In the previous study, we analyzed and reported the main contents in pu-erh tea [20]. Approximately one third (33.13 ± 3.18%) of pu-erh tea consisted of polyphenols, of which theabrownins represented a significant fraction (7.32–10.50%). The amounts of polysaccharides (4.81 ± 0.13%) and caffeine (9.31 ± 0.09%) were also determined in pu-erh tea, also as main components of pu-erh tea. The results reflected the abundance of theabrownins, polysaccharides and caffeine in pu-erh tea. Next we fed rats each a separate fraction as well as pu-erh tea, to identify which was the main assistant of scavenging NO.

An inhibitory effect of pu-erh tea on the LPS-induced iNOS expression in macrophages has been reported in cultured macrophages [21], however there were no in vivo animal studies examining the inhibitory role of pu-erh tea on iNOS, and the mechanism remains unclear. Thus, we designed the following animal experiments to study the effect of pu-erh tea. In each of the ten groups, the rats increased their weight at a constant rate. There were no differences in weight gain among the groups (data not shown). Nitric oxide production was estimated in serum from rats of group1 (water + saline) that served as controls by measuring the stable metabolite NOx (nitrite/nitrate). A normal production of NOx (13.58 ± 1.83 μM, n = 10) was found in these samples (Figure 1). LPS was found to induce a significant increase of NOx in serum (G6–G10 in Figure 1). Among the groups of the rats which were not injected with LPS, the pu-erh tea group (G2), the theabrownins group (G3) and the polysaccharides group (G5) slightly reduced the NO level in serum, compared with the water group (G1). In LPS-treated groups, NO level of G7, G8 and G10 had a significant decline compared with the water consumed LPS group (G6) as shown in Figure 1. These results indicated that pu-erh tea feeding can reduce serum NO levels, possibly through the theabrownins and/or polysaccharide components in pu-erh tea.

In liver homogenates of the rats, the metabolic level of NO was shown as group1 (water + saline) in Figure 2. A basal production of NOx was about 5.12 ± 0.33 μmol/μg of protein in liver. In the groups of rats that were administered with saline as controls, NO production in the liver was slightly inhibited by being fed with pu-erh tea and tea polysaccharides. In Figure 2, the water + LPS group (G6) had a NOx concentration in liver homogenates of 10.25 ± 0.56 μmol/μg of protein. Interestingly, the groups of rats that were fed theabrownins did not significantly reduce liver NO levels as we previously found in the serum. Only the pu-erh tea group (G7 6.11 ± 0.3 μmol/μg of protein) and the polysaccharides group (G10 4.22 ± 0.19 μmol/μg of protein) helped the rats to significantly reduce the level of NO from the liver. Figure 1 showed that the NO in serum was significantly reduced by feeding pu-erh tea, theabrownins and polysaccharides (G7, G8, G10). However, in liver, the theabrownins feeding group (G8) failed to reduce NO production, as shown in Figure 2. One possible explanation is that the theabrownins directly reacted with NO in the serum because of their ability as antioxidants as a kind of polyphenol, which scavenged the dissociative NO in the serum. However, polysaccharides could not scavenge the NO by reacting with NO directly. It likely reduced the NO production by regulating the expression of the enzymes in liver, which was tested by RT-PCR and Western blot subsequently.

The liver is an important organ that produces endogenous NO in the body [22]. Thus, we tested the NOS expression in the liver to investigate whether there was inhibition of NOS expression by pu-erh tea that attenuated NO production in rats. To investigate whether the effect of pu-erh tea on NO production in rat liver tissue was due to the modulation of NOS expression, liver homogenates were examined using RT-PCR. Both eNOS and iNOS are expressed in the liver and were assayed using the primers listed in Table 1. As shown in Figure 3A, treatment with LPS increased iNOS mRNA levels in the liver, while there was no change in eNOS levels in the control or LPS treated groups in Figure 3B. Compared with G6 (water + LPS) as control whose relative mRNA value was 1 in Figure 3A, the rats fed with pu-erh tea or tea polysaccharides for four weeks had less iNOS mRNA. The relative mRNA units of these two groups (G7 and G10) are respectively 0.57 ± 0.09 and 0.48 ± 0.12. These results were in agreement with the data presented in Figure 2, which implied that pu-erh tea inhibited NO production by strongly suppressing iNOS expression at the transcriptional level, and one of its components, polysaccharides, is likely to be the active ingredient.

