Fischer rat thyroid epithelial (FRT) cells co-expressing TMEM16A and the halide sensor YFP-H148Q/I152L were cultured in F12 coon’s medium (Sigma Chemical Co. St. Louis, MO, USA) supplemented with 10% fetal bovine serum (HyClone company, USA), 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine. HT-29 cells were cultured in RPMI 1640 medium (Sigma Chemical Co. St. Louis, MO, USA) supplemented with 10% fetal bovine serum (HyClone company, USA), 100 u/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine. Cells were incubated at 37°C in the presence of 5% CO₂ and 95% humidity air. SA-11 rotavirus was a gift from Professor Verkman in UCSF.

ICR mice (8–10 weeks) were kept with a standard chew diet under specific pathogen-free conditions in a natural day/night circle at Dalian Medical University (Permit Number: SCXK liao 2008-0002). All experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and were approved by the Liaoning Normal University Committee on Animal Research.

CFTR_inh-172 was synthesized as described previously (Garcia et al., 2009). The specific TMEM16A activator E_act and CaCC inhibitor CaCC_inh-A01 were bought from Chembest Research Laboratory Limited (Shanghai). ATP and NaI were purchased from Sangon Biotech (Shanghai) Co., Ltd. Carbachol (CCh) was purchased from EDM Chemicals, Inc. (San Diego, CA, USA). Fluo-4 NW was purchased from Invitrogen (Invitrogen, Carlsbad, CA, USA). Amphotericin B was purchased from Solarbio (Beijing Solarbio Science & Technology Co., Ltd). T16A_inh-A01 was a generous gift from Professor Verkman at UCSF. Other chemicals, unless indicated otherwise, were all purchased from Sigma (Sigma Chemical Co, St. Louis, MO, USA).

To measure the inhibition of TMEM16A by shikonin, FRT cells expressing TMEM16A were seeded in a black walled clear bottom 96 well plate (Costar, Corning, NY, USA) until confluent. The cells were then washed three times with PBS followed by incubation with different concentrations of shikonin for 10 min. Fluorescence data were recorded with a FLUOstar Galaxy microplate reader (BMG Lab Technologies, Inc.) equipped with HQ 535/30M (535 ± 15 nm) emission and HQ500/20X (500 ± 10 nm) excitation filters (Chroma Technology Corp.), and syringe pumps. Fluorescence was recorded continuously for 14 s, and ATP (200 μM) were pumped into the system along with iodide at 2 s. Iodide influx rates (d[I–]/dt) were computed as described in previous study (Kristidis et al., 1992).

HT-29 cells were seeded in a 96-well black-walled microplate, and incubated until confluent. The cells were washed with PBS three times, and then incubated with Fluo-4 NW according to the manufacturer’s protocol for 15 min. Fluo-4 fluorescence was measured with a FLUOstar Optima fluorescence plate reader equipped with syringe pumps and custom Fluo-4 excitation/emission filters (485/538 nm).

Fischer rat thyroid epithelial cells expressing TMEM16A were plated in snapwell inserts and allowed to grow until 95% confluent. Snapwell inserts were mounted in Ussing chambers (Physiological Instruments, San Diego, CA, USA) connected to a VCC MC 6 multi-channel voltage-current clamp via silver/AgCl electrodes with 3 M KCl agar bridges. FRT cell monolayers were stabilized for 30-min in half-Cl^- solution (apical, containing in mM: 65 NaCl, 65 Na Gluconate, 2.7 KCl, 1.5 KH₂PO₄, 0.5 MgCl₂, 2 CaCl₂, 10 Hepes, 10 Glucose, pH 7.4.) and HCO₃^- buffered solution (basolateral, containing in mM: 130 NaCl, 2.7 KCl, 1.5 KH₂PO₄, 0.5 MgCl₂, 2 CaCl₂, 10 Hepes, 10 Glucose, pH 7.4.). After that, the cells were permeablized basolaterally with amphotericin B (250 mg/L) before activation by E_act.

