodin with baicalein has significant therapeutic effects on SAP rats.

Key Words: Emodin, Severe acute pancreatitis

INTRODUCTION

Severe acute pancreatitis (SAP) has high incidence of complications and high mortality. For many years, treating SAP has been difficult. Up to now, some drugs for treating SAP have been used in the clinic, especially sandostatin, an analog to somatostatin has rather excellent effects on inhibiting pancreatic excretion and preventing complications. However, it is too expensive, especially in remote poor areas. So, it is necessary to find some cheap and high effective drugs. Emodin is a main active monomer of rhubarb, which is widely used in traditional Chinese herb as laxative, is a derivative of anthraquinone (3-methyl-1,6,8-trihydroxyanthraquinone). Baicalein is a main active monomer in herb Scutellaria baicalensis (baikal skullcap root), and belongs to glucuronid category[1,2]. A lot of studies have confirmed[3-5] that intravenous injection of emodin has some curative effects on SAP rats. Our previous research on the treatment of SAP rat using intravenous injection of baicalein showed that baicalein could attenuate pancreatic pathological lesions and reduce SAP mortality. From Chinese traditional medicine view, the effect of complex-prescriptions is better than that of simple ones. For this reason, we investigated the curative effect of combined emodin with baicalein on SAP rats and explored its mechanism.

MATERIALS AND METHODS

Main drugs and reagents

Sodium taurocholate purchased from Sigma Company was diluted to 5% solution before application. Emodin from Beijing Biological Products Clinical Laboratory, baicalein extracted from Baikal skullcap root by Pharmaceutical College of Xi’an Jiaotong University, China (purity>98%, HPLC), were prepared to 0.25% and 1% injection solution, respectively. The national invention patents have applied for the formula of Emodin injection solution and Baicalein injection solution. The patent numbers are 200410016809.X and 200410016810.2, respectively. Sandostatin was purchased from Novartis Pharma Company, Switzerland, and the test kit of amylase was obtained from Nanjing Jiancheng Bioengineering Institute. The test kits of TNF-α and IL-6 were purchased from Shanghai Shensu Biotech Company, China.

Experimental rats

Healthy male sanitary SD rats weighing 250-335 g were supplied by Animal Experimental Center of Shanxi Research Institute of Chinese Traditional Medicine, Shanxi Province, China. A total of 112 SAP rats induced by retrograde injection 5% sodium taurocholate into biliary-pancreatic duct, were randomly divided into an untreated group and three treated groups: emodin group combined emodin with baicalein group, and sandostatin group. Each group contained 28 SAP rats. Meanwhile another 28 rats were selected to perform exploratory laparotomy as sham operation (SO) group. The 28 rats in each group were further divided into three subgroups; postoperative 3 h (8 rats), 6 h (8 rats) and 12 h (12 rats) groups. At the above-correspondent time-points these rats were killed by cervical decapitation. Blood and pancreas tissue samples of the killed rats were studied.

Operative procedures and treatment

Before operation, the rats were fasted for 12 h, but allowed to drink water freely. They were anesthetized by 20% urethane intraperitoneal injection (0.5 mL/100 g) as routine. A thigh vein cannulation was set up, and through it NS (sodium chloride) solution was successively supplied at 1 mL/100 g/h speed. After abdomen was opened, according to the calculation of 4 mg/100 g, 5% sodium taurocholate was retrograde injected into the biliary-pancreatic duct at 10 mg/min speed to induce the SAP model. Meanwhile, exploratory laparatomy was performed on the rats in SO group and their pancreas was gently drawn, then the abdomen was closed. After that, emodin (0.25 mg/100 g/6 h), baicalein (2 mg/100 g/6 h) and sandostatin (0.2 μg/100 g/6 h) were transfused into the rats of each correspondent group.