Given the confirmation that pu-erh tea and its polysaccharides could help rats to reduce NO production in liver via down-regulating iNOS expression at mRNA level, we next focused on protein levels of iNOS and eNOS in the liver. Figure 4A shows that if the rats were not injected with LPS, iNOS protein was undetectable, whereas the bands of iNOS were obvious of the LPS groups (G6–G10). Consistent with mRNA expression in Figure 3, the level of eNOS proteins in the ten groups was not affected by the injection of LPS nor regulated by pu-erh tea feeding. Quantitation of the normalized iNOS and eNOS western blot levels using Image J software (version 1.45; National Institutes of Health (NIH): Bethesda, MD, USA, 2011) reveals that the G7 iNOS protein level was reduced by almost half of the control group (G6), and G10 iNOS expression was nearly one third of G6. These results were consistent with Figure 2, which supported our hypothesis that polysaccharides act mainly through inhibiting iNOS expression. Notably, the iNOS mRNA expression of G7 or G10 was not inhibited as strongly as their protein levels, comparing Figure 3 with Figure 4. This suggests another possible mechanism in addition to transcriptional suppression, possibly during the translation or processing of iNOS protein, might also be affected by pu-erh tea.

It is known that after the cells are stimulated by LPS, iNOS expression will be switched on via a specific signal pathway. LPS binds to CD14, a high-affinity LPS receptor. TLR4 interacts with this CD14-LPS complex, and then activates two intracellular signaling cascades, p38 and IKKα, which are upstream of iNOS transcription. At resting state before IKKα is activated, NFκB is bound by IκBα to repress signaling. Activation of IKKα stimulates IκBα phosphorylation, which triggers the ubiquitination and subsequent degradation of IκBα, and results in available NFκB. As a result, the transcription factor NFκB, together with AP1, which is activated by phosphorylated p38, initiates the transcription of iNOS gene [23–25]. To better understand the mechanism by which pu-erh tea and polysaccharides inhibit the expression of iNOS, we tested the expression and activities of several key proteins in the iNOS expression signal pathway. As shown in Figure 5, the cell surface receptor TLR4 and its downstream molecules, p38 and IKKα, were less phosphorylated in liver than pu-erh tea and polysaccharides feeding groups. Meanwhile, as the substrate of the IKKα kinase, more IκBα were under ubiquitination, resulting in more IκBα degradation. These results indicate that the activity of the iNOS expression signaling was restrained by polysaccharides from TLR4 which is the initiation of this pathway. We speculate that the polysaccharides of pu-erh tea may have similar binding epitopes as LPS, which resulted in polysaccharides competitively inhibiting the binding between LPS and CD14, further reducing the activity of TLR4.

TRIzol reagent was obtained from TaKaRa (Tokyo, Japan), First-Strand cDNA Synthesis Kit (Fermentas, Glen Burnie, MD, USA) and the SYBR Universal PCR Master Mix (Tiangen, Beijing, China) were used in the present research. PCR primers used in this study were synthesized by Sangon (Shanghai, China). The monoclonal antibodies used in the study were bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Cell Signaling Technology (Danvers, MA, USA). Lipopolysaccharide (LPS; Escherichia coli serotype O111:B4) and all other chemicals employed in this study were of analytical grade and were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The pu-erh tea extract, polysaccharides, theabrownins and caffeine were made and provided by China Academy of Pu-erh Tea Research (Pu Erh, Yunnan, China).