Male ICR mice were killed by an overdose of intraperitoneal injection of sodium pentobarbital (100 mg/kg). The colon was removed from each animal and washed with ice-cold modified KH solution (containing in mM: 120 NaCl, 5 KCl, 1 MgCl₂, 1 CaCl₂, 10 _D-glucose, 5 Hepes, 25 NaHCO₃, 10 μM indomethacin, pH 7.4.). It was then mounted in Ussing Chambers after stripping off the muscularis. The hemi-chambers were each filled with 5 mL KH solution bubbled with 95% O₂/5% CO₂ at 37°C. Indomethacin (10 μM) was added into both hemi-chambers to prevent prostaglandin generation. Amiloride (10 μM) was added to the mucosal side to inhibit epithelial Na⁺ current.

For the measurement of apical membrane Cl^- current, the basolateral membrane was depolarized with a high potassium concentration solution. After more than 40 min, apical Cl^- current was measured in the presence of a serosal-to-mucosal Cl^- gradient. The apical solution contains (in mM): 107 K-gluconate, 4.5 KCl, 25 NaHCO₃, 1.8 Na₂HPO₄, 0.2 NaH₂PO₄, 5.75 Ca-gluconate, 1.0 MgSO₄ and 12 _D-glucose. The basolateral solution contains (in mM): 111.5 KCl, 25 NaHCO₃, 1.8 Na₂HPO₄, 0.2 NaH₂PO₄, 1.25 CaCl₂, 1.0 MgSO₄, and 12 _D-glucose.

For basolateral K⁺ current measurement, the colonic tissues were placed in a modified KH solution, the apical solution contains (in mM): 116 K-gluconate, 4.7 KCl, 5 CaCl₂, 1.2 MgCl₂, NaHCO₃, 1.2 KH₂PO₄, 11.1 _D-glucose. The basolateral solution contains (in mM): 116 Na-gluconate, 4.7 KCl, 5 CaCl₂, 1.2 MgCl₂, NaHCO₃, 1.2 KH₂PO₄, 11.1 _D-glucose. 100 μM ouabain was added to the basolateral solution to inhibit Na⁺–K⁺-ATPase. Subsequent permeabilization of the apical membrane was with nystatin (200 μg/mL).

In the measurement of Na⁺/K⁺-ATPase activity, the mucosal side of the tissue was permeablized with nystatin (200 μg/mL). All cells and tissues were aerated with 95% O₂/5% CO₂ at 37°C during the experiments. Transepithelial short-circuit currents were recorded by Acquire and Analyze 2.3 software, transepithelial potential was clamped at 0 mV during the whole processes mentioned above.

ICR mice were starved for 24 h and then orally administered shikonin (5.8 μg). After 30 min, 20 mg of 10% activated charcoal diluted in 5% gum arabic was administered orally. After another 30 min, the animals were sacrificed and the small intestines were removed. Peristaltic index was calculated as the ratio of the length that activated charcoal had traveled to the total length of the small intestine.

Neonatal ICR mice (age 4–7 days, weight 2–3 g) were inoculated with 30 μL rotavirus (virus titer 1.2 × 10⁷ pfu/mL) by oral gavage using a polyethylene tube (0.6 mm outer diameter, 0.3 mm inner diameter) and an insulin syringe. The mice were then returned to their mothers and allowed to suckle. Stool samples were collected daily by gentle palpation of the abdomen. In one set of the experiments, the shikonin-treated group received shikonin orally (0.4 and 1.7 μg in 30 μL PBS) the day before virus inoculation, and three times a day until day 3. Control mice received 30 μL PBS alone. The positive control (CaCC_inh-A01) mice received 34 μg (in 20 μL PBS) CaCC_inh-A01 by intraperitoneal injection on the day before virus inoculation and twice a day thereafter until day 3. In another set of experiments, shikonin-treated group received shikonin in PBS on the next day of virus inoculation, and three times a day until day 3. Negative control mice received 30 μL PBS alone. The positive control mice received CaCC_inh-A01 by intraperitoneal injection the day before virus inoculation and twice a day thereafter until day 3.