Observatory indexes and measurement methods

To observe the survival of rats at each time-point, the fluid from the peritoneal cavity was taken using absorbent cotton, weighed before and after operation, and the difference of weights (g) was converted to mL. The tissue samples were fixed in 40 g/L formaldehyde, then sliced and stained with HE. Under microscopy, the pathological lesion scores were evaluated by the double blind method, the assay standard of the scores was acted in methods of Spomam[6] (Table 1). The level of serum amylase was determined by iodine-amylum chromatometry. The serum TNF-α and IL-6 levels were assayed by ABC-ELISA.

Table 1 The standard of pathological score of pancreas.

Histological changes	Classification	Score
Edema	Lobar diaphragm widen	1
	Lobar + interlobular septae widen	2
	Lobar + lobulous + interacinar septae widen	3
Infiltration of inflammatory cell	1-10 leukocyte / HPF	1
	11-20 leukocyte / HPF	2
	>20 leukocyte / HPF	3
Fat necrosis	<2 necrotic cells / per lobule	3
	3-5 necrotic cells / per lobule	5
	>5 necrotic cells / per lobule	7
Parenchymatous necrosis	Necrotic area <5% total area	3
	Necrotic area accout 5-20% total area	5
	Necrotic area>20% total area	7
Hemorrhagic focus	<2 focus / per lobule	3
	2-4 focus / per lobule	5
	>4 focus / per lobule	7

Note: HPF = 400× microscopy.

Statistical analysis

The results were presented as mean±SD. The χ² test was used to compare the difference of survival rates. The difference among various groups was assessed by variance analysis, and P<0.05 was considered statistically significant. Computations were performed with SPSS software.

RESULTS

Survival rate

At 3 and 6 h, the rats in all groups were alive. At 12 h, the survival rates of the rats in SO group and three treated groups were 100%, and 50% in untreated group. So the survival rate was significantly higher in treated groups than in untreated group (P<0.05) (Table 2).

Table 2 Survival rate of each group.

Groups	3 h (n = 8)	6 h (n = 8)	12 h (n = 12)
SO	8	8	12a
Untreated	8	8	6
Emodin	8	8	12a
Combined	8	8	12a
Sandostatin	8	8	12a

^aP<0.05, vs the untreated group.

Ascites volume

The ascites volume at 3 h was not different between untreated group and treated groups (P>0.05). At 6 h, the ascites volume in sandostatin group was significantly less than that in untreated group (P<0.05). At 12 h, the volume was significantly less in three treated groups than in emodin group (P<0.01), and it was even more significant in the combined group and sandostatin groups than in untreated group (P<0.01), but no difference was found between combined and sandostatin groups (P<0.05). However, the ascites volume in untreated and treated groups was more than that in SO group (P<0.001) (Table 3 and Figure 1).

Figure 1 Ascites volume of each group.

Table 3 Ascites volume of each group (mean±SD, mL).

Groups	3 h (n = 8)	6 h (n = 8)	12 h (n = 12)
SO	1.13±0.32	1.15±0.30	1.21±0.32
Untreated	4.51±1.37	7.06±1.91	14.72±2.36
Emodin	4.15±1.32	6.90±1.58	10.54±1.86b
Combined	4.44±1.31	6.29±1.97	8.47±1.23bd
Sandostatin	4.15±1.04	5.40±1.82a	8.26±1.07bd

^aP<0.05;

^bP<0.01 vs untreated group.

^dP<0.01 vs emodin group.

Serum amylase level

The amylase levels at each time-point were lower in SO group than in all treated groups and untreated group (P<0.001). There was no difference among three treated groups (P>0.05) (Table 4 and Figure 2).

Figure 2 Serum amylase level of each group.

Table 4 Serum amylase level of each group (mean±SD, U/L).

Groups	3 h (n = 8)	6 h (n = 8)	12 h (n = 12)
SO	1473.38±363.80	1425.00±304.85	1489.50±176.67
Untreated	4108.50±862.24	5059.63±326.35	5668.17±547.07
Emodin	3406.00±762.16a	4033.38±481.53b	4399.75±577.54b
Combined	3524.38±637.19a	3941.25±435.76b	4225.50±435.70b
Sandostatin	3518.50±636.9a	3641.00±369.02b	4255.00±605.2b

^aP<0.05;

^bP<0.01 vs untreated group.