Polyphenol content analysis was performed under the guidelines of national standards using the ferrous tartrate method [26,27]. Briefly, the tea extraction solution, buffer solution and ferrous tartrate tetrahydrate solution were mixed in 25-mL capacity bottle. Absorbance (A) at 540 nm with a 10 mm quartz cell was used to calculate the extraction of tea polyphenols. Polysaccharides were quantitated using the anthrone-sulfuric acid method using glucose as standard as described [28]. A standard curve was generated with glucan, which was linear between the concentration range of 5 and 30 μg. The calibration curve equation was y = 0.063× + 0.0579 and had a correlation coefficient of R² = 0.9957. Caffeine was quantitated using the lead subacetate method [27]. A standard curve was generated with caffeine, which was linear between the concentration range of 50 and 300 μg. The calibration curve equation was y = 62.911× + 0.0058 and had a correlation coefficient of R² = 0.9997.

Male Sprague-Dawley rats weighing 280 ± 20 g were obtained from the Animal Resource Center of Jilin University. The researches on rats were approved by the Animal Committee of Jilin University. They were kept under controlled temperature (22–24 °C) and illumination (12-h light cycle starting at 7 a.m.). Animals were maintained on standard laboratory chow and water. After acclimation for 6–7 days, animals were randomly divided into 10 groups (10 animals each):

Pu-erh tea aqueous extracts were dissolved in distilled water to make an aqueous solution of 5 mg/mL. Theabrownins, caffeine and polysaccharides were dissolved into distilled water to make aqueous solutions of 1 mg/mL respectively. The rats were administrated separately by gavage feeding three times per day according to the amount mentioned above. After four-week feeding of each group’s extracts mentioned above, treatment rats were administered either 5 mg/kg, i.p., LPS dissolved in sterile saline. Control rats were injected with an equivalent volume of sterile saline (5 mL/kg). Six hours later, animals were sacrificed, having first been subjected to blood and liver sampling.

The blood was collected in heparinized tubes. The liver samples were homogenized in cold 0.9% saline. The homogenates were then centrifuged at 10,000 r/min for 5 min at 4 °C, the supernatant was taken for NO assay and total protein determination. Nitric oxide production was determined by measuring in serum samples and liver homogenates total NOx, the stable end products, using a modification of Griess’s reaction, as previously described [20].

Homogenates of liver tissue were examined for estimation of total protein per well using the Bio-Rad protein microassay. The protein assay was based on Bradford’s dye-binding procedure [29]. Briefly, known concentrations of bovine serum albumin were used as standard curves. Two hundred microliters of sample or standard and 50 μL of Bio-Rad protein assay was added per well in a 96-well microtiter plate and protein was measured with a microplate reader.

Total RNA was isolated from approximately 50–100 mg snap-frozen liver tissue using the TRIzol protocol as suggested by the supplier. The purity of total RNA was determined by the ratio at 260 nm and 280 nm absorbance. 2 mg of total RNA was subjected to reverse transcription using RevertAid™ First-Strand cDNA Synthesis Kit with oligo-dT. 1 μL of the cDNA solution was used for real-time PCR. The genes were amplified in a 25 μL reaction on Bio-Rad Opticon Monitor (Applied Biosystems). The conditions comprised an initial denaturation step at 95 °C for 5 min, followed by 40 cycles each of 95 °C for 30 s, 60 °C for 15 s and 72 °C for 1 min, before being subjected to melting curve analysis. Amplified products were further verified by automated cycle sequencing. The 2^−ΔΔCT method [30,31] was used for calculating the relative expression level. The primer sequences of the proteins were shown in Table 1.

Homogenates of liver tissue were centrifuged at 12,000 g for 20 min at 4 °C. A total of 30 μg protein from each well was separated by 8% (or 10%) SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked at room temperature for 1 h with 5% skim milk in tris-buffered saline with 0.1% Tween20 (TBST), and followed by incubation with primary antibody diluted with 5% skim milk in TBST at 4 °C overnight and then incubated with secondary antibody conjugated to alkaline phosphatase and detected using the ECL chemiluminescent system. Loading differences were normalized using a monoclonal β-actin antibody.

The results were presented as mean ± SE. One-way ANOVA was used for a statistical comparison. (A probability level of 5%, p < 0.05 was considered statistically significant).