To collect the stool samples, the abdomen of the animal was gently palpated. To quantify the water content in the stool, a polymethyl methacrylate (PMMA) slab of 2.0 mm thickness was used in which a 1.0-mm diameter hole was punched to contain a cylindrical 1.57 mm³ volume of stool. The cylindrical stool plug was deposited onto a piece of cellulose acetate membrane (saturated with 0.1% neutral red, then dried) and allowed to contact the paper for 3 min in a humidified atmosphere. The wetted area was quantified by digital imaging.

Mice were sacrificed on day 3, and the ileum were isolated and rinsed with PBS. Specimens were fixed with formalin for 24 h, and then embedded in paraffin, and stained with haematoxylin and eosin.

All data were expressed as mean ± SE or as representative traces. One-way or two-way ANOVA followed by Dunnett’s multiple comparison test was used to compare test and control values, and statistically significances were considered at the p < 0.05 level.

This study was carried out in accordance with the recommendations of “Guide for the Care and Use of Laboratory Animals of the National Institutes of Health” and were approved by the Liaoning Normal University Committee on Animal Research. All surgery was performed under sodium pentobarbital anesthesia, and possible efforts were made to minimize suffering.

TMEM16A was the first CaCC identified, and the effect of shikonin (Figure 1A) on TMEM16A chloride channel activity was studied using a previously set up cell-based fluorescent quenching assay. Short-circuit current measurements were performed in FRT cells expressing TMEM16A. Shikonin inhibited ATP-stimulated TMEM16A chloride channel activity with an IC₅₀ value of 6.5 μM (Figure 1B). Short-circuit current measurement showed that inhibition of TMEM16A chloride currents by shikonin was concentration-dependent, and the highest concentration (10 μM) of shikonin used could only abolish about 65% of the Cl^- current stimulated by E_act (Figure 1C). On the other hand, T16A_inh-A01 (30 μM) completely inhibited E_act-induced TMEM16A-mediated short-circuit current (Figure 1D).

The efficacy of inhibition exerted by shikonin on endogenous gastrointestinal CaCC (CaCC_GI) of mouse colon was investigated. Different concentrations of shikonin were added to the mucosal solution, and 15 min later short-circuit currents were stimulated by cholinergic agonist carbachol. Inhibition of CaCC_GI current by shikonin was concentration dependent, with 5 and 20 μM shikonin reduced the currents by 24 ± 8.7 and 48 ± 10.7%, respectively (Figure 2A), and this was better than the inhibition exerted by 50 μM CaCC_inh-A01 (34 ± 8.8%).

Since CaCCs can be activated by increases in cytoplasmic calcium concentration, we therefore measured the calcium concentration following activation by ATP. It appeared that ATP-induced cytoplasmic calcium concentration was not affected by 5 μM shikonin (Figure 2B).

To exclude the possibility that the reduced transepithelial currents caused by shinkonin were due to inhibition of K⁺ transport, transepithelial currents were measured under depolarization condition. Experiments were performed under the condition that the basolateral membrane was depolarized by high concentration of K⁺. The results indicated that apical application of shikonin (20 μM) and the CaCC specific inhibitor CaCC_inh-A01 (50 μM) still could reduce CCh-induced short-circuit currents by 27.8 ± 2.7 and 30.5 ± 3.3%, respectively, as compared to the DMSO control (Figure 2C), suggesting that shikonin directly inhibited chloride currents.