Serum TNF-α levelL

At each time-point, TNF-α level was significantly lower in SO group than in the untreated group and all treated groups (P<0.001). Among treated groups at 6 h, the TNF-α levels in combined and sandostatin group was markedly lower than in emodin group (P<0.01), no difference was found between the combined and sandostantin groups (P>0.05) (Table 5 and Figure 3).

Figure 3 Serum TNF-α levels of each group.

Table 5 Serum TNF-α levels of each group (mean±SD, pg/mL).

Groups	3 h (n = 8)	6 h (n = 8)	12 h (n = 12)
SO	53.12±6.57	60.00±8.61	62.92±10.43
Untreated	253.25±9.69	353.88±23.67	327.50±25.62
Emodin	185.13±21.35b	306.50±16.83b	249.42±29.86b
Combined	199.38±23.69b	267.50±21.04bd	264.25±40.79b
Sandostatin	194.50±18.46b	269.50±26.87bd	232.58±23.35b

^bP<0.01 vs untreated group.

^dP<0.01 vs emodin group.

Serum IL-6 level

At each time-point, IL-6 level was significantly lower in SO group than in untreated group and all treated groups (P<0.001). At 3 h, the IL-6 levels in combined and sandostatin groups was lower than in untreated group (P<0.01). At 6 and 12 h, the IL-6 levels in three treated groups were lower than those in untreated group (P<0.01). No difference was found between combined and sandostatin groups at all time-points (P>0.05) (Table 6 and Figure 4).

Figure 4 The serum IL-6 levels of each group.

Table 6 Serum IL-6 levels of each group (mean±SD, pg/mL).

Groups	3 h	6 h	12 h
SO	72.75±13.26	65.13±13.91	69.08±12.89
Untreated	252.00±21.21	304.63±29.60	299.67±18.02
Emodin	236.25±25.06	270.63±20.85b	259.75±18.76b
Combined	217.63±22.62b	236.75±12.09bd	224.25±22.30bd
Sandostatin	214.75±24.22b	237.50±21.26bd	212.00±18.78bd

^bP<0.01 vs untreated group.

^dP<0.01 vs emodin group.

Pathological lesions of pancreas

In SO group, there was no significant abnormality in pancreas at all time-points. Mild interstitial edema and inflammatory cell infiltration could be seen in some rats. But in three treated groups and the untreated group, the pathological lesions progressed gradually. However, compared with untreated group, they were obviously attenuated in three treated groups. At 12-h points, the attenuations were more remarkable.

Pathological lesion scores for pancreas

The pathological score at each time-point had no marked difference in SO group, but it was lower in the untreated group and the three treated groups (P<0.001), and was more significantly lower in treated groups than in untreated group at different time-points (P<0.05, P<0.01). There was no difference between combined and sandostatin groups at 6 and 12 h (P>0.05) (Table 7 and Figure 5).

Figure 5 Pathological lesion score for pancreas of each group.

Table 7 Pathological lesion score for pancreas of each group (mean±SD).

Groups	3 h	6 h	12 h
SO	0.50±0.53	0.88±0.64	0.83±0.48
Untreated	7.69±0.84	10.57±2.24	13.50±3.90
Emodin	6.31±1.44b	8.75±0.97a	10.67±0.86b
Combined	6.19±0.96b	6.94±0.73ad	8.38±1.05bd
Sandostatin	5.69±0.96b	6.06±1.29ad	8.17±1.64bd

^aP<0.05;

^bP<0.01 vs untreated group.

^dP<0.01 vs emodin group.

DISCUSSION

SAP is a life-threatening disease with a high incidence of complications. Its pathogenesis has not been completely elucidated, nevertheless, experimental and clinical studies have demonstrated that a lot of factors were involved in the local and systemic complications of SAP, such as systemic inflammatory response syndrome which could lead to multiple organ failure and pancreatic necrosis, and are the major cause of deaths. Among them, an intensive systemic inflammatory response mediated by overproduction of proinflammatory cytokines and pancreatic autodigestion by activated trypsin plays a very important role. In addition, oxidative stress, secondary infection, endotoxemia are also risk factors. So that, blocking these pathogenetic chains should be the key measures in the treatment of SAP.