The efficacy of shikonin-mediated inhibition of CFTR chloride channel activity was also tested ex vivo in freshly isolated mouse colonic mucosa. Short-circuit currents were stimulated by FSK (20 μM), and then shikonin (2–50 μM) was added into the mucosal or serosal solution to investigate the inhibitory effect of shikonin on FSK-stimulated CFTR-mediated Cl^- current. Shikonin did not affect CFTR-mediated short-circuit currents in mouse colonic epithelia (Figure 2D). The remaining currents were abolished by CFTR_inh-172 (100 μM). These results suggested that shikonin directly inhibited CaCC_GI chloride activity in mouse colonic epithelia.

Apical secretion of Cl^- in the enterocyte requires the driving force provided by Na⁺/K⁺-ATPase, Na⁺/K⁺/2Cl^- cotransporter and K⁺ channels located in the basolateral membrane. Thus inhibition of these transporters will indirectly lead to reduced apical chloride secretion in the enterocyte. We therefore analyzed the effect of shikonin on basolateral K⁺ channel and Na⁺/K⁺-ATPase in mouse colonic epithelia. As seen in Figure 3A, after permibilization of apical membrane by nystatin, shikonin decreased CCh-stimulated short-circuit current by 55.0 ± 4.3% compared to DMSO control, whereas the basolateral K⁺ channel inhibitor clotrimazole decreased the current by 82.5 ± 2.8%, suggesting that shikonin significantly inhibited Ca²⁺-activated basolateral K⁺ channel. To measure the activity of Na⁺/K⁺-ATPase, short-circuit current was analyzed after mucosal permeabilization and addition of different concentrations (5–50 μM) of shikonin to the serosal solution. As shown in Figure 3B, shikonin had no effect on Na⁺/K⁺-ATPase-mediated short-circuit current, and the nystatin-stimulated currents were completely abolished by the Na⁺/K⁺-ATPase inhibitor ouabain (500 μM), suggesting that inhibition of chloride transport by shikonin was not via inhibition of basolateral Na⁺/K⁺-ATPase activities.

Enhanced intestinal motility is also a symptom in some infectious diarrhea. TMEM16A is expressed abundantly in the ICC where it generates smooth muscle contraction, thereby mediates gastrointestinal motility (Huang et al., 2009; Hwang et al., 2009; Ferrera et al., 2010). Therefore the effect of shikonin on gastrointestinal motility was investigated (Figure 4A). Oral administration of 5.8 μg shikonin significantly reduced intestinal peristalsis and delayed charcoal movement in mice, yielding a peristaltic index of 34.9 ± 9.1%, compared to 82.3 ± 2.9% induced by PBS (Figure 4B).

The inhibitory effect of shikonin on rotaviral diarrhea in vivo was tested with two protocols as described above. Figure 5A (top) shows the first protocol used to induce rotaviral diarrhea in neonatal mice. Grossly watery stools started on day 1 after rotavirus inoculation, and it lasted for the next 3 days. Comparison of the images of the cellulose acetate membrane taken 3 min after the deposition of the stools with those taken after the removal of the stools showed the wetted area (marked by thin broken circular lines) varied among the different treatments over the 3 days (Figure 5A bottom). The water content decreased in shikonin- and CaCC_inh-A01-treated groups, as shown by the smaller wetted area. Figure 5B compares the wetted area left by the stools on days 1–3 from mice treated with shikonin or CaCC_inh-A01. In the second protocol, shikonin was also effective when administered on day 1, after watery diarrhea had begun, with significantly lower water content in the stools on days 2–3 (Figure 5C). These results demonstrated that shikonin could prevent water diarrhea following rotavirus inoculation in neonatal mice, and the effect was better than CaCC_inh-A01.

Rotavirus infection of the intestine was further verified by histology, which revealed marked vacuolization of enterocytes in the infected animals. Three days after infection, villus tip swelling and large vacuoles were seen in enterocytes lining in the surface of villi in the rotavirus-infected groups (Figure 6). Vacuoles were not seen in the non-infected mice. Shikonin-treated mice also showed vacuolization similar to that in the untreated rotavirus-infected mice, supporting the conclusion that shikonin reduced water diarrhea through antisecretory action rather than preventing rotavirus infection.