Chinese traditional medicine and herbs are the precious treasure of our country, “Qingyi decoction” (Pancreas clearance soup) is an effective traditional prescription in treatment of SAP and has the advantages of a low price and a high therapeutic effect. But during the therapeutic period, patients with SAP always need fasting and gastrointestinal decompression, so oral administration is disadvantageous[7-11]. Rhubarb and Baikal skullcap root are two key herbs in “Qingyi decoction”, and emodin and baicalein are their main effective components. They can be taken in the form of an intravenous injection and their prices are low. Researches have demonstrated that both emodin and baicalein have a wide, range of pharmacodynamic effects, the effects are similar and complementary to each other in many aspects. For example, they could effectively inhibit inflammatory reaction, depress the activity of pancreatins or trypsin, antibiosis, antioxidation, scavenging free-radical and anti-thrombosis. A combination of emodin and baicalein could better block the pathogenesis of SAP on multiple levels (the pharmacodynamics of emodin and baicalein is shown in Table 8). Some studies have confirmed that intravenous injection of emodin alone can improve the prognosis and decrease the mortality of SAP in rats[3-5]. Whether combined emodin with baicalein can further enhance SAP curative effect, and cut down mortality remains to be studied.

Table 8 Pharmacodynamic comparation of emodin and baicalein[12-53].

Pharmacological function	Emodin	Baicalein
Antibiosis	Escherichia coli, Dysentery Bacillus. Aureus staphylococcus	G+,G- bacterium, fungi, spirilla
Anti-inflammation	Inhibiting inflammatory cytokine release, restraining vascular permeability, inhibiting leukotrienes synthesis	Anti-histamin release. anti-arachidonic acid metabolism, restraining vascular permeability, anti-allergy
Immunoregulation	Enhance immunity	(-)
Inhibiting pancreatin activity	Strongly suppressive effect on multiple pancreatins	Only on trypsogen
Protecting liver	Anti-hepatofibrosis, antagonzism of liver damage by CCL4	Anti-hepatofibrosis,
Protecting kidney	Inhibiting kidney compensative hypertrophy. Restraining proliferation of kidney fibroblast, preventing and curing earlier injury of kidney on rats with diabetes	(-)
Protecting pancreas	(+)	(-)
Cholagog	(+)	(-)
Diuresis	(+)	(+)
Antioxidation	(+)	(+)
Scavenging free-radicals	(+)	(+)
Anti-thrombosis	(+)	(+)
Circulatory system	Dilative effect on multiple blood vessel, depressing proliferation of vascular smooth muscle cell	Double effects of vasodilatation and vasoconstriction
Gastrointestinal tract	Double regulation of isolated intestine, anti-gastric ulcer,reducing gastric acid and pepsin, purgate	Curative effect on acute and chronic gastroenteritis
Anti-endotoxemia	(+)	(+)
Fever relieving	(–)	(+)

“+”for positive pharmacological action; “–”for negative pharmacological action.

Our results showed that intravenous injection of emodin alone could significantly decrease the serum level of proinflammatory cytokines such as TNF-α, IL-6, and serum amylase concentration in SAP rat, and combination of emodin with baicalein further reduced the TNF-α and IL-6 levels. It indicated that the combination had better effect on blocking important factors of pathogenesis of SAP. At the same time, the combined group showed better therapeutic effect on reducing ascites volume and attenuating pancreatic pathological lesions than those of emodin alone. Sandostatin is considered to be one of the best drugs in the treatment of SAP. However, our results also showed that the therapeutic effect of a combination of emodin with bacalein was similar to that on SAP rats. Emodin and baicalein are easy to be extracted, isolated and identified, so they show an excellent prospect in the development of some new drugs for treating SAP.

In conclusion, the therapeutic effect of a combination of emodin with baicalein on SAP rats is better than emodin alone, and is worthy to be studied further.